CN111084098A

CN111084098A - Hypsizygus marmoreus H18 and cultivation method thereof

Info

Publication number: CN111084098A
Application number: CN202010002887.3A
Authority: CN
Inventors: 余荣琳; 黄志勇; 胡卫红; 于潇
Original assignee: SHANGHAI XUERONG BIOTECHNOLOGY CO Ltd
Current assignee: SHANGHAI XUERONG BIOTECHNOLOGY CO Ltd
Priority date: 2020-01-02
Filing date: 2020-01-02
Publication date: 2020-05-01
Anticipated expiration: 2040-01-02
Also published as: CN111084098B

Abstract

The invention relates to the field of fungi, in particular to hypsizygus marmoreus H18 and a cultivation method thereof. The novel crab-flavor mushroom variety 'crab-flavor mushroom H18' obtained by systematic breeding can be well adapted to the production mode of liquid strains of the crab-flavor mushroom, and has remarkable effects in the aspects of improving unit yield, optimizing product form and the like: in the culture and cultivation fruiting stage, the yield of the hypsizygus marmoreus H18 is highest, the average yield per unit is more than 282.3 g/1100 mL, and the factory cost can be effectively reduced; in the aspect of agronomic characters, the crab-flavored mushroom H18 of the applied patent variety has dark color and obvious patterns. Fills the blank of the cultivation mode of the liquid spawn of the hypsizygus marmoreus.

Description

Hypsizygus marmoreus H18 and cultivation method thereof

Technical Field

The invention relates to the field of fungi, in particular to hypsizygus marmoreus H18 and a cultivation method thereof.

Background

Hypsizygus marmoreus belonging to the class of Hyphomycetes, the class of Hymenomycetes, the order Agaricales, the family of Tricholomataceae, the genus Hypsizygus, also known as Hypsizygus Marmoreus, Hypsizygus marmoreus, etc., is a large woody saprophytic fungus. In natural environment, generally, autumn grows on dead or living hardwood trees such as beech. The crab-flavored mushroom is a good rare delicious edible mushroom in northern temperate zone. Currently, the yield of the Hypsizygus marmoreus is highest in Japan all over the world. The fruit body grows to a cluster. The surface of the pileus is nearly white to grey brown, and the center of the pileus is often provided with dark marbling. The fungal folds are nearly white, are rounded with stipe and are dense to be slightly thin. When the crab-flavored mushrooms grow laterally, the stipe grows slightly, the spores are printed to be nearly white, and the shape of the spores is wide in an egg shape to be nearly spherical.

The crab-taste mushroom contains rich vitamins and 17 amino acids, wherein the content of lysine and arginine is higher than that of common mushrooms, which is helpful for increasing intelligence and increasing intelligence of teenagers, resisting cancer and reducing cholesterol, particularly, the extract of fruit bodies (namely, the parts above roots) has various physiological active components, wherein, fungal polysaccharide, purine and adenosine can enhance immunity, promote the formation of antibodies and antioxidant components can delay senility, beautify the face and the like, β -1, 3-D glucan extracted from the fruit bodies of the crab-taste mushroom (hypsizigus marmoreus) has high antitumor activity, and the activity of polymeric carbohydrase separated from the hypsizigus marmoreus is much higher than that of other mushrooms, and the hot water extract and the organic solvent extract of the fruit bodies of the hypsizigus marmoreus have the function of removing free radicals in vivo, so that the crab-taste mushroom has the unique efficacies of preventing constipation, resisting cancer, preventing cancer, improving immunity, preventing senility and prolonging life.

The crab-taste mushroom is fresher than oyster mushroom, the meat is thicker than slippery mushroom, the quality is tougher than mushroom, the taste is excellent, the crab-taste mushroom also has unique crab-taste, and the theory that the mushroom is fragrant in tricholoma matsutake and fragrant in Hypsizygus marmoreus is provided in Japan. The hypsizigus marmoreus is introduced in 80 s of China, and is mainly cultivated in Shanxi, Hebei, Henan, Shandong and Fujian on a small scale, and the salted mushrooms are exported to Japan. In recent years, the scale is gradually enlarged, the scale is distributed throughout the country, and the factory production is realized.

Most of the industrialized production of the beech mushrooms in the current market utilizes solid strains to cultivate mushrooms, and the cultivation mode has the defects of longer cultivation period and general yield.

Disclosure of Invention

In view of the above, the invention provides hypsizygus marmoreus H18 and a cultivation method thereof. The novel crab-flavor mushroom variety 'crab-flavor mushroom H18' obtained by systematic breeding can be well adapted to the production mode of the liquid spawn of the crab-flavor mushroom, has obvious effects on the aspects of improving unit yield, optimizing product form and the like, and fills the blank of the cultivation mode of the liquid spawn of the crab-flavor mushroom.

In order to achieve the above object, the present invention provides the following technical solutions:

the systematic breeding of edible fungi, namely selecting excellent natural variation from the existing variety group, and breeding a new variety through comparative identification, wherein the quality of the new variety is excellent. The specific process is as follows: original strain-hypha turning and inoculating-shake flask culturing-fermentation tank culturing-fruiting comparing-sporocarp tissue separating-establishment of preferred strain. The invention obtains a new variety of the hypsizygus marmoreus, namely hypsizygus marmoreus H18, by utilizing crossbreeding and systematic breeding and selecting the excellent medium. The breeding process is shown in figure 1.

The invention provides a beech mushroom with a preservation number of CGMCC No. 18154.

On the basis of the research, the invention also provides application of the Hypsizygus marmoreus in preparing medicines for reducing cholesterol, enhancing immunity, delaying aging and preventing constipation.

In addition, the invention also provides the liquid culture medium of the Hypsizygus marmoreus, wherein 120L of the culture medium comprises:

the invention also provides the culture medium for the Hypsizygus marmoreus, which comprises the following components in parts by mass:

on the basis of the research, the invention also provides a method for breeding the Hypsizygus marmoreus system, which comprises the steps of taking a1c3 obtained by hybridizing H6 with H27, and hybridizing the A1c3 with K8 to obtain a Hypsizygus marmoreus strain; taking the Hypsizygus marmoreus strain, performing hypha turning, culturing, fruiting, selecting fruiting bodies with robust mushroom bodies and good mushroom shapes, performing tissue separation, and inoculating into a PDA culture medium; selecting a strain with high yield, robust mushroom body and good mushroom shape, namely the crab-flavored mushroom.

The invention also provides a method for culturing the Hypsizygus marmoreus, which comprises the step of inoculating the Hypsizygus marmoreus to a culture medium for culture.

In some embodiments of the invention, the culturing comprises hyphal inversion, shake flask culture, fermentor culture, and culture flask culture.

In some specific embodiments of the invention, the hypha inoculation adopts a PDA culture medium, the inoculation amount of the Hypsizygus marmoreus is 1 block, the size of the block is 5 × 3mm, and the number of viable bacteria is 700-800 cfu/g;

the inoculation amount of the hypsizygus marmoreus in shake flask culture is 4 bacterium blocks, the size of each bacterium block is 5 × 3mm, and the number of viable bacteria is 700-800 cfu/g;

the inoculation amount of the hypsizygus marmoreus in the fermentation tank culture is 600mL of bacterial liquid, and the viable count is 750-900 cfu/mL;

the inoculation amount of the hypsizygus marmoreus in the cultivation bottle culture is 20-25mL of bacterial liquid, and the viable count is 750-900 cfu/mL.

In some embodiments of the present invention, the shake flask culture temperature is 21-23 ℃ and the culture rotation speed is 130 rpm/min; the culture time is 8 days;

the culture condition of the fermentation tank is to culture for 6 to 7 days at the temperature of between 22 and 23 ℃;

the cultivation bottle culture comprises the following steps: CO at the temperature of 21-23 ℃ and the humidity of 70-80 percent₂Culturing for 70-80 days under the condition that the concentration is not higher than 4000 ppm.

In some embodiments of the invention, the conditions for inducing budding after scratching are: the temperature is 14-16 ℃, the humidity is 90-95 percent, and CO is₂The concentration is within 2000ppm, the illumination intensity is 50 lux-100 lux, and the bud forcing time is 8-10 days; the conditions for the growth of the sporocarp are as follows: the temperature is controlled to be 14-17 ℃, the humidity is more than 90 percent, and CO is₂The concentration is less than 2000ppm, the illumination intensity is 250-500 lux, and the growth time is 14-17 days.

Compared with the Hypsizygus marmoreus on the market, the Hypsizygus marmoreus H18 of the patent application has the following advantages:

1. preliminarily judging that the crab-flavor mushroom H18 and other tested varieties are different white beech mushroom varieties through an antagonistic experiment;

2. in the stage of culturing and cultivating fruiting, the yield of the 'crab-flavor mushroom H18' of the applied patent variety is the highest, and the average yield per unit is more than 282.3 g/1100 mL, so that the factory cost can be effectively reduced;

3. in the aspect of agronomic characters, the crab-flavored mushroom H18 of the applied patent variety has dark color and obvious patterns.

The test data are recorded in detail in the above technical scheme of the invention. The following is the biological characteristics of the crab-flavor mushroom H18 of the patent application.

Mycelium: the hypsizygus marmoreus H18 hyphae grow vigorously at 20-24 ℃, and the hyphae are dense, have high growth speed and general high temperature resistance.

And (3) mushroom cap: the crab-flavored mushroom H18 is hemispherical in shape when the cap is young, dark brown, gradually flat and light in color and is yellowish brown; dark middle part, dark brown; the cover surface is smooth and has 2-3 circles of stripes; the cover edge is flat or slightly bent downwards and is slightly wavy; the diameter of the pileus is 2-13 cm.

And (3) pleating: the crab-flavored mushroom H18 has white to light yellow crenulated, curvelent, sometimes slightly straight, dense and unequal length, and is separated from the raw material.

Stipe: the crab-flavored mushroom H18 is middle-growing and cylindrical, grows 3-12 cm, is obviously expanded at the lower part when young, is white to grey white, is O.5-3.5 cm thick, is thin at the upper part and thick at the lower part, is almost the same in upper and lower thickness when fully grown, is mostly slightly bent, has yellow brown stripes, is middle-full, and is soft at the inner part when being aged.

Fruiting body: clumps, 15-50 plants per clump are different, with less scatter. The mushroom meat is white, tough and crisp in texture, compact and slightly bitter.

Spore: basidiospores are colorless, smooth, spherical, and white in spore impression. Conidia are white and appear at the end of aerial hyphae when culture conditions are not suitable.

Biological preservation Instructions

Biological material: h18; and (3) classification and naming: hypsizigus marmoreus (Hypsizygus marmoreus); the strain is preserved in the China general microbiological culture Collection center in 2019, 08 and 21 months, and the preservation center addresses are as follows: the institute of microbiology, national academy of sciences No.3, Xilu No.1, Beijing, Chaoyang, Beijing; the preservation number is CGMCC No. 18154.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the process of "Hypsizygus marmoreus H18" selection;

FIG. 2 shows a process flow for producing "Hypsizygus marmoreus H18";

FIG. 3 shows the hyphal growth morphology of the strains; from left to right are: "H6", "K8", "H18" and "H27";

FIG. 4 shows antagonism of strains with each other; the first row, from left to right, is: "H18" - "H6", "H18" - "K8", "H18" - "H27"; the second row, from left to right, is: "K8" - "H6", "H6" - "H27", "K8" - "H27";

FIG. 5 shows fruiting photographs of different Hypsizygus marmoreus strains;

FIG. 6 shows an electrophoretogram of genomic DNA of the test strain "H18" provided by the present invention;

FIG. 7 shows a Hypsizygus marmoreus strain ITS phylogenetic tree;

FIG. 8 shows the biological characteristics of "Hypsizygus marmoreus H18";

FIG. 9 shows the ITS sequence alignment of Hypsizygus marmoreus;

FIG. 10 shows the DNA sequencing result of Hypsizygus marmoreus H18.

Detailed Description

The invention discloses a Hypsizygus marmoreus and a culture method thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The terms:

crab-flavored mushroom: belonging to Basidiomycotina, Hymenomycetes, Agaricales, Tricholomataceae, Hypsizygus marmoreus, etc., and is a large wood saprophytic fungus.

Scratching fungi: the old strain blocks and the fungus skin are removed by a mycelium stimulation machine (or manually), which is an important measure for promoting the mycelium to generate primordium, and the fruiting bodies can be regularly generated from the surface of a culture medium by the mycelium stimulation

Fruiting body: is the spore-forming structure of higher fungi, i.e. fruit body, and is composed of organized mycelium. Basidiomycetes are called Basidiomycetes and ascomycetes are called ascomycetes. Whether sexual reproduction or asexual reproduction, whether simple or complex in structure, the spore-forming structure is called a sporophore.

Primordia: before the edible mushrooms grow out, the hyphae are twisted to form a small particle, and the grown small particle is a mushroom.

Hypha: a single tubular filament, the structural unit of most fungi, and some prokaryotes also have hyphae, such as actinomycetes.

Mycelium: many hyphae aggregate together to form the trophozoite of the fungus, i.e., the mycelium.

Mutation breeding: a breeding method for generating variation in DNA molecules of gene carrier by physical or chemical factors, and selecting and using the variation. Physical factors such as ultraviolet light, x-rays, r-rays, isotope Co60, laser, and the like. Chemical mutagens act directly on DNA molecules and can be generally classified into three main groups according to their chemical properties or mode of action: (1) base analogues such as 5-bromouracil and a-aminopurine; (2) mutagens that change the base structure of DNA, such as nitrous acid, diethyl sulfate, ethyl methanesulfonate, nitrosoguanidine, etc.; (3) the frame shift mutagens, such as acridines, pyridines, polycyclic hydrocarbons, nitrogen heterocyclic carcinogens, mycotoxins, antibiotics, are the most studied acridines.

The protoplast fusion technology is a process of fusing protoplasts of two cells with different genetic traits by a manual method so as to obtain a stable recombinant with parental genetic traits.

Most of the existing hypsizygus marmoreus in the market is obtained by solid culture fruiting, the culture period of the production mode is longer, the yield is general, and aiming at the defects, the invention obtains a new variety of hypsizygus marmoreus, namely hypsizygus marmoreus H18, through systematic breeding, the new variety can be well adapted to the production mode of hypsizygus marmoreus liquid strains, the yield per unit is improved, the form of hypsizygus marmoreus products is optimized, the cultured hypsizygus marmoreus is deep in color, obvious in pattern and good in uniformity, and the blank of the cultivation mode of the hypsizygus marmoreus liquid strains is filled.

The Hypsizygus marmoreus and the raw materials and reagents used in the culture method thereof provided by the invention can be purchased from the market.

The invention is further illustrated by the following examples:

example 1

1 materials and methods

1.1 test strains

H6: hypsizygus marmoreus original strain collected from Japan market, and obtained by tissue isolation

H27: collected in Shanghai market and obtained by tissue isolation

K8: collected in Shanghai market and obtained by tissue isolation

H18: h6, H27 and K8 are subjected to single-spore cross breeding, specifically, a first filial generation of H6 and H27 is subjected to single-spore cross breeding, and then the first filial generation and K8 are subjected to single-spore cross breeding to obtain the H18 strain.

1.2 culture Medium

PDA culture medium: 4% Japan agar medium, natural pH.

Liquid culture medium: 2400g of white granulated sugar, 360g of soybean meal powder, 60g of corn flour, 72g of potassium dihydrogen phosphate, 72g of magnesium sulfate, and adding tap water to 120L, wherein the pH is natural.

Cultivation medium: 46.6 percent of corncob, 11.9 percent of bran, 10.4 percent of cottonseed hull, 22.7 percent of rice bran, 5 percent of corn flour, 1.5 percent of shell powder, 1.8 percent of calcium carbonate, 845 plus or minus 20 grams of bottle filling weight and 64 to 66 percent of water content.

The raw material requirements are as follows: the crab-taste mushroom industrial cultivation raw materials mainly comprise corncobs, cottonseed hulls, bran, rice bran, corn flour and the like. The raw materials must be kept fresh and dry, and the raw materials are free from worm damage and mildew, and the granularity meets the requirements of equipment and technology. Ventilating in the process of storing raw materials, and keeping the storage environment dry.

Mixing raw materials: accurately quantifying according to the formula requirement, fully and uniformly stirring the materials, and adding water until the water content reaches 64-66 percent and the pH value is 6.5-7.0. And adjusting the stirring time according to seasonal changes to prevent the mixture from rancidity, and stirring until bottling is finished.

Bottling: the high-temperature resistant plastic bottle is used, the filling compactness is required to be uniform, the weight of a 1100mL plastic bottle filled with the high-temperature resistant plastic bottle is 845 +/-20 grams, the middle part of the plastic bottle is punched to the bottom of the plastic bottle, and the plastic bottle cover is immediately covered after the plastic bottle is filled with the high-temperature resistant plastic bottle.

And (3) sterilization: autoclaving was carried out immediately after bottling. The sterilization time is that the central temperature of the material must reach 118-121 ℃ and be kept for 30 minutes, and all microorganisms and spores in the culture material must be killed thoroughly.

And (3) cooling: after sterilization, the bottle frame is pushed into a cooling chamber for forced cooling, and the cooling chamber and the inoculation chamber are provided with a purification device and refrigeration equipment.

1.3 liquid seed preparation

And (3) sterilization: after the liquid culture medium is prepared, putting the liquid culture medium into a vertical sterilizing pot for sterilization, wherein the sterilization process comprises the following steps: and keeping the temperature at 121 ℃ for 60 minutes. After sterilization, the liquid culture medium is placed in a clean area for cooling, and inoculation can be carried out after the liquid culture medium is cooled to 18-22 ℃;

inoculation: the ultra-clean workbench is opened for 30min, the ultraviolet lamp is opened for 30min, then a person wears the special clean clothes for inoculation, wears a mask and medical gloves, wears clean zone work shoes, enters the clean zone after air showering, and is operated aseptically on the ultra-clean workbench (note: all tools are sterilized by high-temperature high-pressure steam). The selected plate seeds were inoculated into shake flasks using an inoculation tool.

Tearing off a sealing film of a selected plate strain to be used → tearing off a plate cover by a cover-tearing clamp → clamping the plate by a clamp of a plate-supporting clamp → scraping off aerial hyphae on the surface of the plate by an inoculation hook → uniformly punching by a puncher → fixing the plate in the clamp of the plate-supporting clamp → placing on a tripod → igniting the inoculation hook → placing on a tool rack for cooling → taking off aluminum platinum paper, taking off a bottle stopper of a shake flask by crucible tongs, placing on the tool rack → taking 4 small pieces of mother seeds from the upper part, the lower part, the left part and the right part of the plate by the inoculation hook respectively, placing on the shake flask → the bottle stopper, sealing the aluminum platinum paper → finishing inoculation, and recording strain batch numbers and inoculation dates.

And (3) inoculating hypha by using a PDA (personal digital assistant) culture medium, wherein the inoculation amount of the hypsizygus marmoreus is 1 block, the size of the block is 5 × 3mm, and the number of viable bacteria is 700-800 cfu/g.

The inoculation amount of the Hypsizygus marmoreus in shake flask culture is 4 bacterium blocks, the size of each bacterium block is 5 × 3mm, and the viable count is 700-800 cfu/g.

Culturing: placing the inoculated strains into a shaking table for shake culture, placing the well inoculated shake flasks into a shake culture device for culture, wherein the culture temperature is as follows: 22 +/-1 ℃, culture rotation speed: 130 rpm/min; culturing time: and 8 days, taking out after the culture is finished, and preparing for inoculation and cultivation.

1.4 cultivar preparation

Each strain is made into 10 baskets, 480 bottles are totally reserved, 1 basket is randomly drawn after mycelium stimulation, agronomic characters such as mycelium color change, primordium bud development time, primordium quantity, uniformity, harvesting time and the like are observed, and average unit yield is calculated after all the strains are harvested.

The compost is filled by a bottling machine after being stirred uniformly, wherein 845 +/-20 grams of water is contained in 1100mL plastic bottles per bottle, the water content is adjusted to 64% -66%, the pH is adjusted to 6.4-7.2, and the compost is sterilized at high temperature and kept at 100 DEG CHeating to 121 ℃ for 60min, keeping the temperature for 60min, cooling to below 22 ℃ in a cooling chamber, inoculating the liquid spawn of the Hypsizygus marmoreus with the inoculation amount of 20-25 mL. Then placing the mixture at the temperature of 21-23 ℃, the humidity of 65-70 percent and CO₂Culturing in culture room with concentration of 2000-₂And (4) fruiting in a growth room with the concentration of less than 2000ppm, irradiating the fruiting room with certain light during the period, harvesting after the fruiting standard is reached, and weighing. Various parameters are recorded.

The inoculation operator must meet the requirement of aseptic operation, the temperature in the bottle is controlled below 25 ℃ during inoculation, microscopic examination is used, the liquid strain is ensured to be pollution-free and vigorous, the liquid strain can be used for inoculation cultivation, the inoculation amount of each cultivation bottle is 20-25mL of bacterial liquid, the wet weight of the mycelium is about 2.7g/20mL, and the viable count is about 820 cfu/mL.

1.5 cultivation for growth and fruiting

Culturing: the temperature of the culture room is controlled to be 21 +/-2 ℃, the humidity is maintained to be 70-80 percent, and the concentration of CO2 is controlled to be below 4000 ppm. The culture time is about 75 days.

Scratching fungi: after the culture is mature, mycelium stimulation treatment is carried out. A special mycelium stimulation tool bit is adopted, water is injected after mycelium stimulation, and the water injection amount is 15-20 mL.

Bud forcing: the indoor temperature is controlled to be 14-16 ℃, the humidity is controlled to be 90-95%, the concentration of CO2 is within 2000ppm, and the illumination intensity is 50-100 lux. The bud induction time is 8-10 days.

And (3) fruiting body growth: the temperature is controlled to be 14-17 ℃, the humidity is more than 90%, the concentration of CO2 is less than 2000ppm, the illumination intensity is 250-500 lux, and the light starting time is adjusted according to different growth stages. The growth time is 14-17 days.

Harvesting and packaging: the diameter of the pileus reaches 18mm, the length of the stipe reaches 80mm, and then the mushroom can be harvested. When in collection, the whole is collected, the culture medium is cut off, the whole is packed by an automatic packing machine and then is put into a cold storage, and the temperature of the cold storage is controlled between 3 ℃ and 5 ℃.

Digging a bottle: the harvested bottle frames are timely transported to a bottle digging area, the culture materials are dug, the empty bottle baskets are transported to a bottling area to be reused, and the waste materials are cleared and delivered out of the factory.

Example 2 hypsizygus marmoreus Strain vegetative growth stage hyphal growth characteristics

After the mycelium is cultured for 14 days, the 'H6' aerial mycelium is coarse and short, and has high concentration and whiteness which are more polished; h27 aerial hypha is slender, has slightly low whiteness and grows stolonically; the K8 aerial hyphae are thick, white, vigorous and uniform; the hypsizygus marmoreus H18 has exuberant and strong and uniform aerial hyphae, and the colony morphology is between the strains K8 and H27. As shown in fig. 3.

Example 3 hyphal antagonism between different strains

As can be seen from FIG. 4, the antagonistic lines between different strains are very obvious, which indicates that the strains are very different, and the strains are preliminarily judged to be different Hypsizygus marmoreus strains.

Example 4 comparison of the cultivation yields per unit of cultivation of different strains

In the fruiting stage of cultivation, the strain H27 fails to produce mushroom. As can be seen from Table 1, the yield per unit of the strain "Hypsizygus marmoreus H18" is significantly higher than that of the other two varieties, and is averagely higher than that of 17.45 g/bottle, wherein the most significant difference is that the yield per unit of the variety "H6" is higher than that of the variety "H18" by 28.7 g/bottle than that of the variety "H6".

TABLE 1 yield per unit of different Hypsizygus marmoreus strains

TABLE 2 independent sample Biometrics test results

The yield per unit data of the variety H18 and the varieties H6, H27 and K8 are analyzed by a biometric software spss to obtain a result shown in Table 2, and compared with a control bacterium, the yield per unit of the variety H18 is very different (P is less than 0.01).

Example 5 comparison of agronomic traits of different strains

The quality of the agronomic characters is directly related to the quality of the hypsizygus marmoreus as a commodity and the cost of an edible fungus factory. The yield of the ficus microcarpa hypsizygus marmoreus variety 'hypsizygus marmoreus H18' of the patent application is slightly improved compared with that of the original strain, the average yield per unit is more than 282.3 g/1100 mL, and the strain cap of the 'hypsizygus microcarpa H18' is dark in color, obvious in pattern and uniform.

TABLE 3 comparison of pileus color of different Hypsizygus marmoreus strains

TABLE 4 independent sample Biometrics test results

The color data of the pileus of the variety "H18" and the varieties "H6", "H27" and "K8" were analyzed by the biometric software spss to obtain Table 4, and the pileus of the variety "H18" had a very significant difference (P < 0.01) compared with the control bacteria.

TABLE 5 comparison of pileus patterns of different Hypsizygus marmoreus strains

TABLE 6 independent sample Biometrics test results

The data of the pileus patterns of the cultivars "H18" and "H6", "H27" and "K8" were analyzed by the biometric software spss to obtain table 6, and the pileus pattern of the cultivar "H18" was significantly different from that of the control strain (P < 0.01).

Example 6 molecular identification report of "Hypsizygus marmoreus H18

6.1 test varieties

H6: original strain of Hypsizygus marmoreus introduced in Japan

H27: collected in Shanghai market and obtained by tissue isolation

K8: collected in Shanghai market and obtained by tissue isolation

H18: is prepared from H6, H27 and K8 through cross breeding and systematic selective breeding, and the crab-taste mushroom H18

6.2 test Medium, reagent and preparation method

(1) PDB liquid medium: 200g of peeled potatoes, 20g of glucose and distilled water to a constant volume of 1L.

(2) Genomic DNA extraction reagent:

0.424g of LETS buffer LiCl, 0.372g of EDTA, 0.242g of Tris and 0.5g of SDS (after the reagents are weighed, the reagents are dissolved in 90ml of sterile redistilled water, the pH value is adjusted to 7.8-8.0 by HCl, finally the volume is determined to be 100ml, protease K is added according to the ratio of 50mg to 100 ml)

PCI reagent phenol chloroform isoamyl alcohol (ratio 25: 24: 1)

(3) ITS amplification reaction reagent

The primer sequence is as follows: the ITS region was amplified using the fungal universal primer ITS1F (Gardes & Bruns,1993)/ITS4(White et al 1990) with the following primer sequences:

ITS 15 '-TCCGTAGGTGAACCTGCGG-3' (shown in SEQ ID No. 2)

ITS 45 '-TCCTCCGCTTATTGATATGC-3' (shown as SEQ ID No. 3)

The primers were synthesized by England JunvyBiotechnology GmbH, and prepared into 10 μ M reaction solution, and subpackaged at-20 deg.C for use.

Agarose for electrophoresis (Spanish)

10 XPCR reaction buffer, 25mM MgCl₂TaqDNA polymerase (Promega)

10mM dNTP (Genview split)

(4) DNA gel recovery kit (Axygen)

6.3 Main instrumentation

81-2 model produced by Shanghai Si le Instrument plant with constant temperature magnetic stirrer

Swing bed for Shanghai Yiheng

Sterilized pot SS-325, TOMY SEIKO CO.

Vacuum freeze dryer EDWARDS, 4K2-4L

PCR amplification apparatus Mastercycler Gradient (Eppendorf)

BIO-RAD (bipolar junction ionization-Radar) level plate electrophoresis instrument

Camera system Image Master VDS (Pharmacia Biotech)

6.4 Experimental methods

(1) Hypha culture and extraction of genomic DNA

Inoculating cultured strain (PDA) into PDY liquid culture medium, culturing at 25-28 deg.C under shaking (150r/min) for 7-9 days, collecting mycelium, washing with sterile ultrapure water for 2-3 times, freeze drying, and storing at low temperature. Genomic DNA was extracted using a modified LETS method. The method comprises the following specific steps:

① grinding the lyophilized mycelium in a pre-cooled sterile mortar, adding appropriate amount of LETS buffer, centrifuging at 12000rpm and 4 deg.C for 20min

② adding equal volume of PCI reagent into the supernatant, mixing gently for more than 10min, centrifuging at 12000rpm and 4 deg.C for 10min, and repeating the steps for 3-4 times.

③ collecting supernatant, adding 2/3 volume of-20 deg.C pre-cooled isopropanol, mixing, placing in-20 deg.C refrigerator, centrifuging at 10000rpm and 4 deg.C for 10 min.

④ the pellet was washed 2 times with 75% ethanol, 10mM KAc, 10000rpm, 4 ℃ and centrifuged for 5 min.

⑤ adding pre-cooled 95% ethanol at-20 deg.C into the residue, mixing, centrifuging at 10000rpm and 4 deg.C for 10min, and air drying.

⑥ the precipitate was dissolved by adding an appropriate amount of TE buffer, and 2. mu.l of RNase (1mg/ml) was added in a 37 ℃ water bath for 1 hour to remove RNA.

⑦ mu.l of DNA sample was subjected to electrophoresis on a 1% agarose gel to determine the quality of the DNA.

(2) PCR amplification of ITS regions

Amplification reaction system (25. mu.l): 10 XPCR buffer 2.5. mu.l, MgCl₂2.0. mu.l (25mmol/L), 0.5. mu.l dNTP (10mmol/L), 1. mu.l each of primers ITS1F/ITS4 (10. mu. mol/L), 0.25. mu.l Taq enzyme (5U/. mu.l), 10ng DNA template, and sterile double distilled water. The PCR reaction condition is 94 ℃ for 2 min; 15s at 94 ℃, 30s at 62 ℃, 1min at 72 ℃ and 30 cycles; 5min at 72 ℃.

After the reaction is finished, 10 mul of amplification product is taken, and the amplification product is subjected to 1.2 percent agarose gel electrophoresis, ethidium bromide staining and then is placed in a VDS gel imaging system for photographing.

(3) Purification of PCR amplification products

① the PCR amplification product was subjected to agarose electrophoresis, and the agarose gel containing the band of interest was cut under an ultraviolet lamp.

② the gel was weighed and the gel volume was calculated to be 1mg to 1. mu.l.

③ Buffer DE-A was added in 3 volumes and after uniform suspension heated at 75 deg.C and mixed every 2-3 minutes until the gel was completely melted for about 6-8 minutes (the gel mass must be completely melted to release the DNA from the gel sufficiently).

④ Buffer DE-B was added in an amount of 50% by volume of Buffer DE-A and mixed well.

⑤ the DNA-prep Tube was placed in 2ml Microfuge Tube, and the mixture obtained in step 5 was transferred to the DNA-prep Tube and centrifuged at 12000g for 1 minute.

⑥ discard the filtrate, put the DNA-prep Tube back into the original 2ml Microfuge Tube, add 500. mu.l buffer W1, and centrifuge at 12000g for 1 min.

⑦ the filtrate was discarded, and the DNA-prep Tube was returned to the original 2ml Microfuge Tube, and 700. mu.l of Buffer W2 to which absolute ethanol had been added was added, centrifuged at 12000g for 1 minute, and washed once with 700. mu.l of Buffer W2 in the same manner.

⑧ placing the DNA-prep Tube back into the original 2ml Microfuge Tube, centrifuging at 12000g for 1 minute (9) placing the DNA-prep Tube into another sterilized 1.5ml centrifuge Tube, adding 25 μ l of the eluent preheated at 65 ℃ to the center of the silica membrane, standing at room temperature for 1 minute, and centrifuging at 12000g for 1 minute to elute the DNA.

⑨ the recovery effect was checked by agarose gel electrophoresis using 2. mu.l of sterile pipette tips.

(4) Sequencing of PCR amplification products of ITS region

Sequencing was performed by Shanghai Producer corporation.

(5) Accurate positioning of ITS sequences and construction of phylogenetic trees

The length of the ITS sequence is determined according to the ITS sequence data of the white beech mushroom existing in Genbank. The DNA sequence in the FastA format was subjected to alignment analysis using ClustalX2.1 software. Using Hypsizygus tessulatus ITS sequence obtained after rearrangement as an external group, performing clustering analysis by using neighbor-join methods in MEGA 4.1 software, and establishing a phylogenetic tree based on ITS sequence

6.5 results and analysis

(1) The effect of DNA extraction is shown in FIG. 6.

FIG. 1 shows that the quality of DNA is good and can completely meet the requirements of the test.

(2) ITS sequence alignment: the specific sequence is shown in figure 10 and SEQ ID No.1, and the sequence alignment result is shown in figure 9.

The length of the ITS sequence of the Hypsizygus marmoreus is about 657bp determined by comparative analysis with related sequences in GenBank.

(3) Genetic distance between strains

TABLE 7 genetic distance of Hypsizygus marmoreus

		1	2	3	4	5	6
								1	Clitocybe_amarescens_MH862093.1
2	H6	0.113
								3	H18	0.109	0.003
4	H27	0.107	0.005	0.002
								5	Hypsizygus_marmoreus_JX046031.1	0.107	0.005	0.002	0.000
6	K8	0.111	0.005	0.002	0.003	0.003

As can be seen from Table 7, the tested Hypsizygus marmoreus strains have extremely small genetic distance and small ITS sequence difference, which indicates that the ITS sequences of the strains have relatively slow evolution speed.

(4) Phylogenetic trees, as shown in FIG. 7.

As can be seen from FIG. 7, H18 differs from H6, H27 and K8.

Although the ITS sequence comparison result and the phylogenetic tree of the Hypsizygus marmoreus show that the 4 tested strains have no great difference in ITS sequence and are relatively close in genetic relationship, the difference or even significant difference in the agronomic characters such as appearance, yield and the like of the fruiting bodies of the Hypsizygus marmoreus under the artificial action, namely the difference among varieties cannot be excluded.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Shanghai Xue banyan Biotech Ltd

<120> Hypsizygus marmoreus H18 and cultivation method thereof

<130>MP1935141

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>657

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

tgcggaagga tcattattga ataaacttgt ttgggttgtt gctggctctt aggagcatgt 60

gcacgcctga cacattttta ccacctgtgc accctttgta gacctggaac acctctcgag 120

gcaactcggt ttgaggattg ccgctgctga aaagccggct ttccttgcgt tcccggtcta 180

tgtctttata taccccatga atgtaactga atgtctttaa tgggccttag tgcctttaaa 240

tcaaatacaa ctttcaacaa cggatctctt ggctctcgca tcgatgaaga acgcagcgaa 300

atgcgataag taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacctt 360

gcgctccttg gtattccgag gagcatgcct gtttgagtgt cattaaattc tcaacctttc 420

cagcttttac tagcttggtc aggcttggat gtgggggttg cgggcttctc agaagtcggc 480

tctccttaaa tgcattagcg gaacctttgt tgaccagctc tggtgtgata attatctacg 540

ccattgcgaa gcagctttaa taatggggtt cagcttctaa ccgtcccctt cacgggacaa 600

ctctctgaca ttttgacctc aaatcaggta ggattacccg ctgaacttaa gcatatc 657

<210>2

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

tccgtaggtg aacctgcgg 19

<210>3

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

tcctccgctt attgatatgc 20

Claims

1. The beech mushroom is characterized in that the preservation number is CGMCC No. 18154.

2. Use of the Hypsizygus marmoreus of claim 1 in the preparation of a medicament for lowering cholesterol, enhancing immunity, delaying aging, and preventing constipation.

3. The liquid medium of Hypsizygus marmoreus of claim 1, wherein 120L of the medium comprises:

4. the cultivation medium of the Hypsizygus marmoreus as claimed in claim 1, which comprises the following components in parts by mass:

5. the method for systematically breeding beech mushroom according to claim 1, wherein a1c3 obtained by hybridizing H6 with H27 is taken and then hybridized with K8 to obtain beech mushroom strain; taking the Hypsizygus marmoreus strain, performing hypha turning, culturing, fruiting, selecting fruiting bodies with robust mushroom bodies and good mushroom shapes, performing tissue separation, and inoculating into a PDA culture medium; selecting a strain with high yield, robust mushroom body and good mushroom shape, namely the crab-flavored mushroom.

6. The method for culturing Hypsizygus marmoreus according to claim 1, wherein the Hypsizygus marmoreus according to claim 1 is inoculated to a culture medium for culture.

7. The method of claim 5 or 6, wherein the culturing comprises hyphal inversion, shake flask culture, fermentor culture, and culture flask culture.

8. The method of claim 7, wherein the hypha inoculation is performed by using a PDA culture medium, the inoculation amount of the Hypsizygus marmoreus is 1 block, the block size is 5 x 3mm, and the viable count is 700-800 cfu/g;

9. The method according to claim 7 or 8, wherein the shake flask culture is carried out at a culture temperature of 21-23 ℃ and a culture rotation speed of 130 rpm/min; the culture time is 8 days;

10. The method according to any one of claims 7 to 9, wherein the conditions for inducing budding after scratching are: the temperature is 14-16 ℃, the humidity is 90-95 percent, and CO is₂The concentration is within 2000ppm, the illumination intensity is 50 lux-100 lux, and the bud forcing time is 8-10 days;

the conditions for the growth of the sporocarp are as follows: the temperature is controlled to be 14-17 ℃, the humidity is more than 90 percent, and CO is₂The concentration is less than 2000ppm, the illumination intensity is 250-500 lux, and the growth time is 14-17 days.