CN106613350A - Schizophyllum commune and domestication and cultivation method and application thereof - Google Patents

Abstract

The invention discloses a schizophyllum commune and a domestication and cultivation method and application thereof. The preservation number of the schizophyllum commune HMGIM-S140099 is CCTCC NO: M2016236. The schizophyllum commune HMGIM-S140099 is obtained through separation in Guangdong province, and the strain has very good medical and edible values and has a wide application prospect.

Description

A strain of schizophyllum and its domestication cultivation method and application

Technical field:

The invention belongs to the field of microorganisms, and in particular relates to a schizophyllum strain and its domestication cultivation method and application.

Background technique:

Schizophyllum commune Fr. is commonly known as: Badanchai, white flower, smallpox fungus, white ginseng fungus, tree flower. Belongs to Mycota, Eumycota, Basidiomycetes, Agaricales, Schizophyllaceae, Schizophyllum.

The fruiting body of Schizophyllum is small, fan-shaped or palm-shaped, usually imbricate in stacks, clusters or groups, shaped like small chrysanthemums. The cap is 6-50mm long, 8-30mm wide, and 1-3mm thick. The surface of the cap is white, off-white, fleshy brown to yellowish brown, densely covered with hairs or coarse hairs, with multiple lobes, tough fleshy to soft leathery, The edges are rolled inward. Gills white, off-white, light flesh-colored or purplish brown, unequal in length, with deep grooves in the middle of the frill edge. The fungus flesh is about 1mm thick, white, leathery, flexible, tasteless, strongly shrinks after drying, and returns to its original shape after absorbing water. Fruiting bodies are sessile or have short stalks. Spore print white or pale flesh-colored, basidiospores 5-7×2-3.5 μm, oval or sausage-shaped, smooth, colorless, non-starch.

Distribution: Schizophyllum is widely distributed all over the world, and is distributed in most provinces and cities in my country, and the strain resources are very rich.

Habitat: Scattered to gregarious, often stacked on rotten wood or rotten bamboo

Medicinal effect: When the fruiting body of Schizophyllum fungus is young, it is tender and delicious, with a special strong fragrance, mild in nature, sweet in taste, can nourish and strengthen, strengthen the body, sedative effect, and treat neurasthenia, lassitude, dizziness, tinnitus, sweating, It is a high-grade edible and medicinal fungus, and its metabolites have good medicinal value. Schizophyllum fruiting bodies or mycelium are rich in immune polysaccharides, proteins, biologically active L-type amino acids, various essential amino acids and trace elements, etc. These substances have preventive effects on cardiovascular diseases, liver, kidney and other organ diseases , and has anti-cancer, anti-aging, anti-inflammatory and other effects, has various physiological activities such as improving the body's immunity, anti-bacterial and anti-inflammatory, anti-radiation, etc., and has good edible and medicinal value. At present, schizophyllan has been developed into an anti-tumor agent, which is used in the treatment of cervical cancer, gastric cancer and hepatitis B virus in combination with radiotherapy and chemotherapy.

Invention content:

The first object of the present invention is to provide edible and medicinal schizophyllum (Schizophyllum commune) HMGIM-S140099 from Guangdong, which has been preserved in the China Center for Type Culture Collection on April 29, 2016. (CCTCC for short, Bayi Road, Hongshan District, Wuhan City, Hubei Province, Wuhan University), the preservation number is: CCTCC NO: M2016236.

The second object of the present invention is to provide a domestication cultivation method of Schizophyllum commune (Schizophyllum commune) HMGIM-S140099, which is characterized in that it comprises the following steps:

Under aseptic conditions, inoculate the parent species of Schizophyllum commune HMGIM-S140099 into a bag containing the original seed medium, and culture it in the shade at a constant temperature of 25°C until the mycelium is full of material to obtain the original seed. Seeds are inoculated into bags containing cultivation medium under aseptic conditions, cultivated at a constant temperature of 25°C, with a relative air humidity of 60-70% and protected from light. After the mycelium is covered with cultivation medium, fruiting manage;

The fruiting management is to control the temperature between 23±1°C, the relative air humidity between 60-80%, the illumination of 100-200lux, and control the carbon dioxide content above 2000ppm when the primordium just emerged. , adjust the temperature between 20-23°C, the relative air humidity between 70-85%, the light about 200-300lux, and keep the carbon dioxide content below 1200ppm.

The original seed culture medium comprises 79% sorghum, 20% millet, and 1% CaCO ₃ by mass fraction, and the components are uniformly mixed according to their content, and then water is added to make the water content 55-60%, Thus, a stock culture medium was obtained.

The cultivation material culture medium includes 50% of cottonseed hulls, 38% of sawdust, 10% of bran and 2% of CaCO ₃ by mass fraction. After mixing the components uniformly according to their contents, water is added to make the water content 60%.

The third object of the present invention is to provide the application of Schizophyllum commune HMGIM-S140099 in the preparation of Schizophyllum commune.

The present invention isolates a new bacterial strain Schizophyllum commune HMGIM-S140099 from Guangdong Province. The bacterial strain has very good value for both medicine and food, and has broad application prospects.

Schizophyllum commune (Schizophyllum commune) HMGIM-S140099, the strain has been preserved in the China Center for Type Culture Collection (CCTCC for short, Bayi Road, Hongshan District, Wuhan City, Hubei Province, Wuhan University) on April 29, 2016, and the preservation number is : CCTCC NO: M 2016236.

Description of drawings:

Fig. 1 is the antagonism diagram between Schizophyllum commune HMGIM-S140099 and prior art Schizophyllum commune Sc-1 and Schizophyllum commune Sc-2, wherein S140099, Sc-1 and Sc-2 represent Schizophyllum commune HMGIM-S140099, Schizophyllum commune Sc-1 and Schizophyllum commune Sc-2, respectively.

Figure 2 is a growth chart of Schizophyllum commune HMGIM-S140099 wild in Baiyun Mountain;

Fig. 3 is a growth chart of Schizophyllum commune HMGIM-S140099 after domestication and cultivation.

detailed description:

The following examples are to further illustrate the present invention, rather than limit the present invention.

Example 1:

1. Acquisition and identification of Schizophyllum commune HMGIM-S140099:

The schizophyllum commune HMGIM-S140099 of the present invention is isolated from fallen wood in the broad-leaved forest of Baiyun Mountain, Guangzhou City, Guangdong Province.

Morphological characteristics and ITS sequence of Schizophyllum commune HMGIM-S140099:

Schizophyllum fruiting bodies are born on dead wood, leathery, cap fan-shaped or palm-shaped, 7-15mm long, 8-20mm wide, 2-3mm thick, grayish white on the surface, covered with coarse hairs, and curled inward at the edges. Gills pale flesh-colored, unequal in length, longitudinally split into deep grooves in the middle of the pleats. Bacteria flesh white, leathery, about 1mm. Stipe short to absent. The spore print is pale flesh-colored, and the spores are colorless and transparent under an optical microscope. The spores are oval, 2.57-3.92×5.3-6.43 μm.

The DNA of Schizophyllum commune HMGIM-S140099 mycelium was extracted by Sangon Ezup Column Fungal Genomic DNA Extraction Kit, ITS-PCR was performed with ITS1 and ITS4 as primers, and the sequence of base number 636bp was obtained after the product was sequenced , its nucleotide sequence is shown as SEQ ID NO.1, and its sequence similarity with the previously published Schizophyllum strains such as JX848644.1 and JF439509.1 is as high as 99% through GenBank sequence BLAST comparison analysis. The isolated strain is Schizophyllum commun, named: Schizophyllum commune HMGIM-S140099.

2. Antagonism experiment

Common PDA medium was used to make plate culture medium (9 cm in diameter), with natural pH, and Schizophyllum commune (Schizophyllum commune) HMGIM-S140099, Schizophyllum commune Sc-1 and Schizophyllum commune were inoculated respectively according to the triangular inoculation method. Bacteria (Schizophyllum commune) Sc-2 strain block (approximately 5mm square bacterial block), respectively do 3 repetitions, seal with parafilm, and culture in a constant temperature incubator at 25°C in the dark until the hyphae of the 3 species interact with each other. Fully contact, observe whether there are antagonistic lines, and take pictures for records.

As can be seen in Figure 1, there is obvious antagonism between Schizophyllum commune HMGIM-S140099 and prior art Schizophyllum commune Sc-1 and Schizophyllum commune Sc-2 Because of the different genetic characteristics, the mycelium is immiscible, and the antagonism of the mycelium is one of the visible indicators of this immiscible state. The results showed that the Schizophyllum commune HMGIM-S140099 and There are obvious genetic differences between Schizophyllum commune Sc-1 and Schizophyllum commune Sc-2 in the prior art, and it can be judged that Schizophyllum commune HMGIM-S140099 and the prior art Schizophyllum commune Sc-1 and Schizophyllum commune Sc-2 are different strains.

3. Domestication and cultivation

1. Medium

1) Tissue separation and parent culture medium

Formula: comprehensive PDA (according to mass fraction, including potato 20%+glucose 2%+agar 2%+potassium dihydrogen phosphate 0.3%+magnesium sulfate 0.15%+vitamin B1 trace, solvent is water, that is, its method is to first make potato Wash and peel, then weigh 200g of potatoes and cut into small pieces, add water to boil until rotten (boil for 20-30 minutes, it can be pierced by a glass rod), filter with eight layers of gauze to obtain the filtrate, add 20g of glucose, 20g of agar, 3g of potassium dihydrogen phosphate, 1.5g of magnesium sulfate, a small amount of vitamin B1, then make up to 1L with water, and sterilize.

2) Original culture medium

Formula: according to the mass fraction, it includes 79% of sorghum, 20% of millet and 1% of CaCO ₃ , and the components are uniformly mixed according to their content, and then water is added to make the water content 55-60%.

3) Cultivation medium

The best formula is finally determined by comparing the ingredients and contents of different formulas: in terms of mass fraction, it includes 50% cottonseed hulls, 38% wood chips, 10% bran, and 2% CaCO ₃ . After mixing the above ingredients evenly, add water, Make the moisture content 60%.

2. Cultivation technical parameters

1) Mycelia culture

Obtain the parent species of Schizophyllum commune HMGIM-S140099 after tissue separation and purification under sterile conditions; inoculate the parent species of Schizophyllum commune HMGIM-S140099 into the culture medium containing the original species under sterile conditions Cultivate in a shading bag at a constant temperature of 25°C until the mycelium is full of material to obtain the original species. The original seed is inoculated into the bag containing the cultivation material medium under aseptic conditions, and cultivated at a constant temperature of 25°C and with a relative air humidity of 60-70% in the dark. mushroom management. The whole process takes about 20-25 days.

2) Conditions for fruiting

The temperature is controlled between 23±1°C, the relative humidity of the air is between 60-80%, the light is about 100-200lux, and the carbon dioxide content is controlled above 2000ppm when the primordium just emerges. During the growth period of the fruit body, adjust the temperature between 20-23°C, the relative air humidity between 70-85%, the light of about 200-300lux, and keep the carbon dioxide content below 1200ppm.

3. Mushrooming

1) Growth cycle:

The original seed is inoculated into the cultivation bag 13-15 days after the mycelia grows all over the bag, the primordium emerges after 5-7 days, and the fruiting bodies mature and can be harvested after 5-7 days. The whole growth cycle from inoculation to harvesting of the cultivation bag is about 23- 28 days.

2) Yield: Each fungus bag produces 45-60 grams of mushrooms per tide, and three tides can be produced, and the overall biotransformation rate is about 70-85%.

Fruiting body traits:

4. Determination of crude polysaccharide content

1. Extraction of crude polysaccharide from fruiting body of Schizophyllum commune HMGIM-S140099

Take Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 strain acclimatized and cultivated Schizophyllum fruiting bodies, after crushing, according to the ratio of solid to liquid of 1:30, extract at 100°C for 2 hours, filter, concentrate and use 4 times Volume of absolute ethanol was precipitated overnight and dialyzed to obtain the crude polysaccharide solution of the fruiting bodies of Schizophyllum.

2. Determination of polysaccharide content

According to the People's Republic of China Agricultural Industry Standard (NY/T 1676-2008) Determination of Crude Polysaccharide Content in Edible Fungus The content of crude polysaccharide in the fruit body of Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 (crude polysaccharide/fruit body dry weight × 100% ) to measure. Its polysaccharide content was measured to be 5.17%. According to literature records, schizophyllan (SPG) has anti-tumor, immune regulation, antibacterial, antiviral and other effects. Therefore, the Schizophyllum commune HMGIM-S140099 has good development potential.

sequence listing

<110> Guangdong Provincial Institute of Microbiology (Guangdong Microbial Analysis and Testing Center)

<120> A strain of Schizophyllum and its domestication cultivation method and application

<160> 1

<210> 1

<211> 636

<212>DNA

<213> Schizophyllum commune HMGIM-S140099

<400> 1

TCCGTAGGTG AACCTGCGGA AGGATCATTA ACGAATCAAA CAAGTTCATC TTGTTCTGAT 60

CCTGTGCACC TCATGTAGTC CCAAAGCCTT CACGGGCGGC GGTTGACTAC GTCTACCTCA 120

CACCTTAAAG TATGTTAACG AATGTAATCA TGGTCTTGAC AGACCCTAAA AAGTTAATAC 180

AACTTTCGAC AACGGATCTC TTGGCTCTCG CATCGATGAA GAACGCAGCG AAATGCGATA 240

AGTAATGTGA ATTGCAGAAT TCAGTGAATC ATCGAATCTT TGAACGCACC TTGCGCCCTT 300

TGGTATTCCG AGGGGCATGC CTGTTTGAGT GTCATTAAAT ACCATCAACC CTCTTTTGAC 360

TTCGGTCTCG AGAGTGGCTT GGAAGTGGAG GTCTGCTGGA GCCTAACGGA GCCAGCTCCT 420

CTTAAATGTA TTAGCGGATT TCCCTTGCGG GATCGCGTCT CCGATGTGAT AATTTCTACG 480

TCGTTGACCA TCTCGGGGCT GACCTAGTCA GTTTCAATAG GAGTCTGCTT CTAACCGTCT 540

CTTGACCGAG ACTAGCGACT TGTGCGCTAA CTTTTGACTT GACCTCAAAT CAGGTAGGAC 600

TACCCGCTGA ACTTAAGCAT ATCAATAAGC GGAGGA 636

Claims (5)

1. Schizophyllum commune HMGIM-S140099, the deposit number is: CCTCC NO: M 2016236. 2. A domestication cultivation method of Schizophyllum commune (Schizophyllum commune) HMGIM-S140099, is characterized in that, comprises the following steps: Inoculate the mother species of Schizophyllum commune HMGIM-S140099 in claim 1 into a bag containing the original seed medium under aseptic conditions, and cultivate it in shade at a constant temperature of 25°C until the mycelium is full of material. To obtain the original seed, the original seed is inoculated into the bag containing the cultivation material medium under aseptic conditions, and cultivated at a constant temperature of 25°C and with a relative air humidity of 60-70% in the dark, until the hyphae are covered with the cultivation material medium After that, carry out fruiting management; The fruiting management is to control the temperature between 23±1°C, the relative air humidity between 60-80%, the illumination of 100-200lux, and control the carbon dioxide content above 2000ppm when the primordium just emerged. , adjust the temperature between 20-23°C, the relative air humidity between 70-85%, the light about 200-300lux, and keep the carbon dioxide content below 1200ppm. 3. preparation method according to claim 2 is characterized in that, described original seed culture medium, by mass fraction, comprises sorghum 79%, millet 20%, CaCO3 ₁ %, each component is mixed according to its content Mix evenly, and then add water to make the water content 55-60%, thus obtaining the original seed culture medium. 4. The preparation method according to claim 2, characterized in that, said cultivation material culture medium comprises, by mass fraction, 50% of cottonseed hulls, 38% of sawdust, 10% of bran and 2% of CaCO ₂ %. Its content After mixing the components evenly, add water to make the water content 60%. 5. The application of Schizophyllum commune HMGIM-S140099 according to claim 1 in the preparation of Schizophyllum commune.