CN106613350B - Schizophyllum commune and domestication and cultivation method and application thereof - Google Patents
Schizophyllum commune and domestication and cultivation method and application thereof Download PDFInfo
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Abstract
The invention discloses a schizophyllum commune, and a domestication cultivation method and application thereof. Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 with the preservation number: CCTCC NO: m2016236. The invention separates new strain Schizophyllum commune HMGIM-S140099 from Guangdong province, and the strain has good medical and edible value and wide application prospect.
Description
The technical field is as follows:
the invention belongs to the field of microorganisms, and particularly relates to a schizophyllum commune, and a domestication cultivation method and application thereof.
Background art:
schizophyllum commune Fr) is commonly referred to as: caulis et folium Badiae Sibiricae, flos Lonicerae, smallpox fungus, Ginseng radix alba fungus, and flos Dendrobii. Belonging to the kingdom of fungi (Mycota), the phylum Eumycota, the class Basidiomycetes (Basidiomycetes), the order Agaricales (Agaricales), the family Schizophyllaceae (Schizophyllaceae), the genus Schizophyllum (Schizophyllum).
The schizophyllum commune sporocarp is small in size, is cracked in a fan shape or a palm shape, is generally overlaid with tile-shaped overlapping, clustered or clustered, is similar to a small chrysanthemum flower, has a pileus length of 6-50 mm, a width of 8-30 mm and a thickness of 1-3 mm, has a white surface, a grey color, a meat brown color to a yellow brown color, is densely covered with downy or coarse hair, has a plurality of split lobes, and has a tough flesh color to a soft leather color, is curled inwards at the edge, has a crenulated white color, a grey meat color or a purple brown color with unequal length, is longitudinally split into deep furrows at the middle part of the ruffled edge, has a thickness of about 1mm, and is white, flexible, tasteless, smooth, colorless and non-starchy, and can recover to an original shape after absorbing water, has no handle or short handle, has a spore print with a white color or light meat color, and basidiospores of 5-7 × 2-3.5 mu m.
Distribution: schizophyllum commune is widely distributed all over the world, is distributed in most provinces and markets of China, and is very rich in strain resources.
And (3) habitat: scattered to group, often superposed on dried bean curd sticks or dried bean curd sticks
The drug effect is as follows: when the schizophyllum commune fruiting body is young, the schizophyllum commune fruiting body is tender and delicious, has special aromatic flavor, is mild in nature and sweet in taste, can tonify and strengthen body, strengthen body resistance and consolidate constitution, has a tranquilizing effect, is used for treating neurasthenia, lassitude, dizziness, tinnitus and asthenic perspiration, is a high-grade edible and medicinal fungus, and the metabolite of the fungus has a good medicinal value. The schizophyllum commune sporocarp or mycelium contains rich immune polysaccharide, protein, bioactive L-type amino acid, various essential amino acids, trace elements and the like, has the effects of preventing and treating cardiovascular diseases, liver diseases, kidney diseases and other organ diseases, has the effects of resisting cancer, aging, inflammation and the like, has various physiological activities of improving the immunity of the organism, resisting bacteria, diminishing inflammation, resisting radiation and the like, and has good edible and medicinal values. At present, Schizophyllum commune polysaccharide is developed into an anti-tumor preparation, and is combined with radiotherapy and chemotherapy for treating cervical cancer, gastric cancer, hepatitis B virus and the like.
The invention content is as follows:
the invention aims to provide edible and medicinal dual-purpose Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 which is collected from Guangdong area, is preserved in China center for type culture Collection (CCTCC for short, eight paths in flood mountain area of Wuhan city, Hubei province, Wuhan university) in 2016 and 29 days, and has the preservation number: CCTCC NO: m2016236.
The second purpose of the invention is to provide a domesticated cultivation method of Schizophyllum commune (HMGIM-S140099), which is characterized by comprising the following steps:
inoculating Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 mother strain into a bag filled with an original strain culture medium under aseptic condition, carrying out light-shielding culture at a constant temperature of 25 ℃ until the hyphae is full of the culture medium to obtain the original strain, inoculating the original strain into the bag filled with a culture medium under aseptic condition, carrying out light-shielding culture at a constant temperature of 25 ℃ and an air relative humidity of 60-70%, and carrying out fruiting management after the hyphae grows full of the culture medium;
the fruiting management is that the temperature is controlled to be 23 +/-1 ℃, the relative air humidity is 60-80%, the illumination is 100-.
The stock culture medium comprises 79 percent of sorghum, 20 percent of millet and CaCO by mass31 percent, uniformly mixing the components according to the content, and then adding water to ensure that the water content is 55-60 percent, thereby obtaining the stock culture medium.
The culture medium comprises 50% of cottonseed hulls, 38% of sawdust, 10% of bran and CaCO by mass32 percent, after all the components are evenly mixed according to the content, water is added to ensure that the water content is 60 percent.
The third purpose of the invention is to provide the application of Schizophyllum commune HMGIM-S140099 in the preparation of Schizophyllum commune polysaccharide.
The invention separates new strain Schizophyllum commune HMGIM-S140099 from Guangdong province, and the strain has good medical and edible value and wide application prospect.
Schizophyllum commune (Schizophyllum commune) HMGIM-S140099, which is deposited in China center for type culture Collection (CCTCC for short, eight ways in flood and mountain area of Wuhan city, Hubei province, Wuhan university) in 2016 and 4 and 29 days, has the preservation number: CCTCC NO: m2016236.
Description of the drawings:
FIG. 1 is an antagonistic map between Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 and Schizophyllum commune (Schizophyllum commune) Sc-1 and Sc-2 of the prior art, wherein S140099, Sc-1 and Sc-2 represent Schizophyllum commune (Schizophyllum commune) HMGIM-S140099, Schizophyllum commune (Schizophyllum commune) Sc-1 and Schizophyllum commune (Schizophyllum commune) Sc-2, respectively.
FIG. 2 is a growth diagram of wild Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 in Yunshan;
FIG. 3 is a diagram showing the growth of domesticated Schizophyllum commune HMGIM-S140099.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
firstly, acquisition and identification of Schizophyllum commune HMGIM-S140099:
schizophyllum commune (Schizophyllum commune) HMGIM-S140099 of the present invention was isolated from the hardwoods of the Baiyunshan mountain in Guangzhou, Guangdong province.
Morphological characteristics and ITS sequence of Schizophyllum commune (Schizophyllum commune) HMGIM-S140099:
the Schizophyllum commune fruiting body group grows on dead wood, is leather, is fan-shaped or palm-shaped, is 7-15 mm long, 8-20 mm wide, 2-3 mm thick, is gray in surface, is coated with coarse fluff, is curled inwards at the edge, is light in flesh color and unequal in length, is longitudinally split into deep furrows in the middle of the ruffle, is white in flesh color and tough in leather, is about 1mm short in stipe and is not printed with flesh color by spores, and the spores are colorless, transparent and oval in shape and are 2.57-3.92 × 5.3.3-6.43 mu m under an optical microscope.
Extracting Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 mycelium DNA by using a biological Ezup column type fungus genome DNA extraction kit, carrying out ITS-PCR by using ITS1 and ITS4 as primers, sequencing a product to obtain a sequence with the base number of 636bp, wherein the nucleotide sequence is shown as SEQ ID NO.1, comparing and analyzing the sequence with GenBank sequence BLAST, wherein the sequence similarity of the sequence with Schizophyllum commune strains such as JX848644.1 and JF439509.1 which are published on the sequence is up to 99%, determining that the separated strain is Schizophyllum commune, and naming the separated strain as Schizophyllum commune: schizophyllum commune (Schizophyllum commune) HMGIM-S140099.
Second, antagonistic experiment
Preparing a plate culture medium (diameter is 9 cm) by using a common PDA culture medium, p H naturally, respectively inoculating strain blocks (about 5mm square strain blocks) of Schizophyllum commune (Schizophyllum commune) HMGIM-S140099, Schizophyllum commune) Sc-1 and Schizophyllum commune Sc-2 by a triangular inoculation method, respectively repeating the steps for 3 times, sealing the openings by using a sealing film, culturing in a constant-temperature incubator at 25 ℃ in a dark place until 3 strains of hyphae are fully contacted with each other, observing whether an antagonistic line exists, and photographing and recording.
As can be seen in FIG. 1, significant antagonistic lines were present between Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 and Schizophyllum commune (Schizophyllum commune) Sc-1 and Sc-2 of the prior art, and the genetic characteristics of the mycelia were different, so that the mycelia were not miscible, and the mycelia antagonistic reaction was one of the macroscopic indicators of the immiscible state, indicating that significant genetic differences were present between Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 and Schizophyllum commune (Schizophyllum commune) Sc-1 and Sc-2 of the Schizophyllum commune (Schizophyllum commune) of the prior art, and judging that Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 and Schizophyllum commune (Schizophyllum commune) Sc-2 of the prior art are both different strains.
Third, domestication and cultivation
1. Culture medium
1) Tissue isolation and mother culture medium
The formula is as follows: the comprehensive PDA (calculated by mass fraction, comprising 20% of potatoes, 2% of glucose, 2% of agar, 0.3% of monopotassium phosphate, 0.15% of magnesium sulfate and 0.15% of vitamin B1, wherein the solvent is water, the method comprises the steps of firstly cleaning and peeling the potatoes, then weighing 200g of the potatoes, cutting the potatoes into small blocks, adding water for boiling (boiling for 20-30 minutes and being capable of being punctured by a glass rod), filtering by eight layers of gauze to obtain a filtrate, adding 20g of glucose, 20g of agar, 3g of monopotassium phosphate, 1.5g of magnesium sulfate and a trace amount of vitamin B1 into the filtrate, then supplementing 1L of water, and sterilizing.
2) Stock culture medium
The formula is as follows: comprises 79 percent of sorghum, 20 percent of millet and CaCO by mass fraction 31 percent, evenly mixing all the components according to the content, and then adding water to ensure that the water content is 55-60 percent.
3) Cultivation material culture medium
Through different formula components and content comparison experiments, the optimal formula is finally determined: comprises 50 percent of cottonseed hull, 38 percent of wood flour, 10 percent of bran and CaCO by mass fraction32 percent, after the components are uniformly mixed, water is added to ensure that the water content is 60 percent.
2. Technical parameters of cultivation
1) Hypha culture
Obtaining Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 mother strain after tissue separation and purification under aseptic condition; inoculating Schizophyllum commune HMGIM-S140099 mother strain into a bag filled with stock culture medium under aseptic condition, and culturing in shade at 25 deg.C until the strain is full of material to obtain stock. Inoculating the stock seed into a bag filled with culture medium under aseptic condition, culturing at 25 deg.C under constant temperature and air relative humidity of 60-70% in dark place, and performing fruiting management after the culture medium is full of mycelia. The whole process takes about 20-25 days.
2) Fruiting conditions
The temperature is controlled to be 23 +/-1 ℃, the relative humidity of air is 60-80%, the illumination is about 100-200lux, and the content of carbon dioxide just after the primary reaction is controlled to be more than 2000 ppm. During the growth period of the sporocarp, the temperature is adjusted to be between 20 and 23 ℃, the relative humidity of air is adjusted to be between 70 and 85 percent, the illumination is about 200 and 300lux, and the content of carbon dioxide is kept to be below 1200 ppm.
3. Fruiting status
1) And (3) growth period:
inoculating the stock seed into the cultivation bag for 13-15 days, allowing the mycelium to grow into full bag, allowing primordium to appear after 5-7 days, allowing the fruiting body to mature after 5-7 days, and inoculating the stock seed into the cultivation bag until the whole growth cycle is about 23-28 days.
2) Yield: each fungus bag can produce three tides within 45-60 g per tide, and the total biological conversion rate is about 70-85%.
And (3) fruiting body properties:
fourth, crude polysaccharide content determination
1. Extraction of crude polysaccharide from Schizophyllum commune HMGIM-S140099 fruiting body
Domesticating and cultivating Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 strain to obtain Schizophyllum commune fruiting body, pulverizing, and mixing according to the weight ratio of 1: 30, leaching for 2 hours at the temperature of 100 ℃, filtering, concentrating, precipitating with 4 times of volume of absolute ethyl alcohol overnight, and dialyzing to obtain a crude polysaccharide solution of schizophyllum commune sporocarp.
2. Determination of polysaccharide content
The content of crude polysaccharide in Schizophyllum commune HMGIM-S140099 fruiting body (crude polysaccharide/fruiting body dry weight × 100%) is measured according to the content measurement of crude polysaccharide in edible fungi of the agricultural industry standard (NY/T1676) -2008. the polysaccharide content is 5.17%, and the Schizophyllum commune polysaccharide (SPG) has the effects of resisting tumors, regulating immunity, inhibiting bacteria, resisting viruses and the like according to the literature.
Sequence listing
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
<120> Schizophyllum commune and domestication and cultivation method and application thereof
<160>1
<210>1
<211>636
<212>DNA
<213> Schizophyllum commune (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 (Schizophyllum commune) HMGIM-S140099 with the preservation number: CCTCC NO: m2016236.
2. A domestication cultivation method of Schizophyllum commune HMGIM-S140099 is characterized by comprising the following steps:
inoculating Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 mother strain in claim 1 into a bag filled with a stock culture medium under aseptic conditions, culturing in shade at a constant temperature of 25 ℃ until the stock is full of mycelia, inoculating the stock into the bag filled with a culture medium under aseptic conditions, culturing in the shade at a constant temperature of 25 ℃ and relative air humidity of 60-70%, and performing fruiting management after the stock is full of the culture medium;
the fruiting management is that the temperature is controlled to be 23 +/-1 ℃, the relative air humidity is 60-80%, the illumination is 100-.
3. The method according to claim 2, wherein the stock culture medium comprises, by mass, 79% sorghum, 20% millet and CaCO31 percent, uniformly mixing the components according to the content, and then adding water to ensure that the water content is 55-60 percent, thereby obtaining the stock culture medium.
4. The method as claimed in claim 2, wherein the cultivation medium comprises 50% of cottonseed hull, 38% of wood flour, 10% of bran and CaCO by weight32 percent, after all the components are evenly mixed according to the content, water is added to ensure that the water content is 60 percent.
5. Use of Schizophyllum commune (Schizophyllum commune) HMGIM-S140099 of claim 1 for the preparation of Schizophyllum commune polysaccharide.
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| CN107828664B (en) * | 2017-10-25 | 2020-10-23 | 湖南省农业生物技术研究中心 | Schizophyllum commune XT-1 and cultivation method and application thereof |
| CN107736190A (en) * | 2017-11-13 | 2018-02-27 | 云南菌视界生物科技有限公司 | A kind of waste material after fruiting using schizophyllum commune cultivates the method that high warm lung shape is picked up the ears |
| CN109156266A (en) * | 2018-08-03 | 2019-01-08 | 西北师范大学 | A kind of method schizophyllum commune cultivation culture medium and cultivate schizophyllum commune using it |
| CN109247189A (en) * | 2018-09-29 | 2019-01-22 | 云南菌视界生物科技有限公司 | A kind of white ginseng bacterial strain and its white ginseng low cost cultural method |
| CN113179853B (en) * | 2021-04-28 | 2024-05-07 | 马布平 | White ginseng strain, cultivation site and white ginseng |
| CN114214209B (en) * | 2021-12-27 | 2024-01-09 | 宁波希诺亚海洋生物科技有限公司 | Schizophyllum commune variety capable of producing lactase and application thereof |
| CN114391421A (en) * | 2022-01-06 | 2022-04-26 | 毕节市农投菌业科技有限责任公司 | Cultivation method for continuously fruiting white ginseng fungi |
| CN116590156B (en) * | 2023-06-20 | 2024-01-30 | 南京高新工大生物技术研究院有限公司 | Schizophyllum commune strain with high protein yield and breeding method thereof |
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| WO2016091892A1 (en) * | 2014-12-12 | 2016-06-16 | Wintershall Holding GmbH | Process for the fermentation of fungal strains |
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