CN104480021A - Hirsutella sinensis capable of high-yielding cordyceps polysaccharide and cultivating method thereof - Google Patents

Hirsutella sinensis capable of high-yielding cordyceps polysaccharide and cultivating method thereof Download PDF

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CN104480021A
CN104480021A CN201410664856.9A CN201410664856A CN104480021A CN 104480021 A CN104480021 A CN 104480021A CN 201410664856 A CN201410664856 A CN 201410664856A CN 104480021 A CN104480021 A CN 104480021A
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culture
cordyceps
cordyceps polysaccharide
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高艳华
任明
袁博
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National Power Bio Tech Ltd Jinan
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N1/145—Fungal isolates
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

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Abstract

The invention discloses hirsutella sinensis capable of high-yielding cordyceps polysaccharide and a cultivating method thereof, and belongs to the field of biological science. The strain, with a Preservation No. of CGMCC No.9046, is preserved in China General Microbiological Culture Collection Center (CGMCC) on May 29, 2014. According to the invention, through solid slant culture, and seed and fermented cultivation, mycelia are obtained; then, by carrying out suction filtration and drying on the mycelia, mycelia powder is obtained; and by testing, a situation that the content of effective constituents such as cordyceps polysaccharide, cordycepic acid, cordycepin, and the like in the mycelia powder are all higher than that of natural cordyceps sinensis, especially the content of cordyceps polysaccharide can be up to 10-11% is found, thereby laying a foundation for the further preparation of cordyceps polysaccharide and series products.

Description

One plant height produces China pilose spore and the cultural method thereof of Cordyceps Polysaccharide
Technical field
The present invention relates to bio-science field, be specifically related to China pilose spore (Hisutella sinensis) and cultural method thereof that a plant height produces Cordyceps Polysaccharide.
Background technology
Cordyceps sinensis has another name called cordyceps sinensis, is the Hepialus larva that Cordyceps fungus parasitizes in high mountain steppe, and larva body is ossify, and namely the complex body that forms of the sporophore of Cordyceps fungus and bombys batryticatus sclerotium, belongs to edible fungi.Cordyceps sinensis is a kind of traditional famous and precious tonic Chinese medicine material of China, has the multi-efficiencies such as immunity moderation system function, antitumor, antifatigue.
The active substance that in Cordyceps sinensis, content is the highest is Cordyceps Polysaccharide, because it can effect such as anti-oxidant, antitumor and strengthening immunity and favoring by many people.Cordyceps Polysaccharide be by seminose, semi-lactosi, pectinose, glucose, fucosedeng the saccharan of composition.Cordyceps Polysaccharide has the effect of reinvigoration, strengthening vital QI to eliminate pathogenic factors, can immunity moderation, antitumor, protect kidney, delay senility, the several functions such as antifatigue, adjustment lipid metabolism and carbohydrate metabolism, anti-hepatic fibrosis and radioprotective, be the mcroorganism treasure-house being worth excavation.
Summary of the invention:
The object of this invention is to provide China pilose spore and cultural method thereof that a plant height produces Cordyceps Polysaccharide.China pilose spore (Hisutella sinensis) M-010 is separated to from a strain Cordyceps Sinensis From Tibet sporophore.This bacterial classification, by cultivating step by step, obtains mycelium, finds that in mycelium, Cordyceps Polysaccharide content is far above natural cordyceps after testing.
In order to reach one of above-mentioned purpose, the present invention realizes by following technical measures:
One strain filamentous fungus M-010, apply for strain identification on May 29th, 2014 to China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) and submit preservation to, through identifying that this bacterial strain is China pilose spore (Hisutella sinensis), deposit number: CGMCC No.9046, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Classification And Nomenclature: Cordyceps sinensis Ophiocordyceps sinensis.
RRNA gene ITS1-5.8S-ITS2 region sequence is as follows:
CACTGGAGGGTGTGGTGGTTTCACGGCGTGACCGCCTCCGCGCTCCCGGTGCGAGGTTCTCAGCGAGCTACTGCGCAGGGGAGCCGCGGCGGGGTCGCCACTGCGTTTAGGGGGCGGCGCGGGGCCGTGACCCCCAAGGCCGGGCCCCGCCGCCGCGAGGCGGGGGGCTCGAGGGTTGAGATGACGCTCGGACAGGCATGCCCGCCAGAGTGCTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGGTTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTCATTTGCTTGCTTCTTGACTGAGAGATGCCACTGCGACAGGAGGGTCTGGGGGTCCTCCGGCGGGCGCCCTGGTCCGGGCCCTGGGGCGCCGGGGCGGTCCCGCCGAGGCAACTGCTGTGGTGTTCGCAGGGGGTTTGGGAGTGGTGACTCGATAATGATCCCTCCGCTGGTTCACCAACGGAGACCTTGTTA
In order to reach above-mentioned purpose two, the present invention realizes by following technical measures:
A cultural method for the China pilose spore of high yield Cordyceps Polysaccharide, comprises the steps:
(1) strain inclined plane is cultivated
Mycelium one ring of picking China pilose spore M-010 slant culture, is inoculated in fresh slant medium, is placed in constant incubator quiescent culture, and culture temperature is 15-25 DEG C, and incubation time is 5-15 days; Slant medium selects potato culture;
(2) bacterial classification seed culture
The mycelium of the fresh inclined-plane growth of picking one ring step (1) is in liquid seed culture medium, and culture temperature is 15-25 DEG C, and culture cycle is 5-10 days;
Seed culture medium: glucose 1-3%, peptone 0.2-1.0%, aminoacids complex 0.01-0.1%, potassium primary phosphate 0.1-0.3%, magnesium sulfate heptahydrate 0.02-0.08%, all the other are water, pH nature;
(3) strain fermentation is cultivated
Be inoculated in fermention medium with the inoculum size of 5-20% (V/V) by step (2) seed liquor, culture temperature is 15-25 DEG C, and fermentation period is 7-10 days;
Fermention medium: glucose 2-5%, peptone 0.5-1.5%, aminoacids complex 0.01-0.1%, potassium primary phosphate 0.2-0.5%, magnesium sulfate heptahydrate 0.05-0.1%, all the other are water, pH nature;
(4) suction filtration, to dry step (3) fermentation culture gained mycelium by Plate Filtration, discard filtrate, obtain wet mycelium, 50-70 DEG C dry pulverize after mycelium powder.
The China pilose spore M-010 of described high yield Cordyceps Polysaccharide cultivates through aforesaid method, obtains Cordyceps fungus powder, adopts following method to measure the content of this bacterial classification product Cordyceps Polysaccharide, cordycepic acid, cordycepin three class active substance respectively:
(1) Cordyceps Polysaccharide assay: obtaining Cordyceps Polysaccharide by cultivating the mycelium obtained by water extraction and alcohol precipitation method, adopting Phenol sulfuric acid procedure to measure the amount of Cordyceps Polysaccharide.
(2) cordycepic acid content measures: adopt liquid phase chromatography.
(3) cordycepin content measures: adopt liquid phase chromatography.
Beneficial effect of the present invention:
(1) present invention finds the filamentous fungus M-010 of a strain high polysaccharide, China pilose spore (Hisutella sinensis) is accredited as, deposit number: CGMCC No.9046 through China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC).
(2) China pilose spore M-010 of the present invention is through fluid enlargement culture, and the Cordyceps fungus powder yield of acquisition is high, and effective nutritive ingredient such as Cordyceps Polysaccharide, cordycepic acid, cordycepin is higher than natural cordyceps.
(3) this bacterial strain energy high yield Cordyceps Polysaccharide, output can reach 10-11%, lays a good foundation for Cordyceps Polysaccharide develops further.
Embodiment
Following examples further illustrate content of the present invention, are only for clearly demonstrating example of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, concerning those of ordinary skill in the field, the change done the inventive method, step or condition, variation, amendment or replacement, still belong to scope of the present invention.
The separation and ientification of embodiment 1 China pilose spore
1. isolation medium
Potato 20%, place of production soil 1.8%, peptone 0.3%, glucose 1.5%, agar 2%.
2. the separation of bacterial classification
The grassland, Changtang, Nagqu area of height above sea level 4500m collects Cordyceps sporophore, 75% alcohol is adopted to carry out surface sterilization to the stroma head of Cordyceps sporophore, stroma handle and sclerotium, carry out grinding pulping with the mortar after sterilizing, then carry out conventional gradients dilution, obtain 10 -1, 10 -2, 10 -3, 10 -4, 10 -5totally 5 extent of dilution, draw 0.1mL diluent respectively and coat isolation medium, be inverted in water-impermeable incubator, cultivate 4 days, namely find 10 at 23 DEG C ± 1 DEG C -2, 10 -3, 10 -4extent of dilution flat board on grow white colony.
3. the purifying of bacterial strain
By 10 -2, 10 -3, 10 -4extent of dilution flat board on the single bacterium colony that grows respectively picking on new culture medium flat plate, carry out Secondary Culture 4 days, find bacterium colony spot, form, in the same size, and carry out microscopy by microscope to mycelia, bacterial classification is simple without living contaminants.
4, the slant culture of purifying bacterial strain
Inoculate above-mentioned purifying bacterial strain in potato culture inclined-plane, under 15-25 DEG C of condition, cultivate 5-15 days.
The liquid culture of embodiment 2 China pilose spore
1. substratum
Seed culture medium: glucose 2%, peptone 0.6%, aminoacids complex 0.05%, potassium primary phosphate 0.1%, magnesium sulfate heptahydrate 0.03%, pH nature.
Fermention medium: glucose 2.5%, peptone 0.7%, aminoacids complex 0.06%, potassium primary phosphate 0.2%, magnesium sulfate heptahydrate: 0.05%, pH nature.
2. cultural method
The mycelium of picking one ring fresh inclined-plane growth, in liquid seed culture medium, concussion is cultivated, and culture temperature is 25 DEG C, and cultivate 10 days, mycelium dry weight reaches 0.5% of fermented liquid.
With the inoculum size of 10% (V/V), secondary seed is inoculated in fermention medium, culture temperature is 25 DEG C, and fermentation period is 7 days, and after fermentation ends, mycelium dry weight yield reaches 1.5%.
Embodiment 3
1. substratum
Seed culture medium: glucose 1%, peptone 0.2%, aminoacids complex 0.01%, potassium primary phosphate 0.3%, magnesium sulfate heptahydrate 0.02%, pH nature.
Fermention medium: glucose 2%, peptone 0.5%, aminoacids complex 0.01%, potassium primary phosphate 0.5%, magnesium sulfate heptahydrate 0.1%, pH nature.
2. cultural method
The mycelium of picking one ring fresh inclined-plane growth, in liquid seed culture medium, concussion is cultivated, and culture temperature is 15 DEG C, and cultivate 5 days, mycelium dry weight reaches 0.3% of fermented liquid.
With the inoculum size of 5% (V/V), secondary seed is inoculated in fermention medium, culture temperature is 15 DEG C, and fermentation period is 10 days, and after fermentation ends, mycelium dry weight yield reaches 1.2%.
Embodiment 4
1. substratum
Seed culture medium: glucose 3%, peptone 1.0%, aminoacids complex 0.1%, potassium primary phosphate 0.2%, magnesium sulfate heptahydrate 0.08%, pH nature.
Fermention medium: glucose 5%, peptone 1.5%, aminoacids complex 0.1%, potassium primary phosphate 0.5%, magnesium sulfate heptahydrate 0.1%, pH nature.
2. cultural method
The mycelium of picking one ring fresh inclined-plane growth, in liquid seed culture medium, concussion is cultivated, and culture temperature is 20 DEG C, and cultivate 8 days, mycelium dry weight reaches 0.8% of fermented liquid.
With the inoculum size of 20% (V/V), secondary seed is inoculated in fermention medium, culture temperature is 20 DEG C, and fermentation period is 8 days, and after fermentation ends, mycelium dry weight yield reaches 1.8%.
The effective Analysis of Nutritional of embodiment 5 China pilose spore
China pilose spore is carried out fluid enlargement culture, after suction filtration, oven dry, obtains hypha powder.Respectively the content of the Cordyceps Polysaccharide of this mycelium powder and natural cordyceps, cordycepic acid, cordycepin is detected.
1. Cordyceps Polysaccharide measures
Adopt Phenol sulfuric acid procedure, the Cordyceps Polysaccharide content measuring the mycelium powder of above-described embodiment liquid culture is 10.6%, 10.1% and 11.0%, the content measuring Cordyceps Polysaccharide in natural cordyceps is 7.8%, and mycelium powder improves 35.9%, 29.4% and 41% respectively than Cordyceps Polysaccharide content in natural cordyceps.
2. cordycepic acid measures
Adopt liquid phase chromatography to carry out the mensuration of cordycepic acid, chromatographic column is Inertsil ODS-3 (4.6mm × 250mm, 5 μm), column temperature 30 DEG C.Detector is differential refraction detector.Moving phase is: acetonitrile: water=80:20, gradient elution,
Flow velocity 0.8mL/min.
The content measuring the cordycepic acid of the mycelium powder of above-described embodiment liquid culture is 7.8%, 7.7% and 8.0%, in natural cordyceps, the content of cordycepic acid is 7.5%, and mycelium powder improves 4.0%, 1.1% and 6% respectively than cordycepic acid content in natural cordyceps.
3. cordycepin measures
Adopt liquid phase chromatography to carry out the mensuration of cordycepin, chromatographic column is Inertsil ODS-3 (4.6mm × 250mm, 5 μm), column temperature 30 DEG C.Detector is diode array UV-detector, determined wavelength 260nm.Moving phase acetonitrile: water=80:20, flow velocity 0.8mL/min.
The cordycepin content measuring the mycelium powder of above-described embodiment liquid culture is 0.56%, 0.57% and 0.59%, in natural cordyceps, the content of cordycepin is 0.48%, and mycelium powder improves 16.7%, 18.7% and 22.9% than cordycepin content in natural cordyceps.
In addition, comparison has been carried out, as following table to other every nutritive ingredients of this mycelium powder and natural cordyceps.

Claims (2)

1. a plant height produces the China pilose spore M-010 of Cordyceps Polysaccharide, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number on May 29th, 2014: CGMCC No. 9046.
2. a cultural method for the China pilose spore of high yield Cordyceps Polysaccharide, is characterized in that, comprises the steps:
(1) solid slant culture slant medium selects potato culture; Inoculation China pilose spore bacterial classification M-010, in potato culture inclined-plane, cultivates 5-15 days under 15-25 DEG C of condition;
(2) seed culture inoculation step (1) mycelia is in seed culture medium, under 15-25 DEG C of condition, cultivate 5-10 days;
Seed culture medium: glucose 1-3%, peptone 0.2-1.0%, aminoacids complex 0.01-0.1%, potassium primary phosphate 0.1-0.3%, magnesium sulfate heptahydrate 0.02-0.08%, all the other are water, pH nature;
(3) fermentation culture presses 5-20%(V/V) the kind daughter bacteria liquid of inoculum size inoculation step (2) in fermention medium, under 15-25 DEG C of condition, cultivate 7-10 days;
Fermention medium: glucose 2-5%, peptone 0.5-1.5%, aminoacids complex 0.01-0.1%, potassium primary phosphate 0.2-0.5%, magnesium sulfate heptahydrate 0.05-0.1%, all the other are water, pH nature;
(4) suction filtration, dry and step (3) fermentation culture gained mycelium carried out suction filtration, dry to obtain hypha powder.
CN201410664856.9A 2014-11-19 2014-11-19 Hirsutella sinensis capable of high-yielding cordyceps polysaccharide and cultivating method thereof Pending CN104480021A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105670942A (en) * 2016-03-15 2016-06-15 江苏神华药业有限公司 Method for producing ophiocordyceps sinensis powder through rapid and deep liquid state fermentation
CN108384726A (en) * 2018-04-25 2018-08-10 杭州中美华东制药有限公司 Aweto mycelium and its fermentation medium and fermentation process
CN108611282A (en) * 2018-05-16 2018-10-02 江苏师范大学 The preparation method and application of one plant of Hypocreales fungi, its exocellular polysaccharide
CN109082382A (en) * 2018-06-15 2018-12-25 浙江工业大学 Cordyceps sinensis Hirsutella sinensis ZJB18002 and its application

Citations (1)

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Publication number Priority date Publication date Assignee Title
CN102373190A (en) * 2011-09-09 2012-03-14 浙江工业大学 Relevant enzymes for preparing mannitol by performing anabolism on Chinese caterpillar fungus and hirsutella sinensis, gene and application thereof

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Publication number Priority date Publication date Assignee Title
CN102373190A (en) * 2011-09-09 2012-03-14 浙江工业大学 Relevant enzymes for preparing mannitol by performing anabolism on Chinese caterpillar fungus and hirsutella sinensis, gene and application thereof

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105670942A (en) * 2016-03-15 2016-06-15 江苏神华药业有限公司 Method for producing ophiocordyceps sinensis powder through rapid and deep liquid state fermentation
CN108384726A (en) * 2018-04-25 2018-08-10 杭州中美华东制药有限公司 Aweto mycelium and its fermentation medium and fermentation process
CN108611282A (en) * 2018-05-16 2018-10-02 江苏师范大学 The preparation method and application of one plant of Hypocreales fungi, its exocellular polysaccharide
CN109082382A (en) * 2018-06-15 2018-12-25 浙江工业大学 Cordyceps sinensis Hirsutella sinensis ZJB18002 and its application

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