CN108753625A - A kind of production polysaccharide space Hydnum ST21-3 and its application in improving biological immune activity - Google Patents
A kind of production polysaccharide space Hydnum ST21-3 and its application in improving biological immune activity Download PDFInfo
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- CN108753625A CN108753625A CN201810510343.0A CN201810510343A CN108753625A CN 108753625 A CN108753625 A CN 108753625A CN 201810510343 A CN201810510343 A CN 201810510343A CN 108753625 A CN108753625 A CN 108753625A
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- hydnum
- hericium
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 53
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 53
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 53
- 241000577951 Hydnum Species 0.000 title claims abstract description 36
- 230000005965 immune activity Effects 0.000 title abstract description 7
- 238000004519 manufacturing process Methods 0.000 title abstract description 6
- 240000000588 Hericium erinaceus Species 0.000 claims abstract description 30
- 235000007328 Hericium erinaceus Nutrition 0.000 claims abstract description 30
- 244000126211 Hericium coralloides Species 0.000 claims abstract description 17
- 235000000035 Hericium coralloides Nutrition 0.000 claims abstract description 17
- 244000005700 microbiome Species 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 238000000855 fermentation Methods 0.000 claims description 19
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- 239000001963 growth medium Substances 0.000 claims description 18
- 241000894006 Bacteria Species 0.000 claims description 17
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 13
- 230000001900 immune effect Effects 0.000 claims description 10
- 230000001413 cellular effect Effects 0.000 claims description 9
- 230000036039 immunity Effects 0.000 claims description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
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- 239000012138 yeast extract Substances 0.000 claims description 5
- 239000000470 constituent Substances 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
- 229920002472 Starch Polymers 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
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- 230000002068 genetic effect Effects 0.000 description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 4
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- 108020004463 18S ribosomal RNA Proteins 0.000 description 3
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- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
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- 150000002500 ions Chemical class 0.000 description 3
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- 239000002028 Biomass Substances 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 208000003591 Hepatoerythropoietic Porphyria Diseases 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
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- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
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- 239000007836 KH2PO4 Substances 0.000 description 1
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- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- QTENRWWVYAAPBI-YZTFXSNBSA-N Streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@H]1[C@H](N=C(N)N)[C@@H](O)[C@H](N=C(N)N)[C@@H](O)[C@@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@H]1[C@H](N=C(N)N)[C@@H](O)[C@H](N=C(N)N)[C@@H](O)[C@@H]1O QTENRWWVYAAPBI-YZTFXSNBSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
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- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
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- 230000036737 immune function Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
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- 238000012549 training Methods 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
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- 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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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
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Abstract
The invention discloses a kind of production polysaccharide space Hydnum ST21-3 and its applications in improving biological immune activity.Hydnum (Hericium coralloides) Fullarton-H-ST21-3 provided by the invention is CGMCC No.15379 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.The present invention carries the Hericium erinaceus for returning to ground after space flight using No. 11 spaceships of Heavenly Palace 2 and divine boat, as a contrast with the original Hericium erinaceus in ground, the higher bacterial strain of immunocompetence is selected, practical basis is provided for application of the space Hericium erinaceus mycelium polysaccharides in improving biological immune activity.The present invention has widened space microbe application range to a certain extent simultaneously, has filled up the blank of food space fungi microbe function research and development.
Description
Technical field
The present invention relates to a kind of production polysaccharide space Hydnum ST21-3 and its in improving biological immune activity
Using.
Background technology
Hericium erinaceus Polysaccharides (Hericium erinaceus polysaccharides, HEP) are by glucose, galactolipin, sweet
Dew sugar composition is the glucan of the branch composition of the main chain and the connection of β-(1,6) glycosidic bond that are connected by β-(1,3) glycosidic bond, is monkey
A kind of critical function active material of the head fermented generation of bacterium.Hericium erinaceus Polysaccharides include polysaccharide and zymotic fluid in mycelial cell
In polysaccharide.Wherein hedgehog fungus mycelium polysaccharide other than with immunoregulatory activity, also have hypoglycemic, reducing blood lipid, it is antitumor,
The bioactivity such as anti-oxidant.Hericium erinaceus Polysaccharides immunoregulatory activity is shown：The activity of natural killer cells can be improved, promoted late
The generation (antianaphylaxis) of hair style hypersensitivity, enhances the phagocytic function of macrophage, and to humoral immunity and immune organ
Proliferation function unobvious.This shows that Hericium erinaceus mycelium polysaccharides mainly adjust body by enhancing the activity of immunocyte
Immune function.In addition, many studies demonstrate that, Hericium erinaceus Polysaccharides improve body hypoxia-bearing energy in treatment gastritis, digestive tract ulcer
Power increases heart blood output quantity, accelerates body blood cycle etc. to play an important roll, is a kind of function of food and medicament dual-purpose
Bacterium.
Domestic and foreign scholars are concentrated mainly on the optimization to polysaccharide material extraction conditions and institute to the research of Hericium erinaceus at present
Analysis containing the structure of matter is still in infancy the immunoregulatory activity research of polysaccharide material, and space microorganism
Research is also rested on mostly to the application in terms of space pathogen, saprophytic bacteria and pharmacy, the exploitation for food space microorganism
Still it is in the budding stage.
Invention content
The object of the present invention is to provide a kind of production polysaccharide space Hydnum ST21-3 and its improving biological immune
Application in activity.
Hydnum (Hericium coralloides) Fullarton-H-ST21-3 provided by the invention, in
On April 24th, 2018 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC；Address：
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica；Postcode：100101), deposit number is
CGMCC NO.15379。
The present invention also protect Hydnum (Hericium coralloides) Fullarton-H-ST21-3 or its
Application of the mycelium in preparing product；The purposes of the product is following (1) and/or (2)：
(1) immunologic cellular activity is improved；
(2) immunity of organisms is improved.
The present invention also protects a kind of preparation method of Hericium erinaceus mycelium polysaccharides, includes the following steps：Cultivate coralliform monkey
Head bacterium (Hericium coralloides) Fullarton-H-ST21-3, obtains mycelium；Polysaccharide is extracted from mycelium, is obtained
To the Hydnum mycelium polysaccharides.
In the method, using Hydnum (Hericium described in seed fermentation medium culture
coralloides)Fullarton-H-ST21-3。
In the method, the condition of culture is 28 DEG C, 160r/min shaken cultivations.
The method is concretely：
(a) Hydnum (Hericium coralloides) Fullarton-H-ST21-3 is inoculated in seed hair
It is cultivated in ferment culture medium；
(b) after completing step (a), culture is moved into seed fermentation training according to by the inoculum concentration of 10% (percent by volume)
Base culture is supported, mycelium is collected；Polysaccharide is extracted from mycelium.
In (a), the condition of culture is 28 DEG C, 160r/min shaken cultivations, and the incubation time is 12 days.
In the step (b), the condition of culture is 28 DEG C, 160r/min shaken cultivations, and the incubation time is 5 days.
In the method, collecting mycelial method is specially：Cultivating system is filtered with three layers of sterile gauze, collects bacterium
Filament (after being washed 3-4 times with sterile distilled water after collection, carries out vacuum freeze drying).
In the method, the method that polysaccharide is extracted from mycelium is specially：Powder is broken into after mycelium is lyophilized, with
Boiling waterbath 2h after distilled water mixing, extracting solution is collected after three layers of filtered through gauze (can repeat extracting solution residue after extracting
Merge), extracting solution is subjected to alcohol precipitation (condition can be 4 DEG C of placement 12h) and collects precipitate afterwards, (60 DEG C of constant temperature can be used in precipitation drying
Dry or vacuum freeze drying) after obtain the polysaccharide.
The present invention also protects the Hericium erinaceus mycelium polysaccharides that any description above method obtains.
The present invention also protects application of the Hericium erinaceus mycelium polysaccharides in preparing product；The purposes of the product is such as
Under (1) and/or (2)：
(1) immunologic cellular activity is improved；
(2) immunity of organisms is improved.
It is Hydnum (Hericium coralloides) that the present invention, which also protects a kind of product, active constituent,
Fullarton-H-ST21-3 or its mycelium；The purposes of the product is following (1) and/or (2)：
(1) immunologic cellular activity is improved；
(2) immunity of organisms is improved.
It is the Hericium erinaceus mycelium polysaccharides that the present invention, which also protects a kind of product, active constituent,；The purposes of the product
For following (1) and/or (2)：
(1) immunologic cellular activity is improved；
(2) immunity of organisms is improved.
The present invention also protects a kind of kit being used to prepare Hericium erinaceus mycelium polysaccharides, including Hydnum
(Hericium coralloides) Fullarton-H-ST21-3 and seed fermentation culture medium.
Any description above product concretely food, drug or health products etc..
Any description above seed fermentation culture medium is made of solute and solvent；The solute and its in the seed fermentation
Concentration in culture medium is as follows：Soluble starch 15-25g/L, glucose 25-35g/L, yeast extract 5-15g/L, seven hydration sulphur
Sour magnesium 0.5-0.7g/L, potassium dihydrogen phosphate 2.5-3.5g/L；The solvent is water.
Concentration in the seed fermentation culture medium and its in the seed fermentation culture medium is specific as follows：Solubility is formed sediment
Powder 20g/L, glucose 30g/L, yeast extract 10g/L, bitter salt 0.6g/L, potassium dihydrogen phosphate 3.0g/L.
Solvent in any description above seed fermentation culture medium concretely distilled water.
Any description above improves the burst size that immunologic cellular activity specifically can behave as improving macrophage NO.
The macrophage concretely RAW264.7 cells.
The present invention carries the hedgehog hydnum for returning to ground after space flight using No. 11 spaceships of Heavenly Palace 2 and divine boat
Bacterium, certain biological characters (such as individual morphology, colony characteristics, physiological and biochemical property, immunocompetence), fermenting and producing performance
(such as biomass, product amount, enzyme activity, potency, fermenting speed) is due to by space microgravity effect, high vacuum, extreme temperature
The mutagenesis such as difference, low-intensity magnetic field and high energy particle (electronics, proton, heavy ion) radiation, change gene mutation to a certain extent
Frequency leads to the change of functional activity in turn.
The present invention as a contrast, selects the higher bacterial strain of immunocompetence with the original Hericium erinaceus in ground, is space hedgehog hydnum
Application of the bacterium mycelium polysaccharides in improving biological immune activity provides practical basis.While patent of the present invention is to a certain extent
Space microbe application range has been widened, the blank of food space fungi microbe function research and development has been filled up.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Ground Hericium erinaceus (Hericium erinaceus) GT21：Chinese industrial Organism Depositary, number 14026,
Referred to as ground Hericium erinaceus GT21.
YEPD culture mediums：Yeast extract 10g, peptone 20g, glucose 20g, agar 17g, distilled water are settled to
1000mL, pH6.0.
Seed fermentation culture medium：Soluble starch 20g, glucose 30g, yeast extract 10g, bitter salt 0.6g,
Potassium dihydrogen phosphate 3g, distilled water are settled to 1000mL, natural ph.
Macrophage used is RAW264.7 cells in embodiment, is purchased from ATCC companies of the U.S..
The screening of embodiment 1, space Hericium erinaceus
One, the space flight of bacterial strain
Ground Hericium erinaceus GT21 is returned through No. 11 Spaceship Carryings of Heavenly Palace 2 and divine boat, obtains several plants of space mutagenesis bacterium
Strain.
Two, the activation of bacterial strain
Take freeze in -80 DEG C of ultra low temperature freezers space mutagenesis bacterial strain and ground Hericium erinaceus GT21 glycerol tube preservation bacterium
Kind, a small amount of bacterium solution streak inoculation is dipped in YEPD culture mediums test tube slant with oese, 28 DEG C are cultivated 6 days, after 3 generations of continuous activation
For follow-up test.
Three, the expansion culture of bacterial strain
The space mutagenesis bacterial strain in step 23 generations of activation and ground Hericium erinaceus GT21 is taken to be inoculated in YEPD culture medium triangular flasks oblique
Face is cultivated 6 days in 28 DEG C.
Four, bacterial strain isolates and purifies
After completing step 3, take in the well-grown space mutagenesis bacterial strain in YEPD culture medium triangular flasks inclined-plane and ground hedgehog hydnum
Bacterium GT21, surface spore is rinsed with 100mL sterile salines, is pressed 8 times/sec with sterile homogenizer, is patted 1min, make spore
It is sufficiently separated with mycelium, after being filtered with three layers of sterile gauze, obtains pure spore suspension.Spore suspension 1mL is taken, with 9mL
Sterile saline carries out 10 times of gradient dilutions, takes 10-2Spore dilution is inoculated in YEPD culture medium flat plates with coating method,
It is cultivated 6 days in 28 DEG C.The bacterium colony form of observation space mutagenic strain and ground Hericium erinaceus GT21, picking colony feature and ground
The bacterial strain space mutagenesis bacterial strain single bacterium colony big compared to difference, purifying are incubated at YEPD culture mediums test tube slant, and 4 are cultivated in 28 DEG C
It, obtains the bacterial strain that number is ST21-1 to ST21-10.
Five, the secondary screening of bacterial strain
1, mycelial preparation
Strain to be tested is taken to be inoculated in seed fermentation culture medium, 28 DEG C, 160r/min shaken cultivations 12 days, with sterile homogenizer
By 8 times/sec, 1min is patted, claps and dissipates mycelium pellet, by the inoculum concentration immigration seed fermentation culture medium of 10% (percent by volume), 28
DEG C, 160r/min shaken cultivations 5 days take fermentation system, are filtered with three layers of sterile gauze, collect mycelium, the mycelia that will be obtained
After body is washed 3-4 times with sterile distilled water, vacuum freeze drying.
2, the preparation of mycelium Thick many candies
The mycelium that step 1 is lyophilized is milled to powder, weighs sample 1g, 18mL distilled water, boiling waterbath 2h is added
Afterwards, three layers of filtered through gauze, filter residue repeat extraction 1 time, merge extracting solution, extracting solution is added with distilled water constant volume in 20mL
Absolute ethyl alcohol 300mL, shakes up, in 4 DEG C place 12h after, 10000r/min centrifuge 20min, collect precipitation, with 95% ethyl alcohol from
The heart washs 2 times (centrifugal condition is same as above), will precipitate 60 DEG C of freeze-day with constant temperature until quality constant weight, obtains mycelium Thick many candies.
3, preparation of reagents
(1) preparation of dual anti-solution：It takes the penicillin solid powder of 3mg to be dissolved in the distilled water of 100mL, is made into 30 μ g/
The penicillin solution of mL.10mg streptomysin solid powders are taken to be dissolved in the distilled water of 100mL, the streptomysin for being made into 100 μ g/mL is molten
Liquid.Penicillin solution and Streptomycin Solution are mixed into Uniform in equal volume, as dual anti-solution is saved backup in -20 DEG C.
(2) preparation of PBS (phosphate buffer solution)：Take 0.14mol/LNaCl, 0.0027mol/L KCl,
0.004mol/L Na2HPO4With 0.002mol/L KH2PO4It is isometric to be uniformly mixed, finely tune pH extremely with the sodium hydroxide of 1mol/L
7.40, after being sterile filtered with 0.22 μm of filter membrane, a concentration of 10 μ g/mL of PBS are diluted to distilled water, it is spare.
(3) nitric oxide Griess kits are detected：Green skies company, 4 DEG C of preservations.
4, culture medium is prepared
DMEM complete mediums (percentage by volume)：By 89% (volumn concentration) DMEM high glucose mediums and 10%
(volumn concentration) FBS (fetal calf serum) and the dual anti-solution mixing of 1% (volumn concentration), as DMEM are cultivated completely
Base.
DMEM high glucose mediums are purchased from Shanghai Sheng Gong bioengineering Co., Ltd with FBS (fetal calf serum).
5, the preparation of sample
The accurate mycelium Thick many candies for weighing the preparation of 5mg steps 2 are made into 5mg/ in sterile centrifugation tube with the PBS of pH7.4
The polysaccharide solution of mL centrifuges 30min with 12000r/min, collects supernatant, and sterile working is transferred in 1.5mL sterile centrifugation tubes,
Polysaccharide sample is diluted to 1mg/mL, 500 μ g/mL, 250 μ g/mL with the PBS of pH7.4.
6, the preparation of macrophage suspension
Macrophage of the selection in logarithmic phase, siphons away the culture solution in Tissue Culture Flask, the PBS that 1mLpH7.4 is added is clear
It washes, siphons away supernatant, add 0.25% trypsase, culture bottle is put into 5%CO2Incubator cultivates 6min in 37 DEG C,
The DMEM complete mediums that 1mL is added terminate digestion reaction, blow and beat cell with liquid-transfering gun, cell is made to suspend.By cell suspending liquid
It is transferred in 15mL sterile centrifugation tubes, after centrifuging 5min with 1000r/min, siphons away supernatant, the PBS cleanings of the pH7.4 of 1mL are added
Precipitation centrifuges 5min with 1000r/min, siphons away supernatant, DMEM complete mediums are added, so that cell is suspended, uses blood count
Plate counts, then cell is diluted to 5 × 10 with DMEM complete mediums5Cell/mL, as macrophage suspension.
7, nitric oxide (NO) concentration mensuration
Macrophage suspension prepared by step 6 is added in 48 orifice plates (being purchased from Coster companies), and dosage is 180 μ L,
The polysaccharide sample for the various concentration that 20 μ L steps 5 obtain is added per hole, the PBS (10 μ g/mL) of pH7.4 is negative control, LPS
(10 μ g/mL bacteria lipopolysaccharides, Hua Baitai biologies Co., Ltd) is positive control, in 37 DEG C, 5%CO2In incubator at culture
48h is managed to get macrophage treatment fluid.Sodium nitrite standard items in Griess kits DMEM complete mediums are diluted
At different gradients, in another 48 orifice plate, according to the addition in 200 holes μ L/, it is separately added into sodium nitrite standard items and macrophage is thin
Born of the same parents' HEP treatment fluids then press 200 holes μ L/ additions again, the Griess Reagent Ι examinations being separately added at room temperature in kit
II reagent of agent and Griess Reagent measures absorbance value (A values) under 540nm.The results are shown in Table 1.
The mycelium polysaccharides solution effects of 1 various concentration of table are in the burst size (μm ol/L) of RAW264.7 cells NO
By table 1 as it can be seen that the mycelia Thick many candies stimulation RAW264.7 macrophages of ST21-1 to ST21-10 bacterial strains discharge NO's
Amount has notable difference, and rises with the increase of mycelia polysaccharide solution concentration, wherein the mycelia Thick many candies of ST21-3 bacterial strains
The NO burst sizes of stimulation RAW264.7 macrophages are above other bacterial strains at various concentrations.Due to macrophage NO burst sizes with
Ion vitro immunization activity is positively correlated, therefore the produced mycelia polysaccharide of ST21-3 bacterial strains is to the stimulation of RAW264.7 macrophages
Most strong, NO burst sizes are 47.21 μm of ol/L, are that ground bacterial strain GT21 mycelium polysaccharides act on RAW264.7 cell NO burst sizes
3.91 times, show that ST21-3 bacterial strain ion vitro immunization activity is higher, can become improve biological immune activity most worthy bacterial strain.
Six, the Morphological Identification and molecular biology identification of ST21-3 bacterial strains
1, the colony characteristics of ST21-3 bacterial strains：Bacteria colony white, is slightly in yellowish, felt shape, and surface has many irregular
Bulbous protrusions, aerial mycelium ulotrichy.The back side is gradually changed into yellow, yellowish-brown from bacterium colony center by white, and discoloration is gradual
Expand and deepens.
2, the 18S rDNA of ST21-3 bacterial strains are detected, sequencing result is as shown in the sequence of sequence table 1.18s rDNA identification knots
Fruit shows ST21-3 bacterial strains and similar 99% or more of Hydnum (Hericium coralloides).By morphology
And 18S rDNA identifications, it may be determined that ST21-3 bacterial strains belong to Hydnum.
Seven, the preservation of ST21-3 bacterial strains
It is Hydnum (Hericium coralloides) Fullarton-H-ST21- by ST21-3 Strain Designations
3, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC on April 24th, 2018；
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica；Postcode：100101), preservation is compiled
Number be CGMCC NO.15379.Hydnum (Hericium coralloides)
Fullarton-H-ST21-3 is referred to as Hydnum SST21-3.
Eight, genetic stability
By Hydnum SST21-3 strain passage cultures, genetic stability experiment is carried out, by measuring per a generation not
With influence of the mycelium polysaccharides solution to macrophage RAW264.7 cell NO burst sizes of concentration, genetic stability is judged, tie
Fruit is as shown in table 2.
Influence of the mycelium polysaccharides solution of the 10 generation various concentrations of 2 ST21-3 of table to RAW264.7 cell NO burst sizes
(μmol/L)
As can be seen from Table 2, after ST21-3 passed for 10 generations, NO burst sizes are still within higher level, illustrate various concentration polysaccharide
Solution is in higher level to the immunocompetence of RAW264.7 cells, can judge that Hydnum SST21-3 is being carried substantially
There is genetic stability in terms of high immunobiologic activity.
Embodiment 2, Hericium erinaceus ST21-3 mycelium polysaccharides measure
1, mycelial preparation
Hydnum SST21-3 is taken to be inoculated in seed fermentation culture medium, 28 DEG C, 160r/min shaken cultivations 12 days,
8 times/sec is pressed with sterile homogenizer, pats 1min, claps and dissipates mycelium pellet, seed is moved by the inoculum concentration of 10% (percent by volume)
Fermentation medium, 28 DEG C, 160r/min shaken cultivations 5 days take fermentation system, are filtered with three layers of sterile gauze, collect mycelium,
After obtained mycelium is washed 3-4 times with sterile distilled water, vacuum freeze drying.
2, the preparation of mycelium Thick many candies
The mycelium that step 1 is lyophilized is milled to powder, weighs sample 1g, 18mL distilled water, boiling waterbath 2h is added
Afterwards, three layers of filtered through gauze, filter residue repeat extraction 1 time, merge extracting solution, extracting solution is added with distilled water constant volume in 20mL
Absolute ethyl alcohol 300mL, shakes up, and after 4 DEG C are placed 12h, 10000r/min centrifuges 20min, collects precipitation, will precipitate vacuum refrigeration
It is dry, obtain mycelium Thick many candies.
3, the drafting of standard curve
Taking 105 DEG C of standard glucose, drying to constant weight, is made into the glucose mark product mother liquor of 10mg/mL, then fixed with volumetric flask
Hold and dilutes 200 times to 50 μ g/mL, as glucose standards solution.Absorption 0,0.2,0.4,0.6,0.8,1.0mL standards Portugal respectively
Grape sugar juice is placed in test tube, is mended to 1mL with distilled water, and 5% (mass percent) phenol solution 0.5mL, mixing are added
After be rapidly added the concentrated sulfuric acid (mass fraction 98%) 2.5mL, so that reaction solution is sufficiently mixed using turbula shaker, then by test tube
It is placed in boiling water bath and reacts 15min, water-bath cooling 10min to room temperature measures its suction at 490nm with ultraviolet specrophotometer
Shading value (A values), with a concentration of abscissa of glucose quality, absorbance value is ordinate, draws standard curve.Standard curve
Regression equation is y=0.0152x+0.0208 (R2=0.9995).
4, total sugar content measures
It is polyoses content with the total sugar content that phend-sulphuric acid measures.10mg steps are weighed with the electronic balance of a ten thousandth
Rapid 2 Thick many candies prepared, are dissolved in 50mL distilled water, are then diluted to polysaccharide concentration in standard curve range with distilled water again
It is interior, obtain testing sample solution.In testing sample solution to test tube after taking 1mL to dilute, 5% (mass percent) phenol is added
Solution 0.5mL is rapidly added the concentrated sulfuric acid (mass fraction 98%) 2.5mL after mixing, keeps reaction solution abundant using turbula shaker
Test tube, is then placed in boiling water bath and reacts 15min, water-bath cooling 10min to room temperature is measured with ultraviolet specrophotometer by mixing
Its absorbance value (A values) at 490nm.By the absorbance y values measured substitute into glucose standard curve regression equation in get
To polyoses content x values (mg/mL).The results are shown in Table 3 for Hydnum ST21-3 mycelium polysaccharides.
Small molecule is removed since Thick many candies have been subjected to alcohol precipitation in the preparation, therefore content of reducing sugar is extremely low in Thick many candies, can be with
It ignores.
Mycelia polysaccharide total content=fermented hypha biomass × mycelia polysaccharide content/100.
3 Hydnum ST21-3 mycelium polysaccharides results of table
As shown in Table 3, Hydnum SST21-3 mycelial biomasses are 3.52g/100mL, and polyoses content is
1.08mg/100mL, polysaccharide total amount are 38.17mg/100mL.
Sequence table
<110>Fuller gives birth suddenly object engineering science and technology（Beijing）Co., Ltd
<120>A kind of production polysaccharide space Hydnum ST21-3 and its application in improving biological immune activity
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 648
<212> DNA
<213>Hydnum (Hericium coralloides)
<400> 1
ccttccgtaa gggggcctgc ggaaggatca ttaatgaatt tgaaaggagt tttgttgctg 60
gcttgtcaac ccaggcatgt gcacactccg atctcatcca tcttacacct gtgcaccctt 120
gcgtgggtct gtcggctttg cggtvgttcg ggcttgcgtt ttttctataa acttttatgt 180
agtaacagaa tgtcttaaat gctataaacg catcttatac aactttcaac aacggatctc 240
ttggctctcg catcgatgaa gaacgcagcg aaatgcgata agtaatgtga attgcagaat 300
tcagtgaatc atcgaatctt tgaacgcacc ttgcgcccct tggtattccg agggcacgcc 360
tgttcgagtg tcgtgaaatt ctcaactcaa tcctcttgtt atgagagggc tgggcttgga 420
cttggaggtc ttgccggtgg ttccttcggg accgtcggct cctcttgaat gcatgagtgg 480
atcccttttt gtagggtttg cccttggtgt gataatatct acgccgcggg tagccttgcg 540
cgctggtctg cttctaaccg tccttcggga catgtttttc atctcaactt gacctcgaat 600
caggctgctg ccgctgattt gcccaagcat atcaatacgc gaaggaaa 648
Claims (10)
- Hydnum 1. (Hericium coralloides) Fullarton-H-ST21-3, in Chinese microorganism strain The deposit number of preservation administration committee common micro-organisms center is CGMCC No.15379.
- 2. (Hericium coralloides) Fullarton-H-ST21-3 of Hydnum described in claim 1 or its bacterium Application of the filament in preparing product；The purposes of the product is following (1) and/or (2)：(1) immunologic cellular activity is improved；(2) immunity of organisms is improved.
- 3. a kind of preparation method of Hericium erinaceus mycelium polysaccharides, includes the following steps：Cultivate the Hydnum (Hericium coralloides) Fullarton-H-ST21-3, obtains mycelium, polysaccharide is extracted from mycelium, obtain institute State Hydnum mycelium polysaccharides.
- 4. method as claimed in claim 3, includes the following steps：In the method, using seed fermentation medium culture institute State Hydnum (Hericium coralloides) Fullarton-H-ST21-3；The seed fermentation culture medium is made of solute and solvent；The solute and its dense in the seed fermentation culture medium Degree is as follows：Soluble starch 15-25g/L, glucose 25-35g/L, yeast extract 5-15g/L, bitter salt 0.5- 0.7g/L, potassium dihydrogen phosphate 2.5-3.5g/L；The solvent is water.
- 5. method as described in claim 3 or 4, it is characterised in that：In the method, the condition of culture is 28 DEG C, 160r/ Min shaken cultivations.
- 6. the Hericium erinaceus mycelium polysaccharides that any the method for claim 3 to 5 is prepared.
- 7. application of the Hericium erinaceus mycelium polysaccharides in preparing product described in claim 6；The purposes of the product is as follows (1) and/or (2)：(1) immunologic cellular activity is improved；(2) immunity of organisms is improved.
- 8. a kind of product, active constituent is the Hydnum (Hericium coralloides) of claim 1 Fullarton-H-ST21-3 or its mycelium；The purposes of the product is following (1) and/or (2)：(1) immunologic cellular activity is improved；(2) immunity of organisms is improved.
- 9. a kind of product, active constituent is the Hericium erinaceus mycelium polysaccharides described in claim 6；The purposes of the product is such as Under (1) and/or (2)：(1) immunologic cellular activity is improved；(2) immunity of organisms is improved.
- 10. a kind of kit being used to prepare Hericium erinaceus mycelium polysaccharides, including Hydnum described in claim 1 Seed fermentation culture medium described in (Hericium coralloides) Fullarton-H-ST21-3 and claim 4.
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