CN110447457A - A kind of pycnoporus samguineus new strains and its artificial cultivation method and purposes - Google Patents
A kind of pycnoporus samguineus new strains and its artificial cultivation method and purposes Download PDFInfo
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- CN110447457A CN110447457A CN201910879853.XA CN201910879853A CN110447457A CN 110447457 A CN110447457 A CN 110447457A CN 201910879853 A CN201910879853 A CN 201910879853A CN 110447457 A CN110447457 A CN 110447457A
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- pycnoporus
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- samguineus
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Links
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
-
- A—HUMAN NECESSITIES
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
Abstract
The present invention relates to a kind of new strains and its artificial cultivation method and purposes more particularly to a kind of pycnoporus samguineus new strains and its artificial cultivation method and purposes.Pycnoporus samguineus new strains of the invention pick up from Wuyi Mountain, fujian, it is identified as pycnoporus samguineus, and separation is organized to obtain original strain, it is named as pycnoporus samguineus (Pycnoporus sanguineus) HMGIM-140415, China typical culture collection center is preserved on July 3rd, 2019, deposit number is CCTCC NO:M 2019514.New strains of the invention have been carried out artificial domesticating cultivation, and compared with other bacterial strains of the same race, all show more strong and significant inhibiting rate to tumour and staphylococcus.
Description
Technical field
The present invention relates to a kind of new strains and its artificial cultivation method and purposes more particularly to a kind of pycnoporus samguineus
New strains and its artificial cultivation method and purposes.
Background technique
Currently, the industry development of edible and medical fungi is swift and violent, counted according to edible fungi of china association, China's edible and medical fungi in 2017
Yield reaches 37,120,000 tons, and 3.21% was increased than 2016, and the output value is 2721.92 hundred million yuan, and China accounts for 75% or more the whole world, from
Industry personnel are more than 20,000,000 people, and mushroom industry is come in planting industry in addition to the 5th after grain, dish, fruit, oil, are more than
Tealeaves and silkworm and mulberry.
In today that edible and medical fungi industry flourishes, more and more rare edible and medical fungi kinds progress into people's
The visual field, many original rare kinds are gradually tamed, such as dictyophora phalloidea, Agrocybe chaxingu, from pleat umbrella, hickory chick.But also have large quantities of
Wild edible and medical fungi do not studied due to failing by human knowledge.It was found that at present, there are about more than 300 ten thousand kinds in the world
Fungal species, only 1% species are realized, wherein known about 14000 kinds of macro fungi, the edible mushroom that the country has confirmed that
There are 1789 kinds, 798 kinds of medicinal fungus, and tamed in the middle only less than 100 kinds of wild edible and medical fungi by the mankind, large-scale planting
Kind more only have more than 30 kinds.Research of the mankind apart from macro fungi will be walked with using there are also quite long road.With people
Living standard gradually rises, and the requirement for quality of the life is higher, and macro fungi due to its rich in have the function of nutrition and
Various composition of effect, including fungi polysaccharide, triterpenes, sterol etc. have very good effect for human health, increasingly
It is valued by people.
Pycnoporus samguineus is widely distributed, usually in two summer, autumn season harvestings, dries after adopting.The bacterium has higher medicinal
Value, can antipruritic, hemostasis, myogenic, pleasant etc., fructification contains polyporin (polyporin), and polyporin is to leather orchid
Family name positive and negative bacterium has inhibiting effect, and the bacterium can also be used in anti-inflammatory.
Red fungus economic use is extensive, and medical value is obvious, so more and more people develop and utilize it,
But to the report for so far, not thering is scale to cultivate and apply.
Summary of the invention
Against the above deficiency, the present invention provides a kind of wild rare medicine with anti tumor activity in vitro and bacteriostasis
With bacterium kind pycnoporus samguineus Pycnoporus sanguineus new strains and its artificial domesticating cultivation method and purposes.
The present invention reaches above-mentioned purpose by following scheme:
In a first aspect, pycnoporus samguineus new strains of the invention pick up from Wuyi Mountain, fujian, it is identified as pycnoporus samguineus, and
And tissue separation obtains original strain, is named as pycnoporus samguineus (Pycnoporus sanguineus) HMGIM-140415, in
On July 3rd, 2019 is preserved in China typical culture collection center (abbreviation CCTCC, address are as follows: Wuhan, China), deposit number
For CCTCC NO:M 2019514.
Second aspect, the present invention provide a kind of pycnoporus samguineus (Pycnoporus sanguineus) CCTCC NO:M
2019514 artificial cultivation method, including production parent species, production production parent species, production production kind, cultivation culture and cultivation pipe
Reason, by weight percentage, the culture material includes weed tree sawdust 37-41%, cotton seed hulls 18-20%, corncob 18-20%, wheat
Bran 16-18%, corn flour 2-3%, lime 1-2%.
In the third aspect, the present invention provides a kind of pycnoporus samguineus new strains CCTCC NO:M 2019514 or its extract
Application, for related disease caused by antitumor or antibacterium.
In fourth aspect, the present invention provides a kind of pycnoporus samguineus new strains CCTCC NO:M 2019514 or its extract
Application, be used to prepare the drug of related disease caused by antitumor or antibacterium.
At the 5th aspect, the present invention provides the drug of related disease caused by the antitumor or antibacterium of one kind, including blood
Red samguineus new strains CCTCC NO:M 2019514 or its extract and carrier.
New strains of the invention have been carried out artificial domesticating cultivation, and compared with other bacterial strains of the same race, to tumour and
Staphylococcus all shows more strong and significant inhibiting rate.
Detailed description of the invention
Fig. 1 is the wild subobject graph of the pycnoporus samguineus Pycnoporus sanguineus of embodiment 1.
Fig. 2 is the pycnoporus samguineus subobject graph of the domestication of embodiment 2.
Fig. 3 is the pycnoporus samguineus subobject graph of the domestication of embodiment 2.
Fig. 4 is the ITS sequence of embodiment 1.
Fig. 5 is the scribing line culture subregion schematic diagram of embodiment 4.
Fig. 6 is the suppression staphylococcus aureus schematic diagram of embodiment 4.
Specific embodiment
In a first aspect, pycnoporus samguineus new strains of the invention pick up from Wuyi Mountain, fujian, it is identified as pycnoporus samguineus, and
And tissue separation obtains original strain, is named as pycnoporus samguineus (Pycnoporus sanguineus) HMGIM-140415, in
On July 3rd, 2019 is preserved in China typical culture collection center (abbreviation CCTCC, address are as follows: Wuhan, China), deposit number
For CCTCC NO:M 2019514.
Extracting genome DNA is carried out to the original strain of acquisition and carries out ITS sequencing, by sequencing result in GenBank
Sequence B last is carried out, is found up to close with pycnoporus samguineus Pycnoporus sanguineus (L.) Murrill similitude
100%, combining form identification, the fungus specimen gross feature and microscopic features and Pycnoporus sanguineus describe
Unanimously, qualification result is Pycnoporus sanguineus.
Pycnoporus samguineus Pycnoporus sanguineus belongs to Aphyllophorales, and Polyporaceae is red fungus
The existing name of Trametes sanguinea, fructification is annual, keratin, cap sector, semicircle or kidney shape, overhanging reachable 3cm,
Wide reachable 5cm, base portion thickness is up to 1.5cm；Light reddish brown color, rust brown to yellowish-brown, later period fade when surface is fresh, color after doing
It is almost unchanged；Edge is sharp, and color is shallower, sometimes wavy.Brick-red when vent surface is fresh, color is almost unchanged after doing；Nearly circle
Shape, 5~6 every millimeter；Thin edge, full edge.Sterile edge is obvious, apricot, wide reachable 1mm.Bacterial context light reddish brown color, it is thick reachable
13mm.Tube bronzing is long up to 2mm.3.6~4.4 × 1.7~2 μm of basidiospore, oblong to cylinder is colourless, thin
Wall, smooth, non-starchy, not thermophilic indigo plant.Summer and autumn is singly raw or falls in wood, stub and rotten wood fasciating in a variety of broad leaf trees, causes wood
Material white rot.It is medicinal.Each area is distributed.
Second aspect, the present invention provide a kind of pycnoporus samguineus (Pycnoporus sanguineus) CCTCC NO:M
2019514 artificial cultivation method, including production parent species, production production parent species, production production kind, cultivation culture and cultivation pipe
Reason, by weight percentage, the culture material includes weed tree sawdust 37-41%, cotton seed hulls 18-20%, corncob 18-20%, wheat
Bran 16-18%, corn flour 2-3%, lime 1-2%.
Preferably, the material-water ratio of above-mentioned culture material is 1:1.1-1.3.
Preferably, by weight percentage, the culture material include 39% weed tree sawdust, 20% cotton seed hulls, 20% corncob,
17% wheat bran, 3% corn flour, 1% lime, material-water ratio: 1:1.1-1.3.
Preferably, the cultivation culture includes: that production kind is forwarded in culture material, 25-26 DEG C of constant temperature, shading culture,
Humidity 60%-70%, mycelia cover with bacterium bag and enter cultivation management.
Preferably, the cultivation management includes: to continue shading after-ripening after culture material in the long purseful of the mycelia in cultivating bag
Culture 15 days controls temperature at 23-27 DEG C into fruiting stage, and stronger ventilation amount, and space carbon dioxide content is kept to exist
1% hereinafter, relative air humidity is adjusted to 90% or more, and after 5-7 days, removal cap, mycelia starts to twist together and formed scarlet
Color rice La shape former base；After former base grows to 0.5cm, continue to control temperature between 23 DEG C -27 DEG C, relative air humidity 85-
Between 90%, daily illumination 9 hours, intensity of illumination 300-500lx, and keep the carbon dioxide concentration in air 350~
1500ppm keeps with the humid air, and through 30 days or so, during this period, daily Xiang Yougu sprayed water mist 1-2 times, until fructification is big
Small to be basically unchanged, fructification is mature, harvesting.
Preferably, the production parent species include: that isolated strain transfer to mother culture media is placed in 25 DEG C of constant temperature and is secretly trained
It supports, the picking of Tip Splitting is carried out when mycelia grows and bacterium not yet grows, obtains parent species.
Preferably, the mother culture media is rose bengal medium.
It is further preferred that by weight percentage, the rose bengal medium includes: peptone 0.5%, glucose
1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate (MgSO47H2O) 0.05%, agar 2%, 1/3000 rose-bengal solution 10%,
Chloramphenicol 0.01%, remaining is water.
Preferably, the production production parent species include: that parent species are forwarded to production mother culture media, and it is dark to be placed in 25 DEG C of constant temperature
Culture covers with inclined-plane to mycelia and obtains production parent species.
Preferably, the production mother culture media is to add rich comprehensive PDA.
It is further preferred that by weight percentage, described plus rich comprehensive PDA include: potato 20%, glucose 2%,
Peptone 1%, agar 2%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.15%, vitamin B1 are micro, remaining is water.
Preferably, production production kind include: into production kind culture medium sterile access produce parent species, when inoculation, ensures
It produces in parent species material block embedment original seed material, is placed in 25 DEG C of constant temperature dark cultures, production kind is obtained after mycelia eats full material.
Preferably, by weight percentage, the production kind culture medium includes: 98-99% sorghum and 1-2% calcium carbonate.
It is further preferred that by weight percentage, the production kind culture medium includes: 98% sorghum and 2% calcium carbonate.
It preferably, further include tissue separation strain before the production parent species of the artificial cultivation method.
A kind of artificial cultivation side of pycnoporus samguineus (Pycnoporus sanguineus) CCTCC NO:M 2019514
Parent species, production production parent species, production production kind, cultivation culture and cultivation management are made after method, including tissue separation strain, with weight
Percentages are measured, the culture material includes weed tree sawdust 37-41%, cotton seed hulls 18-20%, corncob 18-20%, wheat bran 16-
18%, corn flour 2-3%, lime 1-2%.
Preferably, the tissue separation strain includes: the pycnoporus samguineus fructification of acquisition back aseptically wine
Behind essence wiping surface, tears, the inside meat bacteria organization of 0.2-0.5mm × 0.2-0.5mm is accessed in a manner of sterile working, is placed in 25
Constant temperature dark culture in DEG C, the strain separated after mycelia covers with inclined-plane.
Preferably, the tissue isolation medium is comprehensive PDA culture medium.
It is further preferred that by weight percentage, the comprehensive PDA culture medium includes potato 20%, glucose
2%, agar 2%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.15%, vitamin B1 are micro.
In the third aspect, the present invention provides a kind of pycnoporus samguineus new strains CCTCC NO:M 2019514 or its extract
Application, for related disease caused by antitumor or antibacterium.
Preferably, the extract is ethyl acetate extract.
Preferably, the tumour is tumour caused by HepG2 cell.
Preferably, the bacterium is staphylococcus, further preferably staphylococcus aureus.
In fourth aspect, the present invention provides a kind of pycnoporus samguineus new strains CCTCC NO:M 2019514 or its extract
Application, be used to prepare the drug of related disease caused by antitumor or antibacterium.
Preferably, the extract is ethyl acetate extract.
Preferably, the tumour is tumour caused by HepG2 cell.
Preferably, the bacterium is staphylococcus, further preferably staphylococcus aureus.
At the 5th aspect, the present invention provides the drug of related disease caused by the antitumor or antibacterium of one kind, including blood
Red samguineus new strains CCTCC NO:M 2019514 or its extract and carrier.
Preferably, the extract is ethyl acetate extract.
Preferably, the tumour is tumour caused by HepG2 cell.
Preferably, the bacterium is staphylococcus, further preferably staphylococcus aureus.
Below in conjunction with specific embodiment, invention is further explained.
Embodiment 1:
The identification of pycnoporus samguineus Pycnoporus sanguineus (L.) Murrill
Liu Yuanchao, Huang Zhiyong collect a pycnoporus samguineus (Preliminary Identification) sample in Wuyi Mountain, fujian, such as Fig. 1 institute
Show.Its PDA pure culture is obtained through tissue isolation, mycelia is collected by Liquid Culture, (40 DEG C) of low temperature drying use liquid nitrogen
Grinding carries out the extraction of DNA genome, obtained DNA solution -20 using Ezup pillar fungal genomic DNA extraction agent box
DEG C refrigeration is spare.
By fungi ribosomes intergenic region universal primer ITS1/ITS4 (ITS1:TCCGTAGGTGAACCTGCGG,
ITS4:TCCTCCGCTTATTGATATGC is synthesized by Sangon Biotech (Shanghai) Co., Ltd.) carry out material ITS-
PCR experiment, amplification carry out in Biometra PCR instrument, and PCR reaction solution forms (totally 50 μ l) are as follows:
Reaction condition are as follows: 94 DEG C of reaction 5min；94 DEG C of reaction 1min, 55 DEG C of reaction 1min, 72 DEG C of reaction 1min, 30 are followed
Ring；The direct inspection of 72 DEG C of reaction 10min.PCR products carries out bidirectional sequencing, is completed by Hua Da gene.
It is as shown in Figure 4 that obtained ITS sequence is sequenced.
The sequencing result of Fig. 4 is subjected to sequence B last, discovery and pycnoporus samguineus Pycnoporus in GenBank
Sanguineus (L.) Murrill similitude is up to nearly 100%, combining form identification, the fungus specimen gross feature and aobvious
Micro- feature and Pycnoporus sanguineus description are consistent, and qualification result is Pycnoporus sanguineus.The strain
E140415 is preserved in China typical culture collection center (address: Wuhan, China), deposit number CCTCC in July, 2019
NO:M2019514.
Embodiment 2
One, culture medium (by weight percentage):
1, isolation medium (comprehensive PDA) is organized:
Potato 20%, glucose 2%, agar 2%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.15%, vitamin B1 are micro-
Amount, remaining is water.
2, mother culture media (rose bengal medium) is purified:
Peptone 0.5%, glucose 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate (MgSO47H2O) 0.05%, agar
2%, 1/3000 rose-bengal solution 10%, chloramphenicol 0.01%, remaining is water.
3, mother culture media (adding rich comprehensive PDA) is produced:
Potato 20%, glucose 2%, peptone 1%, agar 2%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.15%,
Vitamin B1 is micro, remaining is water.
4, production kind culture medium:
98-99% sorghum, 1-2% calcium carbonate.
5, culture material:
39% weed tree sawdust, 20% cotton seed hulls, 20% corncob, 17% wheat bran, 3% corn flour, 1% lime, material-water ratio: 1:
1.1-1.3。
Two, method:
1, tissue separation strain:
Tissue isolation medium is made, test tube is dispensed, in 0.11MPa atmospheric pressure, 121 DEG C of high temperature and pressure moist heat sterilizations
30min takes out cooling and is put into inclined-plane.The wild naematoloma fasciculare fructification of acquisition back aseptically uses 75% wipes of alcohol
It after wiping surface, tears, the inside meat bacteria organization of 0.2-0.5mm × 0.2-0.5mm is accessed in a manner of sterile working.It is placed in 25 DEG C of trainings
Support case in constant temperature dark culture, after mycelia covers with inclined-plane after can then transfer, the time covered with probably -15 days 10 days it
Between.
2, production purifying parent species:
Pure medium is made by formula, test tube is dispensed, in 0.11MPa atmospheric pressure, 121 DEG C of high temperature and pressure moist heat sterilizations
30min, the strain for separating postoperative infection bacterium are transferred.It is placed in constant temperature dark culture in 25 DEG C of incubators, is grown to mycelia
And bacterium carries out picking and the switching of Tip Splitting when not yet growing, and obtains purifying parent species.
3, production production parent species:
Production production mother culture media, dispenses test tube, in 0.11MPa atmospheric pressure, 121 DEG C of high temperature and pressure moist heat sterilizations
30min takes out cooling sterile working access separation successfully purifying parent species.It is placed in constant temperature dark culture in 25 DEG C of incubators, to bacterium
Filament length obtains production parent species after expiring behind inclined-plane can then transfer.The time that production parent species cover with is probably between -20 days 15 days.
4, production production kind
The sorghum for weighing required ratio, it is wet overnight through bubble, it is mixed into calcium carbonate in proportion, is fitted into 250ml conical flask, rolls over
It closes per bottled siccative 100-150g, obtains production kind culture medium, sealed with silica gel plug, in 0.147MPa atmospheric pressure, 128 DEG C of high temperature
Culture medium is shaken loose rear sterile working access production parent species after taking-up is cooling by high pressure moist heat sterilization 90min.Ensure to produce when inoculation
In parent species material block embedment production kind material.It is placed in constant temperature dark culture in 25 DEG C of incubators, (20 days or so) then after mycelia eats full material
It can be used as production kind to use in access cultivating bag.
5, cultivation culture
The compost for claiming domestication's culture medium to take required ratio is sufficiently mixed and adds water (water content 55-65%), dress
Enter 17cm × 35cm transparent polypropylene strain bag resistant to high temperature.Equivalent every packed siccative 400-420g.Small wood is used after installing material
It burrows in Bag Material, hole is deep to a bag bottom, then covers upper plastic hoop in sack, buckles the cultivation that matched lid makes to get one
Train material bag.In 0.147MPa atmospheric pressure, 128 DEG C of high temperature and pressure moist heat sterilization 90min, sterile working access production after cooling is taken out
Kind.Ensure to expect when inoculation in block embedment culture material.After inoculation in 25 DEG C ± 1 DEG C, the culturing room of relative air humidity 60-70%
It is protected from light culture.(18 days or so) can then enter cultivation management after mycelia eats full material.
6, cultivation management (including after-ripening management, former base are formed, sporophore growth)
(1) after-ripening management
After culture material in the long purseful of the mycelia in cultivating bag, continue shading After-mature cultivation 15 days, into fruiting stage.
(2) former base is formed
Temperature is controlled at 23-27 DEG C, and stronger ventilation amount, keeps space carbon dioxide content 1% hereinafter, air is opposite
Humidity is adjusted to 90% or more, after 5-7 days, removes cap, the horizontally-arranged placement (between bag and bag should there are gaps) of cultivating bag,
Mycelia starts to twist together and formed cerise rice La shape former base at this time.Relative humidity is kept at this time, not directly towards spraying water in former base.
(3) the sporophore growth phase
After former base grows to 0.5cm, continue control temperature between 23 DEG C -27 DEG C, relative air humidity 85-90% it
Between, daily illumination 9 hours, intensity of illumination 300-500lx, and 350~1500ppm of the carbon dioxide concentration in air is kept, it keeps
It is with the humid air.Through 30 days or so, fructification full maturity.During this period, daily Xiang Yougu sprays water mist 1-2 times, until son is real
Body size is basically unchanged, and is illustrated that fructification has become mature, should be harvested at this time.About pass through 35 to fructification maturation from former base is grown
It.As shown in Figures 2 and 3.
Three, fruiting situation
1, fruiting phase: the kind fruiting phase is 81 days, one tide of picking.
2, yield: each every damp 9.87-13.69 grams of fruiting of mushroom bag, average bag yield is 11.99 grams, and biological transformation ratio exists
3.43% or so.
3, fructification character: fructification is fan-shaped or shape as one wishes, storied to Dan Sheng, is in cerise.
4, compared with wild state, kind fructification individual after domestication increases 2-3 times, and shape rounding produces
It measures higher.
Embodiment 3 inhibits tumour cell function
One, experimental method
1, the preparation of ethyl acetate extract
Red fungus bacterial strain carries out solid fermentation, and culture medium is rice and water.Culture will cover with mycelial big after 45 days
Rice culture medium ethyl acetate soaking time ultrasonic extraction after 10 hours, ultrasonic 100min extracts secondary, merges Secondary Organic
Phase.After the organic phase gathered is filtered, obtained liquid will be filtered and rotated with Rotary Evaporators to dripless state, it is low
Warm preservation (4 DEG C) is spare.While other wild strains that ferment equally are operated as control.Using taxol as positive control, concentration
For 0.5mg/ml.
2, tumor cell culture
Tumour cell HepG2 (people is carried out with the DMEM culture solution containing penicillin, streptomysin and 10% fetal calf serum (FBS)
Liver cancer) culture.By the DMEM cell suspension inoculation containing 10%FBS in tissue culturing plates with 96 hole, cell concentration is 2 ×
104cells/ml.Culture is placed in 37 DEG C, 5%CO2Incubator for tissue culture in cultivate 4 hours it is stand-by.
3, inhibit tumour cell experiment
(1) sample preparation
Ethyl acetate extract is dissolved with DMSO, is configured to the mother liquor that concentration is 20mg/ml, with containing 10% fetal calf serum
(FBS) mother liquor is diluted to the sample solution of 100ug/ml by DMEM culture solution.
(2) it is loaded
The cell culture fluid in 96 orifice plates is carefully sucked, the sample solution of final concentration of 100ug/ml is added.Every sample three
A multiple holes.It is arranged zeroing hole (culture medium) simultaneously, control wells (cell, culture solution).
(3) it cultivates
It is placed in 37 DEG C, 5%CO2It is cultivated 44 hours in incubator for tissue culture.
(4) inhibiting cancer cell ability is observed
It is observed using mtt assay.After culture 44 hours, 20ulMTT is added in every hole, continues to cultivate 4h.After shaken well,
It is read at 490nm with microplate reader, calculates its inhibiting rate.Sample is repeated twice experiment.
(5) IC50 is calculated
It repeats (1)-(4), it is with the DMEM culture solution containing 10% fetal calf serum (FBS) that mother liquor is dilute when (1) concentration is prepared
It is interpreted as 15.625,31.25,62.5,125,250ug/ml gradient sample solution.Its semilethal rate is calculated after testing.
4, data are analyzed
It is counted using the dedicated statistical software of SPSS.21, using paired t-test, P < 0.05 has significant difference.
Two, experimental result
The dedicated statistical software of SPSS.21 is counted, and the results are shown in Table 1.
Table 1:
Table 1 the results show that under 100 μ g/ml concentration, red fungus is 96.70% to the inhibiting rate of HepG2, for
The IC50 value of HepG2 is 0.34ug, shows it with strong extracorporeal suppression tumor cell effect.The control experiment carried out simultaneously
In, effect of the extract of other most of wild mushrooms without inhibiting tumour cell.In other parallel laboratory tests, it is similarly blood
The ethyl acetate extract of the rice fermented product of the strains A 140247 of red samguineus Pycnoporus sanguineus is in 100 μ
It is 46.9% to the inhibiting rate of HepG2 under g/ml concentration, the external inhibition tumour that this has also proved the new strains in the present invention is thin
Born of the same parents' HepG2 effect is more significant.
4 fermentation liquid Antibacterial Activity of embodiment
One, experimental method
1, the preparation of culture medium
Bacterium culture medium: nutrient agar/broth bouillon；
Nutrient agar/broth bouillon: beef extract 3g, peptone 10g, NaCL 5g, agar 15g add water to be settled to
1000ml tune PH is 7.4 (agar is not added in nutrient broth).
2, the preparation of sample
The preparation of reference substance: taking ammonia benzyl mycin frozen stock solution, is diluted to the reference substance solution that concentration is 5 μ g/mL with sterile water,
Ultraviolet-sterilization 30min, for use.
The preparation of test sample: red fungus bacterial strain carries out solid fermentation, and culture medium is rice and water.It will be grown after culture 45d
Full mycelial rice medium ethyl acetate soaking time ultrasonic extraction, ultrasonic 100min after 10 hours, extract it is secondary,
Merge Secondary Organic phase.After the organic phase gathered is filtered, will filter obtained liquid with Rotary Evaporators rotate to
Dripless state, low-temperature preservation (4 DEG C) are spare.10mg/mL is configured to when experiment, the ethyl acetate solution containing 1%DMSO uses
Asepsis injector and membrane filtration degerming, for use.
3, the preparation of bacteria suspension
(1) staphylococcus aureus frozen stock solution is taken out from -80 DEG C of refrigerators, plate streaking inoculation obtains single colonie；
Note: smooth, round and smooth oese is selected, contains bacterium sample on a small quantity by aseptic manipulation picking.As shown in Figure 5.1) first
It draws the area A: plate is placed in by alcolhol burner flame, catch culture ware lid with left index finger and thumb, other three fingers hold culture
Culture dish is opened towards flame in ware bottom, and the right hand holds bacteria-containing oese, first leggiero draws 3~4 continuously in parallel in the area A
Bacterium source of the line as preliminarily diluted, remaining bacterium sample on burning-off oese；2) remaining area is drawn: by the oese after calcination in plate
Culture medium edge is cooling once, and the area B is made to go to scribing position, oese is moved to the area B by the area A (bacterium source area), immediately
Upper 6~7 fine and close lines are leggiero drawn in the area B, then again similarly to operate more parallel lines in the area C and D zoning,
And keep the lines in the area D parallel with the area A (but cannot be with the line contact in the area A or the area B)；3) constant temperature incubation: by scribed plate
It is cultivated 2~3 days to 37 DEG C；4) a small amount of thallus of picking is preliminary point to test tube slant after cultivation from typical single colonie
From it is purebred.
(2) it is seeded in nutrient broth medium with aseptic inoculation ring picking single colonie, 37 DEG C, 220r/min shaken cultivation
For 24 hours, the bacteria suspension for trying bacterium is obtained.
(3) it draws and obtains 10 with sterile distilled water gradient dilution for the bacteria suspension for trying bacterium-1~10-8The bacterium of times original content
Liquid；
(4) the 200 μ L of bacterium solution for drawing each concentration respectively is uniformly coated on nutrient agar panel, and 37 DEG C are cultivated for 24 hours, Mei Genong
Degree is repeated 3 times；
(5) by colony counting method, the concentration of bacterium solution is determined, for use.
4, the measurement of inhibition zone
Using pre-add bacterium solution pour plate method, a certain amount of bacterium is injected into the plating medium for having cooled to 50 DEG C or so
Liquid makes bacteria concentration 10 in culture medium5~106Cfu/mL shakes up, and pour plate (about 30mL/ plate), horizontal rest is to be solidified
It is stand-by afterwards, it is uniformly punched on plate containing bacterium with sterilized punch, carefully chooses culture medium in hole, aseptically inhale
200 μ L of reference substance/test sample is taken to squeeze into hole, stationary culture 1 day under the conditions of being placed in 37 DEG C measures inhibition zone size.It is positive
Control is the ammonia benzyl mycin of 10 μ g/mL.
Two, experimental result:
The results are shown in Table 2.
Antibacterial circle diameter (x ± s, n=3) of 2 E140415 of table to staphylococcus aureus
| Sample | Antibacterial circle diameter (mm) |
| Positive control | 19.79±1.7 |
| Pycnoporus samguineus E140415 | 19.39±0.86 |
As a result as shown in fig. 6, in Fig. 6, the upper left corner is blank, and the upper right corner is positive control, and the lower left corner is blood red close hole
Bacterium E140415, the lower right corner are certain W140054 of another check variety Pholiota.
In more than the 600 wild edible and medical fungi kind ethyl acetate extracts equally detected using 10mg/mL concentration
In, it is one of effect 10 samples the most apparent that apparent inhibition zone, which is presented, in strain of the present invention, also, bacterial strain of the present invention
Sample has strong bacteriostatic activity to staphylococcus aureus.
In conclusion the red fungus bacterial strain in the present invention, which is one, carries out the new varieties not yet to conduct a research, pass through
Pure culture is isolated in field acquisition, carries out artificial cultivation, and from the point of view of functional experiment, ethyl acetate extract has external inhibit
Tumor effect is obvious, inhibit staphylococcus aureus significant effect, after cultivation economical character preferably etc. characteristics, be one have open
The strain of hair prospect.
The above, preferable specific embodiment only of the invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Design is subject to equivalent substitution or change, should be covered by the scope of protection of the present invention.
Claims (10)
1. a kind of pycnoporus samguineus new strains, which is characterized in that the pycnoporus samguineus new strains are pycnoporus samguineus
(Pycnoporus sanguineus) HMGIM-140415, deposit number are CCTCC NO:M 2019514.
2. a kind of artificial cultivation method of pycnoporus samguineus new strains CCTCC NO:M 2019514, which is characterized in that including system
Make parent species, production production parent species, production production kind, cultivation culture and cultivation management, by weight percentage, the culture material packet
Include weed tree sawdust 37-41%, cotton seed hulls 18-20%, corncob 18-20%, wheat bran 16-18%, corn flour 2-3%, lime 1-
2%.
3. artificial cultivation method according to claim 2, which is characterized in that by weight percentage, the culture material packet
Include 39% weed tree sawdust, 20% cotton seed hulls, 20% corncob, 17% wheat bran, 3% corn flour, 1% lime, material-water ratio: 1:1.1-
1.3。
4. artificial cultivation method according to claim 2, which is characterized in that the cultivation culture includes: to turn production kind
It is connected in culture material, 25-26 DEG C of constant temperature, shading culture, humidity 60%-70%, mycelia covers with bacterium bag and enters cultivation management.
5. artificial cultivation method according to claim 4, which is characterized in that the cultivation management includes: in cultivating bag
The long purseful of mycelia in after culture material, continue shading After-mature cultivation 15 days, into fruiting stage, control temperature at 23-27 DEG C, and
Stronger ventilation amount keeps space carbon dioxide content 1% hereinafter, relative air humidity is adjusted to 90% or more, through 5-7 days
Afterwards, cap is removed, mycelia starts to twist together and formed cerise rice La shape former base；After former base grows to 0.5cm, continue to control temperature
Between 23 DEG C -27 DEG C, between relative air humidity 85-90%, daily illumination 9 hours, intensity of illumination 300-500lx, and protect
350~1500ppm of the carbon dioxide concentration in air is held, keeps with the humid air, through 30 days or so, during this period, daily Xiang Yougu
Spray water mist 1-2 times, until fructification size is basically unchanged, fructification is mature, harvesting.
6. according to the artificial cultivation method any in claim 2 to 5, which is characterized in that the production parent species include: by
Isolated strain transfer is placed in 25 DEG C of constant temperature dark cultures, carries out when mycelia grows and bacterium not yet grows to mother culture media
The picking of Tip Splitting, obtains parent species；And/or
The production production parent species include: that parent species are forwarded to production mother culture media, 25 DEG C of constant temperature dark cultures are placed in, to mycelia
It covers with inclined-plane and obtains production parent species；And/or
Production production kind includes: the sterile access production parent species into production kind culture medium, and when inoculation ensures production parent species material
Block is embedded in original seed material, is placed in 25 DEG C of constant temperature dark cultures, and production kind is obtained after mycelia eats full material.
7. artificial cultivation method according to claim 6, which is characterized in that the mother culture media is rose-bengal culture
Base, by weight percentage, the rose bengal medium include: peptone 0.5%, glucose 1%, potassium dihydrogen phosphate
0.1%, magnesium sulfate (MgSO47H2O) 0.05%, agar 2%, 1/3000 rose-bengal solution 10%, chloramphenicol 0.01%,
Remaining is water；And/or
The production mother culture media is to add rich comprehensive PDA, and by weight percentage, described plus rich comprehensive PDA includes: potato
20%, glucose 2%, peptone 1%, agar 2%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.15%, vitamin B1 are micro,
Yu Weishui；
By weight percentage, the production kind culture medium includes: 98-99% sorghum and 1-2% calcium carbonate.
8. the application of a kind of pycnoporus samguineus new strains CCTCC NO:M 2019514 or its extract, which is characterized in that be used for
Prepare the drug of related disease caused by antitumor or antibacterium；Preferably, the extract is ethyl acetate extract.
9. application according to claim 8, which is characterized in that the tumour is tumour caused by HepG2 cell；And/or
The bacterium is staphylococcus, preferably staphylococcus aureus.
10. a kind of drug of related disease caused by antitumor or antibacterium, which is characterized in that including the new bacterium of pycnoporus samguineus
Strain CCTCC NO:M 2019514 or its extract and carrier.
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