CN107242026A - A kind of cultural method and its culture medium of white Antrodia camphorata fructification - Google Patents
A kind of cultural method and its culture medium of white Antrodia camphorata fructification Download PDFInfo
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- CN107242026A CN107242026A CN201710286295.7A CN201710286295A CN107242026A CN 107242026 A CN107242026 A CN 107242026A CN 201710286295 A CN201710286295 A CN 201710286295A CN 107242026 A CN107242026 A CN 107242026A
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- 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
- A01G7/00—Botany in general
- A01G7/04—Electric or magnetic or acoustic treatment of plants for promoting growth
- A01G7/045—Electric or magnetic or acoustic treatment of plants for promoting growth with electric lighting
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- 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
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05C—NITROGENOUS FERTILISERS
- C05C11/00—Other nitrogenous fertilisers
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F5/00—Fertilisers from distillery wastes, molasses, vinasses, sugar plant or similar wastes or residues, e.g. from waste originating from industrial processing of raw material of agricultural origin or derived products thereof
- C05F5/006—Waste from chemical processing of material, e.g. diestillation, roasting, cooking
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/14—Measures for saving energy, e.g. in green houses
Abstract
The present invention provides a kind of cultural method of white Antrodia camphorata fructification, comprises the following steps：(1) white Antrodia camphorata fructification is subjected to section and obtains fructification section；(2) the fructification section obtained step (1) carries out purifying agaric using bacterium culture medium and obtains white Antrodia camphorata strain；(3) culture medium is prepared；The white Antrodia camphorata strain is connect into bacterium to be cultivated to the culture medium；And white Antrodia camphorata strain of (4) docking bacterium on culture medium carries out growing environment regulation culture.The application cultivates white Antrodia camphorata using artificial cultivation method, and white Antrodia camphorata fructification is cultivated using culture dish, and carries out growing environment regulation culture, for shortening incubation time, and lifts culture success ratio and yield.
Description
Technical field
The invention belongs to edible and medical fungi technical field of cultivation, be related to a kind of white Antrodia camphorata fructification cultural method and its
Culture medium.
Background technology
Antrodia camphorata (Antrodiacinnamomea) belongs to non-folding Zoopagales, bracket fungus material, antrodia karst, antrodia kind, is many
Year raw gill fungus mushroom.Antrodia camphorata is the distinctive strain in Taiwan, in the hollow treehole for being only grow on Taiwan endemic species cinnamomum kanehirai, and growth
Slowly.
Antrodia camphorata contains abundant active material, mainly there is physiologically active composition known to Antrodia camphorata fructification：Many vinegar bodies
(polysaccharides), triterpenes (triterpenoids), Sudismase (suproxide dismutase, SOD),
Adenosine (adenosines), ergosterol (ergosterols) etc.；Wherein, triterpene compound has multiple biological activities, such as
Liver protecting, regulation immune, angiogenesis inhibiting, antitumor, anti-inflammatory, anti-oxidant, hypotensive etc..Lot of documents shows, ox
Antrodia has multiple biological activities, and Antrodia camphorata fructification is in treatment hepatopathy (hepatic disease), nameless sores or boils, heating
(fever), diabetes, hypertension, relieve the effect of alcohol and all displays such as anticancer have high development potentiality.
The problem of being difficult, and have by environmental pollution because wild Antrodia camphorata rare numbers are obtained, thus it is many in recent years
Antrodia camphorata is cultivated with artificial cultivation method, there are liquid cultivation, solid state rheology in main production Antrodia camphorata breeding method system at present
Method and linden cultivation.
Antrodia camphorata sporophore shape is rich and changeful, has tabular, mitriform, horse-hof shape or tower-like, nascent is cerise, gradually long
Be changed into reddish tan, it is filbert or khaki.Wild white Antrodia camphorata is very rare, and is cultivated with artificial cultivation method white
Color Antrodia camphorata is more rare, therefore white Antrodia camphorata commercially more rare and preciousness.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of cultural method of white Antrodia camphorata fructification, its be from
Wild Antrodia camphorata is smoothly detached out white Antrodia camphorata strain and is seeded to the culture dish containing culture medium, further by culture medium
Composition and its content make appropriate adjustment, and the stimulation of culture environment to cultivate the cultural method of white Antrodia camphorata fructification.
Another technical problem to be solved by this invention is to provide a kind of culture medium for being used to cultivate white Antrodia camphorata fructification.
In order to solve the above technical problems, present invention employs following technical scheme realization：
A kind of cultural method of white Antrodia camphorata fructification of the present invention, comprises the following steps：
(1) white Antrodia camphorata fructification is subjected to section and obtains fructification section；
(2) the fructification section obtained step (1) carries out purifying agaric using bacterium culture medium and obtains white Antrodia camphorata
Strain；
(3) culture medium is prepared；The white Antrodia camphorata strain is connect into bacterium to be cultivated to the culture medium；And
(4) white Antrodia camphorata strain of the docking bacterium on culture medium carries out growing environment regulation culture.
Further, wherein the purifying agaric method in the step (2) includes：
Fructification section is inoculated in the culture dish containing bacterium culture medium, after the transplanting of 4~6 times, nothing is confirmed
Miscellaneous bacteria occurs that white Antrodia camphorata strain can be obtained；The bacterium culture medium at least includes：Mass concentration is 1~3%w/w Ma Ling
Potato grape sugar extract (potato dextrose broth, PDB) and the agar that mass concentration is 0.5~1.5%w/w
(agar)。
Wherein, potato glucose extract (potato dextrose broth, PDB), can use potato glucose
Meat soup.
Further, culture medium is prepared in the step (3) includes：
Each constituent of mixed culture medium to scale, the constituent of the culture medium includes：Mass concentration be 2~
4%w/w potato glucose extract, mass concentration is 2~4%w/w Semen Tritici aestivi extract (Malt extract), quality
Concentration be 0.1~0.3%w/w nitrogen source, mass concentration be 0.5~0.6%w/w agar (agar), and mass concentration be 5~
7%w/w sucrose (sucrose)；Wherein, the nitrogen source can be used：Amino acid, peptone, yeast extract or beef extraction
Thing etc., preferably use Amino acid (amino acids)；After each constituent is mixed, it is put into one and is sterilized to multiple
In container and add reverse osmosis water stir make its dissolving (reverse osmosis water addition so that each constituent reach needed for it is dense
Degree is defined)；Culture medium stoste after dissolving is put into Sterilization Kettle；Impose a condition as 121 DEG C, carry out at sterilizing within 15~30 minutes
Reason, culture dish is poured into by the culture medium solution after sterilizing, is stood and be can obtain solid medium after cooling.
Further, white Antrodia camphorata fructification is cut into slices in the culture dish containing bacterium culture medium in the step (2)
Middle culture, cultivation temperature is controlled at 22~30 DEG C.
Further, growth ring is carried out to the white Antrodia camphorata strain for connecing bacterium on culture medium in the step (4)
Border regulation culture includes：Low temperature stimulation culture, knife wound stimulate culture and light to stimulate at least one of culture.
It is preferred that, low temperature stimulation is carried out to the white Antrodia camphorata strain for connecing bacterium on culture medium in the step (4)
Culture, wherein 4 DEG C to 15 DEG C of low temperature stimulation temperature range.The low temperature stimulation culture be by its cultivation temperature range set be 4
~15 DEG C of temperature to winter of simulating Antrodia camphorata under natural environment, to carry out low temperature stimulation to Antrodia camphorata strain, promote
Secondly secondary metabolism thing is produced.
It is preferred that, knife wound stimulation is carried out to the white Antrodia camphorata strain for connecing bacterium on culture medium in the step (4)
Culture, including：Multiple 1~2 centimeter of scar is manufactured in white Antrodia camphorata using blade or syringe needle, for secondary metabolites synthesis
And the increase of weight.
It is preferred that, light stimulation is carried out to the white Antrodia camphorata strain for connecing bacterium on culture medium in the step (4)
Culture, including light stimulation culture is carried out under photoenvironment to the white Antrodia camphorata strain for connecing bacterium on culture medium；Its
In, the photoenvironment selects feux rouges or blue light, and the illumination intensity of the photoenvironment is 5 μm of ol/S.m2To 20 μm of ol/S.m2。
Stimulated by light, to stimulate secondary metabolites hyperplasia, improve the content of the active ingredient of Antrodia camphorata.
It is preferred that, when the photoenvironment is feux rouges, the illumination intensity of the photoenvironment is 12 μm of ol/S.m2To 20 μ
mol/S.m2；Or
When photoenvironment is blue light, the illumination intensity of the photoenvironment is 8 μm of ol/S.m2To 16 μm of ol/S.m2。
Another object of the present invention is to there is provided a kind of culture medium for being used to cultivate white Antrodia camphorata fructification, the training
Foster base at least includes：
Potato glucose extract, its mass concentration is 2~4%w/w；
Semen Tritici aestivi extract (Malt extract), its mass concentration is 2~4%w/w；
Nitrogen source, its mass concentration is 0.1~0.3%w/w, wherein, the nitrogen source can be used：Amino acid, peptone, yeast
Extract or beef extract；It is preferred that, nitrogen source can use Amino acid (amino acids)；
Agar (agar), its mass concentration is 0.5~0.6%w/w；With
Sucrose (sucrose), its mass concentration is 5~7%w/w.
The cultural method of white Antrodia camphorata fructification and its beneficial effect of culture medium of the present invention is also resided in：
The application cultivates white Antrodia camphorata fructification using culture dish, and carries out growing environment regulation culture (low temperature thorn
Swashing culture, knife wound stimulates culture and light to stimulate at least one of culture), it is trained for shortening incubation time, and being lifted
Power and yield.The application is added in culture dish using homemade culture medium and white Antrodia camphorata strain is cultivated, and
Regulation culture is carried out by growing environment, to shorten incubation time by culture medium, and stimulates to promote two by growing environment
Secondary metabolism thing is produced, lifting culture success ratio and yield, when can shorten culture by the stimulation of basal culture medium combination growing environment
Between while, greatly increase the content of the active ingredient of Antrodia camphorata.The application cultivates white Cinnamomum kanahirai hay using artificial cultivation method
Sesame, and it is more polynary to cultivate the Antrodia camphorata of obtained Antrodia camphorata composition species contrast prior art cultivation.
Brief description of the drawings
By reading the detailed description of hereafter preferred embodiment, various other advantages and benefit is common for this area
Technical staff will be clear understanding.Accompanying drawing is only used for showing the purpose of preferred embodiment, and is not considered as to the present invention
Limitation.In the accompanying drawings：
Fig. 1 is white Antrodia camphorata fructification culture flow chart；
Fig. 2 is to carry out white Antrodia camphorata cultures with three groups of different culture medias, determine the white Antrodia camphorata average weight of each group with
And 95% ethanol extraction yield result schematic diagram；
Fig. 3 is to carry out the white Antrodia camphorata of low temperature stimulation with two kinds of different temperatures to grow, and determines the white Antrodia camphorata of each group and is averaged
The result schematic diagram of weight, 95% ethanol extraction yield；
Fig. 4 is to stimulate whiter Antrodia camphorata to grow with different illumination intensity, the white Antrodia camphorata average weight of measure each group,
The result schematic diagram of 95% ethanol extraction yield；
Fig. 5 is the presence or absence of to stimulate relatively whiter Antrodia camphorata to grow with knife wound, determine the white Antrodia camphorata average weight of each group,
The result schematic diagram of 95% ethanol extraction yield；
Fig. 6 is the HPLC chromatography collection of illustrative plates of white Antrodia camphorata alcoholic extract；
Fig. 7 chromatographs collection of illustrative plates for the HPLC of commercially available linden culture Antrodia camphorata (red) alcoholic extract.
Embodiment
The exemplary embodiment of the disclosure is more fully described below with reference to accompanying drawings.Although showing the disclosure in accompanying drawing
Exemplary embodiment, it being understood, however, that may be realized in various forms the disclosure without should be by embodiments set forth here
Limited.On the contrary, these embodiments are provided to facilitate a more thoroughly understanding of the present invention, and can be by the scope of the present disclosure
Complete conveys to those skilled in the art.
Further to illustrate the present invention to reach the technological means and effect that predetermined goal of the invention is taken, below in conjunction with
Accompanying drawing and preferred embodiment, to the cultural method and its culture medium according to white Antrodia camphorata fructification proposed by the present invention specifically
It is bright as follows：
The allotment and screening of culture medium are carried out first：The constituent of the culture medium of the present invention includes：Mass concentration be 2~
4%w/w potato glucose extract, mass concentration is 2~4%w/w Semen Tritici aestivi extract (Malt extract), quality
(nitrogen source can be used the nitrogen source that concentration is 0.1~0.3%w/w：Amino acid, peptone, yeast extract or beef extract；It is excellent
Choosing, nitrogen source can use Amino acid (amino acids), and mass concentration is 0.5~0.6%w/w agar (agar), and quality
Concentration is 5~7%w/w sucrose (sucrose)；Its concocting method is：According to each constituent of aforementioned proportion mixed culture medium,
After each constituent is mixed, be put into one in sterilization container and can add reverse osmosis water so that each constituent reaches to multiple
Required concentration is defined, and stirring dissolves it；Culture medium stoste after dissolving is put into Sterilization Kettle；Impose a condition as 121
DEG C, carry out sterilization treatment within 15~30 minutes, the culture medium solution after sterilizing is poured into culture dish, standing can be consolidated after cooling
State culture medium.Different proportion is used to allocate culture medium and cultivated with screening and culturing medium below.
Embodiment 1
Step 1：White Antrodia camphorata fructification is subjected to section and obtains fructification section；Specifically blade can first be used
After 75% alcohol disinfecting, the white Antrodia camphorata mushroom body on wood is cut, mushroom body is wrapped using clean gauze or polybag.
Before strain isolation, using 75% alcohol wipe and after irradiating UV light 20 minutes, you can be put into white Antrodia camphorata mushroom body sterile
Grasp seat platform.During strain isolation, using the cotton or gauze for speckling with 75% alcohol by mushroom body surface wipes one time；Removed with scalpel
Mushroom body surface skin, and it is divided into the hymenium of 0.5 centimeter of size to cut into slices the inside pulp.
Step 2：The section of obtained fructification is subjected to purifying agaric using bacterium culture medium and obtains white Antrodia camphorata strain；
It can specifically include：Obtained fructification is cut into slices and is inoculated in the potato glucose containing mass concentration is 1~3%w/w
Extract (potato dextrose broth, PDB) and mass concentration for 0.5~1.5%w/w agar (agar) bacterium
In the culture dish for planting culture medium.Each culture dish puts one piece of fructification section, and closes the lid.Above-mentioned culture dish is placed in 20
Cultivated at~25 DEG C, when mycelia length is to about 1~3 centimeter, the mycelia of periphery is cut and transplanted and is put in new culture dish, by 4
Confirm that no miscellaneous bacteria occurs that white Antrodia camphorata strain can be obtained after the transplanting of~6 times.
Step 3：Prepare culture medium (formula one)；The white Antrodia camphorata strain is connect into bacterium to be trained to the culture medium
Support；Culture medium for cultivating white Antrodia camphorata fructification includes following component：Potato glucose extract PDB (Potato
Dextrose Broth), its mass concentration is 4%w/w；Sucrose, its mass concentration is 5%w/w；Amino acid (amino
Acids), its mass concentration is 0.3%w/w；Agar (agar), its mass concentration is 0.5%w/w.Nitrogen source is not limited to Amino acid.
After above-mentioned constituent is mixed in proportion, be put into one in sterilization container and can add 1000ml reverse osmosis water with even to several
Dissolving.It is subsequently placed into Sterilization Kettle；Impose a condition and sterilized for 121 DEG C, 15~30 minutes.Culture medium solution after sterilizing is fallen
Enter culture dish, every culture dish about 12~27ml.Stand and can obtain solid medium after cooling, by the white ox separated
The implantation of antrodia strain connects bacterium on the culture medium, in 22~30 DEG C of environment culture, and cultivated days are 180 days.
Embodiment 2
Step 1 and step 2 be the same as Example 1；
Step 3：Prepare culture medium (formula two)；The white Antrodia camphorata strain is connect into bacterium to be trained to the culture medium
Support；Culture medium for cultivating white Antrodia camphorata fructification includes following component：PDB (Potato Dextrose Broth), its
Mass concentration is 2%w/w；Wheat extract (Malt extract), its mass concentration is 2%w/w；Sucrose, its mass concentration
For 7%w/w；Amino acid (amino acids), its mass concentration is 0.2%w/w；Agar (agar), its mass concentration is
0.6%w/w.After above-mentioned constituent is mixed in proportion, be put into one to it is several can be in sterilization container and to add 1000ml inverse
Water is permeated with even dissolving.It is subsequently placed into Sterilization Kettle；Impose a condition and sterilized for 121 DEG C, 15~30 minutes.By the training after sterilizing
Foster based sols pour into culture dish, every culture dish about 15~25ml.Stand and can obtain solid medium after cooling, will isolate
The white Antrodia camphorata strain implantation come connects bacterium on the culture medium, in 22~30 DEG C of environment culture, and cultivated days are 180 days.
Embodiment 3
Step 1 and step 2 be the same as Example 1；
Step 3：Prepare culture medium (formula three)；The white Antrodia camphorata strain is connect into bacterium to be trained to the culture medium
Support；Culture medium for cultivating white Antrodia camphorata fructification includes following component：Wheat extract (Malt extract), its matter
Amount concentration is 4%w/w；Sucrose, its mass concentration is 6%w/w；Amino acid (amino acids), its mass concentration is 0.1%
w/w；Agar agar, its mass concentration is 0.5%w/w.After above-mentioned constituent is mixed in proportion, be put into one to it is several can
In sterilization container and 1000ml reverse osmosis water is added with even dissolving.It is subsequently placed into Sterilization Kettle；Impose a condition for 121 DEG C, 15~30
Minute is sterilized.Culture medium solution after sterilizing is poured into culture dish, every culture dish about 15~25ml；Stand after cooling i.e.
It can obtain solid medium.The white Antrodia camphorata strain separated implantation is being connect into bacterium on the culture medium, at 22~30 DEG C
Environment culture, cultivated days be 180 days.
The fructification that the medium culture of embodiment 1,2 and 3 obtained after 180 days will be respectively adopted and uses weight, 95%
The detection method of ethanol extraction yield is detected.The detection method is：After the original piece harvesting of Antrodia camphorata fructification, remove culture medium
It is put into oven for drying；Impose a condition as 60 DEG C, 200 minutes；Weighed after drying with Libra.Former piece after drying takes 5g to play powder addition
Extracted about 1~2 hour, then drained with the machine concentration that is concentrated under reduced pressure with ultrasonic vibrating after 95% 40 times of ethanol volume；Scale
Re-computation extract weight and the ratio of former piece weight.
Detect that the weight for the fructification that embodiment 1,2 and 3 is obtained and 95% ethanol are extracted respectively by above-mentioned detection method
Rate；As a result it is as shown in Figure 2.
In the white Antrodia camphorata fructification that three kinds of different formulations are grown as shown in Figure 2, the PDB formulas in embodiment 1
(formula one), average dry weight is 0.9g, and 95% ethanol extraction yield is 28%.Formula PDB in embodiment 2 takes out plus wheat
Go out thing (formula two)；Average dry weight is 1.18g, and 95% ethanol extraction yield is 30%.Formula wheat in embodiment 3 is extracted out
Thing (formula three)；Average dry weight is 0.85g, and 95% ethanol extraction yield is 25%.Show that the Media Components of embodiment 2 are excellent
In embodiment 1 and embodiment 3.
Embodiment 4：The low temperature stimulation culture of white Antrodia camphorata growing environment regulation and control
The carry out low temperature stimulation culture of quality the best embodiment 2 is selected from 3 kinds of culture mediums in embodiment 1~3.
Fig. 1 is white Antrodia camphorata fructification culture flow chart, as shown in Figure 1：
Step in step 1, step 2 and step 3 be the same as Example 2；
Step 4：By implanted white Antrodia camphorata strain in totally three groups of the culture dish of the culture medium (formula two), put to 22
After being cultivated 90 days in~30 DEG C of environment, first group of control group；Lasting culture harvests drying, in addition two groups to after cultivating 180 days
Culture dish is respectively put into cultivated 3~5 days in 4 DEG C and 15 DEG C of environment after, culture dish is put back to again and puts 22~30 DEG C of environment extremely
Culture harvests drying after 180 days.Obtained fructification will be cultivated under three groups of different conditions, extracted using weight, 95% ethanol
The detection method of rate is detected, its weight and 95% ethanol extraction yield are measured respectively.The detection method is：Antrodia camphorata is real
After body original piece harvesting, remove culture medium and be put into oven for drying；Impose a condition as 60 DEG C, 200 minutes；Weighed after drying with Libra.
Former piece after drying takes 5g to beat powder and add after 95% ethanol, 40 times of volumes and is extracted about 1~2 hour with ultrasonic vibrating, then
With being concentrated under reduced pressure, machine concentration is drained；Weighing calculates extract weight and the ratio of former piece weight；As a result it is as shown in Figure 3.
Fig. 3 result is different low temperature stimulation, and first group is 15 DEG C；Second group is 4 DEG C, as a result shows that first group is averaged
Dry weight is 1.18g, and 95% ethanol extraction yield is 33%；Second group of average dry weight is 1.18g, 95% ethanol extraction yield
For 36%.Show that low temperature stimulation culture weight is constant, but ethanol extraction yield increases by 1% and 2% respectively；And 4 DEG C of effect compares 15
Result obtained by DEG C is high.
Embodiment 5：White Antrodia camphorata growing environment regulation and control-knife wound stimulates culture
The carry out stimulation culture of quality the best embodiment 2 is selected from 3 kinds of culture mediums in embodiment 1~3.
Step in step 1, step 2 and step 3 be the same as Example 2；
Step 4：By implanted white Antrodia camphorata strain in the culture dish of the culture medium, put into 22~30 DEG C of environment
After culture 90 days, the culture dish that one of which (stimulation group) has been grown into white Antrodia camphorata takes out, with blade or syringe needle white
Multiple 0.5~1 centimeter of scar are manufactured on color Antrodia camphorata；Simulated hexapod bites to stimulate secondary metabolites GCMS computer, puts back to afterwards
It is further cultured for into 22~30 DEG C of environment to 180 days, another group (non-stimulated group) is cultivated in 22~30 DEG C of environment to 180
My god, two groups of white Antrodia camphorata fructifications obtained are respectively adopted the weight, the detection method of 95% ethanol extraction yield and carried out
Detection, measures its weight and 95% ethanol extraction yield respectively；As a result it is as shown in Figure 4.
Fig. 4 results are the experiment that knife wound is stimulated.First group is average dry weight and 95% ethanol without knife wound stimulation group
Extraction yield is respectively 1.2g and 30%.Second group is that knife wound is stimulated；Its average dry weight of mushroom body and 95% second that scar is stimulated
Alcohol extraction yield is respectively 1.53g and 35%, and dry weight and ethanol extraction yield increase respectively compared with first group without knife wound experiment
27% and 16%.Secondary metabolites GCMS computer and the increase of weight that the stimulation of display knife wound contributes to.
Embodiment 6：White Antrodia camphorata different illumination intensity stimulates culture
The carry out stimulation culture of quality the best embodiment 2 is selected from 3 kinds of culture mediums in embodiment 1~3.
Step in step 1, step 2 and step 3 be the same as Example 2；
Step 4：By implanted white Antrodia camphorata strain in the culture dish of the culture medium, put into 22~30 DEG C of environment
Culture is to carrying out light stimulation after the 75th day.This experiment periods is divided into five groups, respectively control group；Without a licence optical culture, first group with
Feux rouges；Illumination intensity is 20 μm of ol/S.m2, second group is feux rouges；Illumination intensity is 12 μm of ol/S.m2, the 3rd group is blue light；According to
Luminous intensity is 16 μm of ol/S.m2, and the 4th group is blue light；Illumination intensity is 8 μm of ol/S.m2.In addition to control group, each group is passed through 15 days
Stop irradiation after irradiation, put into 22~30 DEG C of environment, continue to cultivate to 180 days.Five groups of white Antrodia camphoratas obtained are sub
Entity is respectively adopted the weight, the detection method of 95% ethanol extraction yield and detected, its weight and 95% second are measured respectively
Alcohol extraction yield；As a result it is as shown in Figure 5.
Fig. 5 tests for light stimulation, and control group average weight is 1.2g；95% ethanol extraction yield is 30%.Light stimulation
First group to the 4th group, experimental result average weight is respectively 1.18g, 1.16g, 1.17g, 1.19g, with control group without too big difference
It is different.In the result of the light stimulation of 95% ethanol extraction yield, by the average 95% ethanol extraction of first group to the 4th group experimental result
Rate is respectively 36%, 32.5%, 34.8%, 31.5%.Compared with 95% ethanol extraction yield of control group respectively increase by 20%,
8.3%th, 16%, 5%.As a result its average weight of group of irradiation stimulation is shown without too big difference, and 95% ethanol extraction yield
There is increase.Wherein feux rouges illumination intensity is 20 μm of ol/S.m2Resulting ethanol extraction yield is highest.
The preparation of the Antrodia camphorata alcoholic extract of embodiment 7, and white Antrodia camphorata extract fingerprint map analyzing
The white Antrodia camphorata for taking the Antrodia camphorata fructification (red) and example 4 of 3 grams of commercially available linden culture to be cultivated respectively
Fresh goods, it is lyophilized with freeze dryer (temperature is less than -45 DEG C, and vacuum is less than 300mTorr).Dry product is crushed with pulverizer again, is fallen respectively
Enter in serum bottle, plus 10 times of volumes 95% alcohol and stirrer, with cold soaking mode stir extraction 3-5 days.Pumping filtering (No.1
Filter paper), weighed after concentration, drying, as Antrodia camphorata alcoholic extract, and being stored in proof cabinet.
By the white Antrodia camphorata extract fingerprint map analyzing of the Antrodia camphorata extract of above-mentioned preparation；Specifically include：
1st, by the white Antrodia camphorata extract of the Antrodia camphorata extract of above-mentioned preparation respectively with methanol back dissolving, concentration 5mg/mL.
2nd, centrifuge, 10000rpm, 10mins, take supernatant to carry out HPLC analyses.
3rd, efficient liquid phase chromatographic analysis instrument：
Pump (Pump):Spectra SYSTEM P1000,
Automatic sampler (Auto injector):Spectra SYSTEM AS1000,
Detecting instrument (Detector):FINNIGAN SURVEYOR PDA Plus Detector.
Liquid phase chromatographic analysis condition:
Chromatograph tubing string：Agilent, Eclipse XDB-C18,4.6mm*150mm, 5 μm
Detecting instrument：PDA(UV 254nm)；
Sample injection amount：5μL；
Chromatographic flow rates：0.8mL/min；
HPLC purges with condition (such as table 1)：
Table 1
Analysis result white Antrodia camphorata extract such as Fig. 6, sells Antrodia camphorata fructification (red) alcoholic extract of linden culture
Thing such as Fig. 7.Compare Fig. 6 and Fig. 7 HPLC chromatography collection of illustrative plates and bibliography (Taiwan endemic species Antrodia camphorata (mushroom)
(Antrodiacinnamomea) fructification standard after correct scientific name judgement key, 2013.08.Taiwan edible medicinal mushroom class life
Skill association) the feature chromatography Pu Feng that HPLC chromatographs the Antrodia camphorata fructification for the linden culture that collection of illustrative plates is indicated is announced, can be obvious
Find out that white Antrodia camphorata extract has the feature chromatography Pu Feng (such as table 2) of the Antrodia camphorata fructification of linden culture, it is white in addition
Antrodia camphorata extract also has some chromatography Pu Fengs different from the Antrodia camphorata fructification of linden culture, and (holdup time divides in such as Fig. 4
Wei 4.2,6.5,6.8,12.7 and 20.5min etc.), white Antrodia camphorata its composition species obtained by the display present invention is more more
Member.
Table 2
| Compound | RT(min) |
| Antcin A | 32.2 |
| Antcin B | 25.7 |
| Antcin C | 17.3 |
| Antcin K | 9.0 |
| Antcin H | 19.4 |
| Dehydrosulphurenic acid | 22.6 |
| Dehydroeburicoic acid | 40.1 |
The foregoing is only the preferred embodiments of the present invention, have to the present invention it is illustrative, it is and non-limiting.This area skill
Art personnel should be understood that without departing from the scope of the present invention, when the technology contents using the disclosure above make it is a little
The equivalent embodiment of equivalent variations is changed or is modified to, as long as being the content without departing from technical solution of the present invention, according to the present invention
Technical spirit any simple modification, equivalent variations and the modification made to above example, still fall within the technology of the present invention side
In the range of case.And it will be understood by those skilled in the art that it can be fitted in the scope of the claims in the present invention
The change of answering property, but all should fall under the scope of the present invention.
Claims (10)
1. a kind of cultural method of white Antrodia camphorata fructification, it is characterised in that：The cultural method comprises the following steps：
(1) white Antrodia camphorata fructification is subjected to section and obtains fructification section；
(2) the fructification section obtained step (1) carries out purifying agaric using bacterium culture medium and obtains white Antrodia camphorata strain；
(3) culture medium is prepared；The white Antrodia camphorata strain is connect into bacterium to be cultivated to the culture medium；And
(4) white Antrodia camphorata strain of the docking bacterium on culture medium carries out growing environment regulation culture.
2. the cultural method of white Antrodia camphorata fructification according to claim 1, it is characterised in that：Wherein described step
(2) the purifying agaric method in includes：
Fructification section is inoculated in the culture dish containing bacterium culture medium, after the transplanting of 4~6 times, confirmed without miscellaneous bacteria
Appearance can obtain white Antrodia camphorata strain；The bacterium culture medium at least includes：Mass concentration is 1~3%w/w potato
Grape sugar extract and the agar that mass concentration is 0.5~1.5%w/w.
3. the cultural method of white Antrodia camphorata fructification according to claim 1, it is characterised in that：In the step (3)
Preparing culture medium includes：
Each constituent of mixed culture medium to scale, the constituent of the culture medium at least includes：Mass concentration be 2~
4%w/w potato glucose extract, mass concentration be 2~4%w/w Semen Tritici aestivi extract, mass concentration be 0.1~
0.3%w/w nitrogen source, mass concentration is 0.5~0.6%w/w agar, and the sucrose that mass concentration is 5~7%w/w；Its
In, the nitrogen source can be used：Amino acid, peptone, yeast extract or beef extract；After each constituent is mixed, put
Enter one to it is multiple can be in sterilization container and adding reverse osmosis water and stirring dissolves it；Culture medium stoste after dissolving is put
Enter Sterilization Kettle；Impose a condition as 121 DEG C, carry out sterilization treatment within 15~30 minutes, the culture medium solution after sterilizing is poured into culture
Ware, stands and can obtain solid medium after cooling.
4. the cultural method of the white Antrodia camphorata fructification according to claim 1 or 2 or 3, it is characterised in that：
The section of white Antrodia camphorata fructification is cultivated in the culture dish containing bacterium culture medium in the step (2), culture temperature
Degree control is at 22~30 DEG C.
5. the cultural method of the white Antrodia camphorata fructification according to claim 1 or 2 or 3, it is characterised in that：The step
(4) carrying out growing environment regulation culture to the white Antrodia camphorata strain for connecing bacterium on culture medium in includes：Low temperature stimulation is trained
Support, knife wound stimulates culture and light stimulates at least one of culture.
6. the cultural method of white Antrodia camphorata fructification according to claim 5, it is characterised in that：In the step (4)
Low temperature stimulation culture is carried out to the white Antrodia camphorata strain for connecing bacterium on culture medium, wherein low temperature stimulation temperature range is 4
DEG C to 15 DEG C.
7. the cultural method of white Antrodia camphorata fructification according to claim 5, it is characterised in that：In the step (4)
Carrying out knife wound to the white Antrodia camphorata strain for connecing bacterium on culture medium stimulates culture, including：Using blade or syringe needle white
Color Antrodia camphorata manufactures multiple 1~2 centimeter of scar, the increase for secondary metabolites GCMS computer and weight.
8. the cultural method of white Antrodia camphorata fructification according to claim 5, it is characterised in that：In the step (4)
Carrying out light to the white Antrodia camphorata strain for connecing bacterium on culture medium stimulates culture, including connects bacterium on culture medium to described
White Antrodia camphorata strain carried out under photoenvironment light stimulate culture；Wherein, the photoenvironment selects feux rouges or blue light,
The illumination intensity of the photoenvironment is 5 μm of ol/S.m2To 20 μm of ol/S.m2。
9. the cultural method of white Antrodia camphorata fructification according to claim 8, it is characterised in that：When the photoenvironment
During for feux rouges, the illumination intensity of the photoenvironment is 12 μm of ol/S.m2To 20 μm of ol/S.m2；Or
When photoenvironment is blue light, the illumination intensity of the photoenvironment is 8 μm of ol/S.m2To 16 μm of ol/S.m2。
10. a kind of culture medium for being used to cultivate white Antrodia camphorata fructification, it is characterised in that：The culture medium at least includes：
Potato glucose extract, its mass concentration is 2~4%w/w；
Semen Tritici aestivi extract, its mass concentration is 2~4%w/w；
Nitrogen source, its mass concentration is 0.1~0.3%w/w, wherein, the nitrogen source can be used：Amino acid, peptone, yeast extraction
Thing or beef extract；
Agar, its mass concentration is 0.5~0.6%w/w；With
Sucrose, its mass concentration is 5~7%w/w.
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| TW107107813A TW201839124A (en) | 2017-04-27 | 2018-03-08 | Culture medium and method for culturing fruiting bodies of antrodia cinnamomea |
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| CN113367252A (en) * | 2020-03-09 | 2021-09-10 | 张腾龙 | Use of nutritional composition |
| CN114568207A (en) * | 2022-03-11 | 2022-06-03 | 青岛浩然海洋科技有限公司 | Dish-type culture process for Antrodia camphorata |
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Also Published As
| Publication number | Publication date |
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| CN107242026B (en) | 2020-06-02 |
| TW201839124A (en) | 2018-11-01 |
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