CN108530549B - Method for extracting polysaccharide from ganoderma lucidum mycelia - Google Patents
Method for extracting polysaccharide from ganoderma lucidum mycelia Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, in particular to a method for extracting polysaccharide from ganoderma lucidum mycelia. Mixing dried ganoderma lucidum mycelia with water, uniformly mixing the dried ganoderma lucidum mycelia and the water by a high-speed homogenizer, then carrying out high-pressure homogenizing dispersion for 1-5 times at 500-1500 bar, and centrifuging to obtain a supernatant containing polysaccharide. Compared with the traditional water extraction method, the method can improve the yield of the ganoderma lucidum polysaccharide, shorten the extraction time of the ganoderma lucidum polysaccharide, and improve the oxidation resistance and the anti-inflammatory effect of the ganoderma lucidum polysaccharide.
Description
Technical field
The present invention relates to microorganisms technical field more particularly to a kind of methods for extracting polysaccharide in ganoderma lucidum mycelium.
Background technique
Ganoderma lucidum Ganoderma lucidum (Curtis:Fr.) P.Karst. is that Basidiomycetes Polyporaceae Ganoderma is true
Bacterium is exactly the rare medicinal fungi in China since ancient times.It can treat a variety of diseases, and strengthening by means of tonics is strengthened the body resistance to consolidate the constitution, and be conducive to machine
Body keeps or restores the rare Chinese medicine of stable state.Modern medical research has found that: major physiological active constituent is ganoderma lucidum polysaccharide in ganoderma lucidum
And ganoderic acid, they have the drug effects such as antitumor, reduction blood cholesterol level, anti-inflammatory.In recent years, people pass through a system
Column research discovery ganoderma lucidum polysaccharide also has antitumor, anti-aging, hypoglycemic, reducing blood lipid, anti-blood other than having immunoregulatory activity
Bolt promotes the effect of biosynthesis of nucleic acid and protein.Just because of there is so more bioactivity, ganoderma active polysaccharide is just
It is quickly developed out as drug and health food.
However Wild ganoderma resource is limited, the Ganoderma Lucidum fructification period is long, and no matter wild or cultivating ganoderma is sub
Entity, polyoses content is all lower, only 2% or so of its dry weight, and Polyose extraction yield is low, high production cost, limitation
The extensive use of ganoderma lucidum polysaccharide.The artificial cultivation period is long (2 months or more), and productivity is low, and is subject to seasonal restrictions, thus shadow
The development and production of ganoderma lucidum polysaccharide are rung.The fructification of artificial cultivation is traditionally mostly used to carry out the extraction of ganoderma lucidum polysaccharide, due to it
Production cycle is long, climate influences big, yield and unstable quality, and the reason of because of water, air and soil, causes heavy metal
Enrichment.
Therefore, it is intended that the ganoderma lucidum mycelium obtained to liquid fermentation and culture extracts, the so achievable period is short by (2~3
It), at low cost, the big polysaccharide preparation of yield, to greatly improve the production capacity of ganoderma lucidum polysaccharide.But, on the one hand, liquid hair
Ferment obtains ganoderma lucidum mycelium and is not easy to；On the other hand, mycelium ganoderic acid is present in ganoderma lucidum mycelium cell wall, extracts
It is not easy to dissolve out in journey, and the method for sufficiently broken wall always inevitably destroys the activity of polysaccharide.Therefore, research extraction efficiency height, polysaccharide
The high extract method of activity is still the hot spot of this field research.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that providing a kind of side for extracting polysaccharide in ganoderma lucidum mycelium
The activity of method, this method high income, and gained polysaccharide is higher.
The method of polysaccharide includes: in extraction ganoderma lucidum mycelium provided by the invention
Dry ganoderma lucidum mycelium is mixed with water, disperses 5min through 5000rpm high speed homogenization, then 500~1500bar
High-pressure homogeneous dispersion 1~5 time, is centrifuged, and the supernatant containing polysaccharide is obtained.
In the embodiment of the present invention, the ganoderma lucidum mycelium of the drying and quality-volume ratio of water are 1g:10~30mL.
In some embodiments, the ganoderma lucidum mycelium of the drying and quality-volume ratio of water are 1g:30mL.
In the embodiment of the present invention, the condition of the high-pressure homogeneous dispersion is that 1000bar disperses 3~5 times or 1500bar points
It dissipates 1~2 time.
In some specific embodiments, the condition of the high-pressure homogeneous dispersion is that 1000bar disperses 2 times.
In some specific embodiments, the condition of the high-pressure homogeneous dispersion is that 1000bar disperses 3 times.
In some specific embodiments, the condition of the high-pressure homogeneous dispersion is that 1000bar disperses 4 times.
In some specific embodiments, the condition of the high-pressure homogeneous dispersion is that 1000bar disperses 5 times.
In some specific embodiments, the condition of the high-pressure homogeneous dispersion is that 1500bar disperses 1 time.
In some specific embodiments, the condition of the high-pressure homogeneous dispersion is that 1500bar disperses 2 times.
Experiments indicate that pressure is higher, the yield for the ganoderma lucidum polysaccharide extracted from Ganoderma lucidum mycelium is higher.In 500bar
More with cycle-index under the conditions of 1000bar, the yield of ganoderma lucidum polysaccharide is higher.But under the conditions of 1500bar, recycle twice just
The yield of ganoderma lucidum polysaccharide can be increased to 100%, however repeatedly circulation, the yield of polysaccharide decline instead later, this may be
Polysaccharose substance is caused to decompose due under the action of continuous high pressure, generating a large amount of thermal energy.
Also, the oxidation resistance of the Ganoderma lucidum mycelium extracting solution extracted by high pressure homogenizer will be much higher than the hot formulation of tradition
The Ganoderma lucidum mycelium extract of acquisition.With the increase of pressure and cycle-index, the oxidation resistance of Ganoderma lucidum mycelium extract is continuous
Increase, but under the conditions of 1500bar, with the increase of cycle-index, oxidation resistance constantly declines.Possible cause is high pressure work
Under, the temperature of Material constantly rises, and bioactive substance is caused to inactivate, and oxidation resistance is caused to decline.In addition, with pressure
It is continuously increased with the anti-inflammatory ability of the increase of cycle-index, Ganoderma lucidum mycelium extract, but under the conditions of 1500bar, with following
The increase of ring number, oxidation resistance constantly decline.
In the embodiment of the present invention, the speed of the high speed homogenization dispersion is 5000rpm, and the time of each homogeneous is 5min.
In some embodiments, the temperature of the high-pressure homogeneous dispersion is room temperature.
In the embodiment of the present invention, the revolving speed of the centrifugation is 3000rpm, and the time is 10 minutes.
In the embodiment of the present invention, the ganoderma lucidum mycelium the preparation method comprises the following steps: by the lucidum strain through overactivation by 1~
3cm2The inoculum concentration of/100mL culture medium is inoculated into fructus hordei germinatus leaching powder liquid fermentation medium, and 3~7 are cultivated at 25~30 DEG C
It, obtains the fermentation liquid containing ganoderma lucidum mycelium.
The lucidum strain is the lucidum strain that solid medium culture obtains.Strain is collected according to surface area.
The fructus hordei germinatus leaching powder liquid fermentation medium includes: fructus hordei germinatus leaching powder 1.5w/v%~2.5w/v%, glucose 1.5w/
V%~2.5w/v%, peptone 0.07w/v%~0.13w/v%, yeast extract 0.07w/v%~0.13w/v%, solvent are water.
The culture ganoderma lucidum mycelium by way of liquid fermentation, training method is simple, incubation time only need 3~7 days i.e.
It can.Meanwhile mycelium is well dispersed, and cell wall is relatively thin, and it is easily broken, facilitate extraction.With traditional ganoderma lucidum fruitbody cultural method
It compares, obtaining Ganoderma lucidum mycelium by liquid fermentation method has easy to operate, and cultivation cycle is short, the advantages of facilitating extraction.
In the embodiment of the present invention, the supernatant obtains ganoderma lucidum polysaccharide through dry, or obtains after ethanol precipitation, washing
Ganoderma lucidum polysaccharide.
The ethanol precipitation is precipitated using the ethyl alcohol of three times volume, and the temperature of the precipitating is 5 DEG C, and the time is 48 small
When.
Precipitating, the ultracentrifugal revolving speed are taken after further including the steps that high speed centrifugation between the ethanol precipitation and washing
For 3000rpm, the time is 10 minutes.
The washing includes using water then heavy with the ethyl alcohol of three times water volume after precipitating that centrifugation obtains redissolved with water
It forms sediment, the temperature of precipitating is 5 DEG C, and the time is 8 hours, turns centrifugation after ten minutes through 3000rpm；It is redissolved again with water, then with three
The ethanol precipitation of times water volume, the temperature of precipitating are 5 DEG C, and the time is 8 hours, turn centrifugation after ten minutes through 3000rpm；Precipitating with
After ethyl alcohol cleaning, the dry ganoderma lucidum polysaccharide for obtaining solid.
Ganoderma lucidum polysaccharide made from the method for the invention.
Ganoderma lucidum polysaccharide made from the method for the invention is preparing the application in cosmetics or health care product.
In the embodiment of the present invention, the cosmetics or health care product have effects that anti-inflammatory and/or oxidation resistant.
The method of polysaccharide includes: to mix dry ganoderma lucidum mycelium with water in extraction ganoderma lucidum mycelium provided by the invention
It closes, disperses 5min through 5000rpm high speed homogenization, then the high-pressure homogeneous dispersion of 500~1500bar 1~5 time, is centrifuged, is contained
There is the supernatant of polysaccharide.Compared to traditional water extraction, the yield of ganoderma lucidum polysaccharide can be improved, traditional hot water extraction procedure
Extraction efficiency is 88.04%, high pressure homogenization method method 1000bar, and the method extraction efficiency that circulation is 3 times is 100.66%；Shorten
The extraction time of ganoderma lucidum polysaccharide, traditional hot water extraction procedure extraction time use high pressure homogenizer at least at 2 hours or more
The extraction time of extraction can foreshorten to a few minutes.
Due to using high pressure homogenization technique, during the extraction process since by high pressure, Strong shear power is hit, cavity effect etc.
Effect, cause the reduction of the molecular weight of macromolecular polysaccharide, resolve into micromolecular polysaccharide, improve the anti-oxidant energy of ganoderma lucidum polysaccharide
Power and anti-inflammatory effect.The oxidation resistance of the extracting solution of traditional hot water extraction procedure is only 42.78%, high pressure homogenization method
Method 1000bar, the oxidation resistance for the method extracting solution that circulation is 3 times are 68.07%；The extracting solution of traditional hot water extraction procedure
Anti-inflammatory ability be only 21.1%, high pressure homogenization method method 1000bar, circulation 3 times method extracting solution anti-inflammatory ability
It is 28.4%, the effect is through statistical analysis, and there are significant difference, p < 0.05.
Detailed description of the invention
Fig. 1 shows the total content of ganoderma lucidum polysaccharide in Ganoderma lucidum mycelium；
Fig. 2 shows the yield of ganoderma lucidum polysaccharide under different pressures and different cycle-indexes；
Fig. 3 shows the oxidation resistance of ganoderma lucidum polysaccharide under different pressures and different cycle-indexes；
Fig. 4 shows the anti-inflammatory ability of ganoderma lucidum polysaccharide under different pressures and different cycle-indexes.
Specific embodiment
The present invention provides a kind of method of polysaccharide in extraction ganoderma lucidum mycelium, those skilled in the art can be used for reference herein
Content is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to those skilled in the art
It is it will be apparent that they are considered as being included in the present invention for member.Method and application of the invention has passed through preferably real
It applies example to be described, related personnel can obviously not depart from the content of present invention, in spirit and scope to methods herein and answer
With being modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The instrument that the present invention uses is all common commercially available product, can all be bought in market.Wherein, the system of Ganoderma lucidum mycelium fermentation liquid
Preparation Method are as follows:
Lucidum strain through overactivation is pressed into 1~3cm2The inoculum concentration of/100ml culture medium is inoculated into fructus hordei germinatus leaching powder liquid hair
In ferment culture medium, is cultivated at 25 DEG C~30 DEG C 3~7 days, both obtain Ganoderma lucidum mycelium fermentation liquid, wherein affiliated fructus hordei germinatus leaching powder liquid
Fermentation medium includes: fructus hordei germinatus leaching powder 1.5w/v%~2.5w/v%, glucose 1.5w/v%~2.5w/v%, peptone
0.07w/v%~0.13w/v%, yeast extract 0.07w/v%~0.13w/v%, solvent are water.
Below with reference to embodiment, the present invention is further explained:
Polysaccharide total content measures in 1 ganoderma lucidum mycelium of embodiment
1, Ganoderma lucidum mycelium fermentation liquid by obtaining Ganoderma lucidum mycelium after centrifugation, dry in 60 DEG C of baking ovens by Ganoderma lucidum mycelium；
2, Ganoderma lucidum mycelium 10g is taken, the water of 100ml is sequentially added according to the ratio of 1:10, after mixing at Ganoderma lucidum mycelium
Mixed liquor；
3, mixed liquor is placed in 80 DEG C of hot water, is stirred, ultrasonication 20min；
4, it is centrifuged after being ultrasonically treated, supernatant is taken out, detect ganoderma polyoses content according to phend-sulphuric acid；
5, the precipitating after being centrifuged is repeated 5 times according to step 2~4, is extracted polysaccharide 6 times in total；
6, the total content of polysaccharide in Ganoderma lucidum mycelium is calculated according to the content for extracting polysaccharide every time.
Phend-sulphuric acid detects the content of polysaccharide in ganoderma lucidum polysaccharide
For polysaccharide under the action of sulfuric acid, hydrolysis generates monosaccharide, and rapid dehydration generates alditol derivative, then contracts with phenol
Synthesize orange-yellow compound, and colour stable.At wavelength 490nm and in certain concentration range, absorbance contains with polysaccharide
It measures in a linear relationship, so as to utilize spectrophotometer measurement its absorbance, and utilizes standard curve quantitative determination sample
Polyoses content.Experimental method are as follows: the configuration of glucose standard, concentration 100ppm；90% phenol solution is configured, is stored standby
With；90% phenol solution is diluted to 6%, is faced with now matching；The standard curve of glucose is made, specific additive amount is as follows:
1 standard curve additive amount of table
The glucose 1ml of various concentration is respectively added in different test tube with ground stoppers first, is added respectively in ice bath
The concentrated sulfuric acid of 3.0ml is slowly added dropwise in the phenol solution of 0.5ml after concussion, be advisable with micro heat release, jumps a queue in boiling water after shaking up
20min is placed, cooling 20min in cold water after taking-up.After having reacted, by 6 samples be respectively added in 96well dish in
Absorption photometric value is measured at 490nm wavelength.Using concentration of glucose as X-axis, absorbance is Y-axis, draws the standard curve of glucose.
Acquire regression equation.The Ganoderma lucidum mycelium extracting solution for taking 1ml, is diluted to a certain concentration, according to above-mentioned test method, measures blank
The absorption photometric value of sample and extracting solution calculates the concentration of polysaccharide in Ganoderma lucidum mycelium extracting solution according to glucose standard curve.
Experimental result such as table 1 and Fig. 1:
The total content of 2 Ganoderma lucidum mycelium polysaccharide of table
To sum up experimental result it is found that extract 3 times after ganoderma lucidum polysaccharide concentration be 22ppm, it is at a fairly low, that is, extract 3
Ganoderma lucidum polysaccharide can be extracted completely substantially after secondary, the total ganoderma polyoses content that can be calculated extraction is 0.1840g, yield
It is 1.84%.
Embodiment 2
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2) take Ganoderma lucidum mycelium 100g, the water of 3000ml be added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 500bar, and cycle-index is 1 time；
4) high speed centrifugation obtains supernatant.
Embodiment 3
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2) take Ganoderma lucidum mycelium 100g, the water of 3000ml be added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 500bar, and cycle-index is 2 times；
4) high speed centrifugation obtains supernatant.
Embodiment 4
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2) take Ganoderma lucidum mycelium 100g, the water of 3000ml be added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 500bar, and cycle-index is 3 times；
4) high speed centrifugation obtains supernatant.
Embodiment 5
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2) take Ganoderma lucidum mycelium 100g, the water of 3000ml be added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 500bar, and cycle-index is 4 times；
4) high speed centrifugation obtains supernatant.
Embodiment 6
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2) take Ganoderma lucidum mycelium 100g, the water of 3000ml be added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 500bar, and cycle-index is 5 times；
4) high speed centrifugation obtains supernatant.
Embodiment 7
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2) take Ganoderma lucidum mycelium 100g, the water of 3000ml be added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 1000bar, and cycle-index is 1 time；
4) high speed centrifugation obtains supernatant.
Embodiment 8
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2 take Ganoderma lucidum mycelium 100g, and the water of 3000ml is added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 1000bar, and cycle-index is 2 times；
4) high speed centrifugation obtains supernatant.
Embodiment 9
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2) take Ganoderma lucidum mycelium 100g, the water of 3000ml be added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 1000bar, and cycle-index is 3 times；
4) high speed centrifugation obtains supernatant.
Embodiment 10
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2) take Ganoderma lucidum mycelium 100g, the water of 3000ml be added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 1000bar, and cycle-index is 4 times；
4) high speed centrifugation obtains supernatant.
Embodiment 11
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2) take Ganoderma lucidum mycelium 100g, the water of 3000ml be added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 1000bar, and cycle-index is 5 times；
4) high speed centrifugation obtains supernatant.
Embodiment 12
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2) take Ganoderma lucidum mycelium 100g, the water of 3000ml be added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 1500bar, and cycle-index is 1 time；
4) high speed centrifugation obtains supernatant.
Embodiment 13
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2) take Ganoderma lucidum mycelium 100g, the water of 3000ml be added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 1500bar, and cycle-index is 2 times；
4) high speed centrifugation obtains supernatant.
Embodiment 14
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2) take Ganoderma lucidum mycelium 100g, the water of 3000ml be added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 1500bar, and cycle-index is 3 times；
4) high speed centrifugation obtains supernatant.
Embodiment 15
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2) take Ganoderma lucidum mycelium 100g, the water of 3000ml be added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 1500bar, and cycle-index is 4 times；
4) high speed centrifugation obtains supernatant.
Embodiment 16
1) Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare；
2) take Ganoderma lucidum mycelium 100g, the water of 3000ml be added, using high-shear homogenizer under 5000rpm speed homogeneous 5min
Afterwards, after mixing at Ganoderma lucidum mycelium mixed liquor；
3) pressure of high pressure homogenizer is set as 1500bar, and cycle-index is 5 times；
4) high speed centrifugation obtains supernatant.
Comparative example 1
1, Ganoderma lucidum mycelium is obtained after the centrifugation of Ganoderma lucidum mycelium fermentation liquid, Ganoderma lucidum mycelium is dried in 60 DEG C of baking ovens, spare.
2, Ganoderma lucidum mycelium 100g is taken, the water of 3000ml is added, after mixing at Ganoderma lucidum mycelium mixed liquor.
3, mixed liquor is placed in 80 DEG C of hot water, is stirred, ultrasonication 20min.
4, it is centrifuged after being ultrasonically treated, supernatant is taken out, detect ganoderma polyoses content according to phend-sulphuric acid.
Polyose extraction efficiency, Activity determination
According to embodiment 1 as a result, being detected to the bioactivity of polysaccharide extract rate and polysaccharide.
1, the method for oxidation resistance detection are as follows:
1., DPPH dissolved with MeOH, concentration 0.15mM
2., the preparation of sample
3., in 96well dish respectively by following materials add:
3 oxidation resistance of table detects material addition
| Sample | Sample blank | Control |
| Sample 100ul | Sample 100ul | Sample solvent 100ul |
| DPPH solution 150ul | MeOH 150ul | DPPH solution 150ul |
4., fully shake mixing after in dark place react 30min
5., with microplate reader absorption photometric value is tested at wavelength 517nm
6., calculate using absorption photometric the clearance rate of DPPH free radical, calculation formula is as follows:
The anti-inflammatory aptitude tests of 3.1 Ganoderma lucidum mycelium polysaccharide extraction liquids
2, LPS induces RAW264.7 cell to discharge NO inhibiting effect
1., this experiment using LPS induction RAW264.7 cell generate inflammatory reaction, discharge NO (nitric oxide).
Griess assay-NO content test method
NO is easily oxidized to NO in vivo or in aqueous solution2, in acid condition, NO and diazonium salt sulfanilamide (SN) generation diazonium are anti-
It answers, and generates diazonium compound, coupling reaction further occurs with naphthylethenyl diamines for the latter, and the product which generates is dense
Degree has linear relationship with NO concentration, there is maximum absorption band at 540nm.
1.1) RAW264.7 cell is drawn into DMEM training base with liquid-transfering gun and dispels cell dispersion, utilize Hemacytometer
Count cell, then using DMEM come diluting cells, being diluted to concentration is 5 × 104cells/ml。
1.2) dilute after cell solution be inoculated into 96well respectively, each hole be 100ul, i.e., 5 × 103cells/
well。
1.3) at 37 DEG C, 5%CO2Incubator in cultivate 24 hours.
1.4) sample to be tested and blank sample prepare: sample to be tested is trained base with DMEM and is diluted, and the concentration after dilution is respectively as follows:
1% (concentration is tested by the MTT of front, and result is nontoxic), specific as follows:
4 anti-inflammatory power of table detects material addition
| Title | Control | Sample |
| Sample composition | LPS 10ppm | LPS 10ppm+1%Sample |
1.5) after cell culture 24 hours, the whether complete adherent growth of cell is observed, if the complete adherent growth of cell
Original training base is removed, is washed with DPBS by words.
1.6) after removing DPBS, it is separately added into the ready sample in front.The solubility of sample is according to toxotest
As a result safe concentration 1% (20ul/well) is selected
1.7) after sample is added, 37 DEG C are put into, 5%CO2Incubator in cultivate 20 hours.
1.8) after 20 hours, on the culture medium to new 96Well that pipettes 50 μ l, the 1%Griess of same volume is added
Reagent after being sufficiently mixed, reacts 10 minutes in dark place.
1.9) absorption photometric value of ELISAreader test sample at 540nm is utilized.
NO Release Inhibition (%)=((ODControl-ODSample)/ODControl) × 100%
Wherein: ODControlThe absorption photometric value of blank sample
ODSampleThe absorption photometric value of sample
As a result such as table 5, antioxidant activity such as Fig. 3, anti-inflammatory activity cream Fig. 4:
5 yield of table and Activity determination result
To sum up for test result it is found that pressure is higher, the yield for the ganoderma lucidum polysaccharide extracted from Ganoderma lucidum mycelium is higher (Fig. 2).
Cycle-index is more under the conditions of 500bar and 1000bar, and the yield of ganoderma lucidum polysaccharide is higher.But under the conditions of 1500bar, follow
The yield of ganoderma lucidum polysaccharide can be increased to 100% twice by ring, but later repeatedly circulation if, the yield of polysaccharide instead under
Drop, it may be possible to cause polysaccharose substance to decompose due under the action of continuous high pressure, generating a large amount of thermal energy.
It compares traditional hot water extraction procedure and high pressure homogenization method method and extracts Ganoderma lucidum mycelium extract, it is high-pressure homogeneous to mention
Take method that can quickly obtain the higher extracting solution of polysaccharide yield.The extraction efficiency of traditional hot water extraction procedure is
88.04%, high pressure homogenization method method 1000bar, the method extraction efficiency that circulation is 3 times are 100.66%；High pressure homogenization method side
Method can obtain the higher extracting solution of oxidation resistance, and the oxidation resistance of the extracting solution of traditional hot water extraction procedure is only
42.78%, high pressure homogenization method method 1000bar, the oxidation resistance for the method extracting solution that circulation is 3 times are 68.07%；High pressure is equal
Matter extracting method can obtain the better extracting solution of anti-inflammatory effect, the anti-inflammatory energy of the extracting solution of traditional hot water extraction procedure
Power is only 21.1%, high pressure homogenization method method 1000bar, and the anti-inflammatory ability for the method extracting solution that circulation is 3 times is 28.4%.
Through statistical analysis, the extraction efficiency and bioactivity of each embodiment all it is extremely significant be better than comparative example 1, p <
0.01；The extraction efficiency highest of embodiment 9~11 and embodiment 13, there are significant difference compared with other embodiments, p <
0.05；Wherein, it is poor all to there is conspicuousness with other embodiments in terms of the bioactivity highest of embodiment 13, especially anti-inflammatory activity
Different, p < 0.05, antioxidant activity is significantly better than Examples 1 to 9, embodiment 12 and embodiment 14~16, p < 0.05.
The above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (6)
1. a kind of method for extracting polysaccharide in ganoderma lucidum mycelium, which is characterized in that
Every 100g dry ganoderma lucidum mycelium is mixed with 300mL water, disperses 5min through 5000rpm high speed homogenization, then 1500bar
High-pressure homogeneous dispersion 2 times, 3000rpm is centrifuged 10min, obtains the supernatant containing polysaccharide.
2. the method according to claim 1, wherein the ganoderma lucidum mycelium the preparation method comprises the following steps: will be through making a living
The lucidum strain of change presses 1~3cm2The inoculum concentration of/100mL culture medium is inoculated into fructus hordei germinatus leaching powder liquid fermentation medium, in 25
DEG C~30 DEG C at cultivate 3~7 days, obtain the fermentation liquid containing ganoderma lucidum mycelium.
3. the method according to claim 1, wherein the supernatant obtains ganoderma lucidum polysaccharide, Huo Zhejing through dry
Ganoderma lucidum polysaccharide is obtained after ethanol precipitation, washing.
4. ganoderma lucidum polysaccharide made from any one of claims 1 to 3 the method.
5. ganoderma lucidum polysaccharide made from any one of claims 1 to 3 the method is preparing the application in cosmetics or health care product.
6. application according to claim 5, which is characterized in that the cosmetics or health care product have anti-inflammatory and/or antioxygen
The effect of change.
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