CN105062899B - One plant of two spore intends the extraction and application of Aode mushroom bacterial strain and its Thick many candies - Google Patents
One plant of two spore intends the extraction and application of Aode mushroom bacterial strain and its Thick many candies Download PDFInfo
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Abstract
The present invention relates to fungi field, discloses extraction and application that one plant of two spore intends Aode mushroom bacterial strain and its Thick many candies.It is that two spores intend Aode mushroom Hymenopellis raphanipes SU41 that two described spores, which intend Aode mushroom Strain Designation, and is preserved in China typical culture collection center, address on April 29th, 2015：Wuhan University of Wuhan City of Hubei China province, its preserving number are CCTCC NO：M 2015269.Obtained polysaccharose substance is extracted from its liquid fermentation mycelium, there is significant antioxidation through Multitest detection, be with a wide range of applications in terms of natural is prepared.
Description
Technical field
The present invention relates to fungi field, particularly relates to one plant of two spore and intends the extraction of Aode mushroom bacterial strain and its Thick many candies and answer
With.
Background technology
Oxidation reaction and human body it is metabolic closely bound up.When human body is carrying out metabolic reaction using oxygen
It is inevitably to produce some unstable free radicals, when internal free radical reaches to a certain degree, people will be damaged
Body health.In order to mitigate or eliminate the harm of oxidation reaction, the especially natural antioxidant of antioxidant, increasingly by
Pay attention to.Natural antioxidant derives from fruit or vegetables mostly, in diseases such as prevention heart disease, cancers, and delays to decline
Always, promoting mental, beauty, skin care etc. has significant effect.Macro fungi is the natural anti-oxidation of great exploitation potential
Agent resource, the active material of new removing interior free yl, and the development of modern medicine, healthcare industry are found from macro fungi
Direction.
The content of the invention
Present invention one side provides one plant of two spore and intends Aode mushroom (Hymenopellis raphanipes) bacterial strain, life
It is entitled：Two spores intend Aode mushroom (Hymenopellis raphanipes) SU41, and are preserved in Chinese allusion quotation on April 29th, 2015
Type culture collection, address：Wuhan University of Wuhan City of Hubei China province, its preserving number are CCTCC NO：M 2015269.
Two described spores intend its ITS sequence of Aode mushroom SU41 bacterial strains as shown in SEQ ID NO.1.
Two spores that the second aspect of the invention is to provide described in one side of the invention intend Aode mushroom strain liquid hair
The preparation method of zymotic fluid, comprises the following steps：Sterilized wheat bran-dextrose culture-medium is poured into sterile petri dish be made it is flat
Plate；After culture medium solidifying, aseptically, per flat board, access activated spawn block, is beaten after culture with card punch from flat board
The activated spawn block after culture is taken, access is equipped with the triangular flask of wheat bran-dextrose broth, and one is produced after shaking table culture
Level shaking flask strain；One-level shaking flask strain is accessed into wheat bran-dextrose broth for second-level shake flask fermentation, shaking table afterwards
Cultivate and produce the two spores plan Aode mushroom strain liquid zymotic fluid；Described wheat bran-dextrose culture-medium composition, with parts by weight
It is designated as：50 parts of wheat bran, 20 parts of glucose, 3 parts of potassium dihydrogen phosphate, 1.5 parts of magnesium sulfate, 15 parts of agar, 1000 parts of water；Described wheat
Bran-dextrose broth composition, is designated as with parts by weight：50 parts of wheat bran, 20 parts of glucose, 3 parts of potassium dihydrogen phosphate, sulfuric acid
1.5 parts of magnesium, 1000 parts of water.
Further, condition of culture of the activated spawn block on plating medium is：28 DEG C, 10- is cultivated under dark condition
12d；Condition needed for shaking table culture is：120r/min, 28 DEG C, cultivate 10-12d under dark condition；One-level shaking flask strain accesses
Volume be 5% of wheat bran-dextrose broth volume in second-level shake flask.
It is thick that two spores that the third aspect of the invention is to provide described in one side of the invention intend Aode mushroom SU41 bacterial strains
The extracting method of polysaccharide, comprises the following steps：
(1) two spores are intended into the centrifugation of Aode mushroom SU41 strain liquids zymotic fluid, abandoning supernatant, precipitation is obtained with distillation water washing
To pure mycelium, constant weight is dried to；
(2) first mycelium is crushed, adds distillation flooding, collected after centrifugation supernatant；
(3) merge supernatant and the concentrate that is concentrated under reduced pressure to obtain, add 95% ethanol of 3 times of volumes of concentrate, alcohol is analysed at 4 DEG C
24h, abandoning supernatant, add after standing 30min with the isometric absolute ethyl alcohol of initial concentration liquid, abandoning supernatant, precipitation
Washing 3 times, freeze-drying, produce two spores and intend Aode mushroom SU41 Thick many candies.
Further, in the step (1), described precipitation uses distillation water washing 2-4 times.
Further, in the step (2), the quality of the distilled water of addition is 50 times of mycelia weight, repeats extraction 3
It is secondary；50-70 DEG C of temperature is controlled in leaching process, extracts 2-4h.
Further, it is concentrated under reduced pressure into the 1/300 of supernatant volume in the step (3)；Washing precipitation reagent used according to
Secondary is absolute ethyl alcohol, acetone and ether.
It is thick that two spore that the fourth aspect of the invention is to provide described in third aspect of the present invention intends Aode mushroom SU41
Application of the polysaccharide in terms of antioxidant is prepared.
Beneficial effects of the present invention：
The spore of new strains two of the present invention intends Aode mushroom Hymenopellis raphanipes SU41, from wild son
Separate and obtain in entity, and bacterial strain preservation has been carried out on April 29th, 2015, be preserved in China typical culture collection center,
In Wuhan University of Wuhan City of Hubei China province, its deposit number is CCTCC NO for address:M 2015269.Intend from two described spores
The polysaccharose substance extracted in Aode mushroom SU41 bacterial strains, is verified through test of many times, is had preferable antioxidation, is being prepared day
It is with a wide range of applications in terms of right antioxidant.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1 is that two spores intend Aode mushroom SU41 colony growth situations under different carbon source；
Fig. 2 is that two spores intend Aode mushroom SU41 colony growth situations under different nitrogen sources；
Fig. 3 is the influence that different temperatures intends two spores Aode mushroom SU41 mycelial growths；
Fig. 4 is that two spores intend Aode mushroom SU41 colony growth situations under different temperatures；
Fig. 5 is the influence that different pH value intend two spores Aode mushroom SU41 mycelial growths；
Fig. 6 is that two spores of different PH processing intend Aode mushroom SU41 colony growth situations；
Fig. 7 is that two spores intend elimination effect of the Aode mushroom SU41 bacterial strains Thick many candies to ultra-oxygen anion free radical；
Fig. 8 is that two spores intend elimination effect of the Aode mushroom SU41 bacterial strains Thick many candies to hydroxy radical；
Fig. 9 is that two spores intend elimination effect of the Aode mushroom SU41 bacterial strains Thick many candies to DPPH free radicals.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other under the premise of creative work is not made
Embodiment, belong to the scope of protection of the invention.
First, the acquisition of bacterial strain
Specimen in use collection is tested from Nanchang City, Jiangxi Province suburb.Wild fructification is taken, is put into desinfection chamber, uses sterilized water
Rinse 3 times, carry out surface sterilization, the vertical partial application in the middle part of cap with the scalpel sterilized immediately, tear, picking cap and bacterium
One fritter bacterial context of handle intersection, on transposing to wheat bran-glucose slant medium, plus tampon, under 28 DEG C, dark condition
Culture 3 days, it is seen that villiform mycelia is grown on tissue block, two spores is produced and intends Aode mushroom bacterial strain.
2nd, morphological feature
Fructification bacteria cover diameter 3-8.5cm, it is open and flat, it is central raised or like umbilical；Cap is smooth, and light brown or dark brown is extremely
Crineous, there is dark radial striped.Bacterial context white is thin.Lamella white, curved life, wider, Length discrepancy.Stem is closely cylindric, long
6-17cm, thick 0.3-1.2cm, light brown, dipped beam is slided, and has a vertical stripe, epidermis gristle matter, internal fiber matter and soft, and base portion is swollen
It is big and prolong generation rhizoid.Spore print white.Load has 2 stigmas, and 14-20 × 10-18 μm of basidiospore (13-) is wide oval near
It is spherical.The mycelia of flat board culture is pure white, and aerial hyphae is dense.
3rd, biological characteristics
Condition of culture：
(1) influence of the carbon source to mycelial growth：
The composition of basal medium is：Carbon source 20g, soy peptone 2g, potassium dihydrogen phosphate 0.46g, dipotassium hydrogen phosphate 1g,
Magnesium sulfate 0.5g, agar 20g, water 1000mL, carbon source select maltose, sucrose, mannitol, starch, lactose and glucose respectively,
Access after two spores intend Aode mushroom bacterial strain, 28 DEG C, cultivate 12d under dark condition.Two spores intend Aode mushroom growing state under 6 kinds of carbon sources
Each different, when two spores intend Aode mushroom using maltose as carbon source, the colony diameter for growing 12d is maximum；Thereafter it is starch, sugarcane successively
Sugar, glucose, mannitol, lactose (table 1)；Significance of difference test result shows, influence of the maltose to mycelial growth rate
For other carbon sources, there is significant difference (table 1)；Simultaneously from the point of view of growth potential, during using maltose as carbon source, bacterium colony mycelia
It is dense, growth potential is vigorous (Fig. 1).As can be seen here, the most suitable carbon source that two spores intend Aode mushroom mycelial growth is maltose.
The different carbon source of table 1 to two spores intend Aode mushroom mycelial growth influence ()
* the colony diameter of flat board culture 12 days；* same columns letter is different to represent significant difference.
(2) influence of the nitrogen source to mycelial growth
The composition of basal medium is：Glucose 20g, nitrogen source 2g, potassium dihydrogen phosphate 0.46g, dipotassium hydrogen phosphate 1g, sulfuric acid
Magnesium 0.5g, agar 20g, water 1000mL.Nitrogen source difference selective chlorination ammonium, peptone, ammonium sulfate, yeast extract, beef extract, access two
Spore intends Aode mushroom bacterial strain, 28 DEG C, cultivates 12d under dark condition.Two spores intend Aode mushroom can grow in 5 kinds of nitrogen sources for examination.
When using ammonium chloride as carbon source, the colony diameter for growing 12d is maximum；Thereafter it is yeast extract, ammonium sulfate, beef extract, albumen successively
Peptone (table 2).Significance of difference test result shows, during using ammonium chloride, yeast extract, ammonium sulfate, beef extract as nitrogen source, mycelia is given birth to
The no significant difference of influence of long speed, but peptone and other several nitrogen sources compare, then have significant difference (table 2)；From
From the point of view of growth potential, when using yeast extract and beef extract as nitrogen source, bacterium colony mycelia is dense, growth potential is vigorous；With ammonium chloride and sulfuric acid
When ammonium is nitrogen source, bacterium colony mycelia is sparse, and growth potential is weaker (Fig. 2).As can be seen here, yeast extract and beef extract are that two spores intend Aode mushroom
The suitable nitrogen source of mycelial growth.
The different nitrogen sources of table 2 to two spores intend Aode mushroom mycelial growth influence ()
* the colony diameter of flat board culture 12 days；* same columns letter is different to represent significant difference.
(3) influence of the different temperatures to mycelial growth
Using wheat bran-dextrose culture-medium, two spores of access intend Aode mushroom bacterial strain, and 12d is cultivated under dark condition.Cultivation temperature
For 20 DEG C when, mycelial growth is slower；With the rise of temperature, mycelial growth rate is progressively accelerated, and reaches most at 24 DEG C
Big value；When temperature is more than 28 DEG C, mycelial growth rate slows down, and at 32 DEG C, mycelia hardly grows (Fig. 3).Significant difference
Property test result show that the 28 DEG C and 32 DEG C influence difference to mycelial growth rate of temperature is not notable, but both and other temperature
There is significant difference between processing；From the point of view of growth potential, at 24 DEG C, bacterium colony mycelia is sparse, and at 28 DEG C, bacterium colony mycelia is dense
Close (Fig. 4).It follows that the optimum temperature that two spores intend Aode mushroom mycelial growth is 28 DEG C.
The composition of the wheat bran-dextrose culture-medium is：Wheat bran 50g, glucose 20g, potassium dihydrogen phosphate 3g, magnesium sulfate
1.5g, agar 15g, water 1000g；Described wheat bran boiling half an hour, filtering, take filtered juice.
(4) influences of the different pH to mycelial growth
Using wheat bran-dextrose culture-medium, two spores of access intend Aode mushroom bacterial strain, cultivate 12d under dark condition, as a result table
Bright, two spores intend Aode mushroom can grow in pH5.0-8.5, and significance of difference test result shows, each pH value is to mycelial growth
The influence difference of speed is not notable (Fig. 5)；From the point of view of growth potential, colony growth is all very vigorous (Fig. 6) under different pH value.Thus may be used
Know, two spores intend Aode mushroom mycelial growth to acid or alkali environment without strict demand.
Based on the above results, the most suitable carbon source that two spores intend Aode mushroom mycelial growth is maltose, and suitable nitrogen source is yeast
Cream and beef extract, most suitable cultivation temperature is 28 DEG C, to acid or alkali environment without strict demand.
4th, the identification of bacterial strain
The genomic DNA of obtained strains enters performing PCR amplification by universal primer ITS4 and ITS5, and acquisition size is 722bp
Specific DNA segment, sequence information is referring to sequence table SEQ ID NO.1.
According to above qualification result, the Strain Designation of gained intends Aode mushroom Hymenopellis for two spores
RaphanipesSU41, and bacterial strain preservation has been carried out on April 29th, 2015, China typical culture collection center is preserved in,
In Wuhan University of Wuhan City of Hubei China province, its preserving number is CCTCC NO for address:M 2015269.
5th, two spores intend the preparation method of Aode mushroom strain liquid fermentation mycelium
Sterilized wheat bran-dextrose culture-medium is poured into 9cm sterile petri dish flat board is made；Treat culture medium solidifying
Afterwards, aseptically, the access activated spawn block per flat board, 10-12d is cultivated under 25-28 DEG C, dark condition.Use card punch
The activated spawn block for taking 5 pieces of diameter 5mm, 500mL of the access equipped with 200mL wheat bran-dextrose broth are beaten from flat board
Triangular flask, 10-12d is cultivated under 120r/min, 25-28 DEG C, dark condition, produce one-level shaking flask strain；Afterwards 500mL's
Load wheat bran-dextrose broth 100mL in triangular flask, after sterilizing, access one-level shaking flask strain carries out second-level shake flask hair
Ferment；The volume of one-level shaking flask strain access is 5% of wheat bran-dextrose broth volume in second-level shake flask, in 120r/
Min, 25-28 DEG C, cultivate 10-12d under dark condition, produce two spore and intend Aode mushroom bacterial strain mycelium fermentation broth.
The composition of above-mentioned wheat bran-dextrose broth is：Wheat bran 50g, glucose 20g, potassium dihydrogen phosphate 3g, sulfuric acid
Magnesium 1.5g, water 1000g；Described wheat bran boiling half an hour, filtering, take filtered juice.
6th, two spores intend the extraction of Aode mushroom SU41 bacterial strain Thick many candies
(1) two spores are intended into the centrifugation of Aode mushroom SU41 bacterial strain fermentation liquors, abandoning supernatant, precipitation is obtained pure with distillation water washing
Mycelium, 60 DEG C are dried to constant weight, and mycelium crushes, and crosses 20 mesh sieves；
(2) erinaceus mycelium powder 40g is weighed, by 1:For 50 material water quality than adding distilled water, 50-70 DEG C extracts 2-4h, from
The heart, supernatant is obtained, repeat extraction 3 times；
(3) above-mentioned supernatant is merged, be concentrated under reduced pressure into supernatant original volume 1/300 obtains concentrate, adds liquid and concentrates 3 times
95% ethanol of volume, alcohol analysis 24h, abandons supernatant at 4 DEG C, adds and stands 30min with volume of concentrate identical absolute ethyl alcohol
After abandon supernatant, precipitate and washed successively 3 times with absolute ethyl alcohol, acetone, ether, is freeze-dried, produce two spores and intend Aode mushroom SU41
Bacterial strain Thick many candies.
7th, two spores intend Aode mushroom SU41 strain liquid fermentation mycelium Thick many candies Antioxidative Activity Determinations
(1) two spore is intended removing of the Aode mushroom SU41 strain liquid fermentation mycelium Thick many candies to ultra-oxygen anion free radical and made
With
2.8mL 0.1mol/L Tris-HCL buffer solutions (pH8.2) are added in a series of test tube, are then added
Two spores of 0.1mL different qualities concentration (0.6,0.8,1.0,2.0,4.0,6.0,8.0mg/mL) intend Aode mushroom liquid fermentation mycelia
The sample solution of body Thick many candies, it is well mixed, 10min is incubated in 25 DEG C of water-baths, then adds 25 DEG C of water-bath pre-temperatures
3mmol/L pyrogallol solution 0.1mL, are rapidly added after mixing in dry quartz colorimetric utensil, are surveyed at 325 nm every 30s
A fixed OD value, reaction 4.5min terminate.Pyrogallol solution is replaced to be managed as zeroing using isometric 10mmol/L HCl, it is empty
White group replaces sample with isometric deionized water, and VC is as positive control.The regression equation of curve is changed over time as absorbance,
Its slope is the autoxidation speed V of pyrogallol, calculates clearance rate of the sample to ultra-oxygen anion free radical according to the following formula, and count
Calculate its EC50Value, its result is as shown in Fig. 7 and table 3, table 4.
Clearance rate (%)=[(VBlank-VSample)]/VBlank× 100%
VBlankFor blank group mouse thymus cells speed (Δ OD/min), VSampleFor sample or the adjacent benzene three of positive control
Phenol autoxidation speed (Δ OD/min).
From Fig. 7 and table 3, table 4, two spores, which intend Aode mushroom liquid fermentation mycelium Thick many candies, has removing superoxide anion
The activity of free radical；Its Scavenging activity shows dose dependent, in 0.6-8mg/mL concentration range, with the increasing of concentration
Add, sample is consequently increased to the clearance rate of ultra-oxygen anion free radical；Its EC50It is worth for 1.72mg/mL.
(2) two spores intend scavenging action of the Aode mushroom SU41 strain liquid fermentation mycelium Thick many candies to hydroxy radical
20mmol/L sodium salicylates 0.3mL and 1.5mmol/L ferrous sulfate 1.0mL are pipetted respectively in each test tube, then
Two spores for being separately added into different quality concentration (0.6,0.8,1.0,2.0,4.0,6.0,8.0mg/mL) intend Aode mushroom liquid fermentation
Mycelium Thick many candies 1.0mL, it is eventually adding 0.7mL 6mmol/L H2O2, rapid to mix, 37 DEG C of water bath with thermostatic control 1h, in 510nm ripples
Its OD value is determined under length.Using distilled water as blank control, VC is as positive control.Sample is calculated as follows to hydroxy radical
Clearance rate：
Clearance rate (%)=[(A0-A1)/A0)] × 100%
A0For the OD values of blank control, A1For added with the OD values after sample or positive control.
From Fig. 8 and table 3, table 4, two spores, which intend Aode mushroom liquid fermentation mycelium Thick many candies, has removing hydroxyl ion certainly
By the activity of base, EC50It is worth for 1.96mg/mL；Its Scavenging activity shows dose dependent, in 0.6-8.0mg/mL concentration model
In enclosing, with the increase of concentration, sample is consequently increased to the clearance rate of hydroxyl ion free radical.
(3) two spores intend scavenging action of the Aode mushroom SU41 strain liquid fermentation mycelium Thick many candies to DPPH free radicals
Take 1.5mL distilled water to be added in 1.5mLDPPH solution, it is rapid to mix, at room temperature lucifuge stand 30min after
Its OD value is surveyed under 517nm；Two spores of different quality concentration (0.6,0.8,1.0,2.0,4.0,6.0,8.0mg/mL) are taken to intend Order
Mill bacteria liquid fermentation mycelium Thick many candies solution 1.5mL is added in 1.5mLDPPH solution, and rapid to mix, lucifuge is quiet at room temperature
30min is put after surveying its OD value under 517nm.Positive control is used as using VC.The clearance rate to DPPH free radicals is calculated as follows：
Clearance rate (%)=[(A0-A1)/A0] × 100%
A0For the OD values (blank control) of DPPH solution in itself, A1For the DPPH solution added with sample or positive control
OD values.
From Fig. 9 and table 3, table 4, two spores, which intend Aode mushroom liquid fermentation mycelium Thick many candies, has removing DPPH free radicals
Activity, EC50It is worth for 1.47mg/mL；Its Scavenging activity shows dose dependent, in the range of finite concentration, with concentration
Increase, sample is consequently increased to the clearance rate of DPPH free radicals.
The spore of table 3 two intend liquid fermentation mycelium Thick many candies antioxidation activity in Aode mushroom SU41 effect ()
The spore of table 4 two intends the IC of Aode mushroom liquid fermentation mycelium Thick many candies50(mg/mL)
In addition, from table 4, although using different assay methods, two spores intend Aode mushroom liquid fermentation mycelium
The EC of Thick many candies50It is more or less the same, although illustrating to measure method difference, the principle of reaction is different, and two spores intend Aode mushroom liquid
Fermentation mycelium Thick many candies show preferable antioxidation activity, and antioxidation activity is basically identical.
More than experiment from many aspects to illustrate that two spores are intended extracting to obtain in Aode mushroom SU41 liquid fermentation mycelium thick
Polysaccharide has good antioxidation, is with a wide range of applications in terms of antioxidant is prepared.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
God any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection with principle.
Claims (8)
1. one plant of two spore intends Aode mushroom (Hymenopellis raphanipes) bacterial strain, it is named as：Two spores intend Aode mushroom
(Hymenopellis raphanipes) SU41, and China typical culture collection center is preserved on April 29th, 2015,
Address：Wuhan University of Wuhan City of Hubei China province, its preserving number are CCTCC NO：M2015269；Its ITS sequence such as SEQ ID
Shown in NO.1.
2. two spore as claimed in claim 1 intends the preparation method of the liquid fermentation liquid of Aode mushroom bacterial strain, it is characterised in that：It will go out
Wheat bran-the dextrose culture-medium for crossing bacterium pours into flat board is made in sterile petri dish, and aseptically, per flat board, access right will
Ask two spores described in 1 to intend Aode mushroom SU41 bacterial strain activated spawn blocks, beaten after culture with card punch from flat board and take strain block, access
One-level shaking flask strain is produced in triangular flask equipped with wheat bran-dextrose broth, after shaking table culture；Afterwards by one-level shaking flask
Strain is accessed in wheat bran-dextrose broth for second-level shake flask fermentation, and two spore is produced after shaking table culture and intends Austria
Moral mushroom strain liquid zymotic fluid；Described wheat bran-dextrose culture-medium composition, is designated as with parts by weight：50 parts of wheat bran, glucose
20 parts, 3 parts of potassium dihydrogen phosphate, 1.5 parts of magnesium sulfate, 15 parts of agar, 1000 parts of water；Described wheat bran-dextrose broth
Composition, is designated as with parts by weight：50 parts of wheat bran, 20 parts of glucose, 3 parts of potassium dihydrogen phosphate, 1.5 parts of magnesium sulfate, 1000 parts of water.
3. two spore as claimed in claim 2 intends the preparation method of the liquid fermentation liquid of Aode mushroom bacterial strain, it is characterised in that activation
Strain block condition of culture on plating medium is：28 DEG C, 10-12d is cultivated under dark condition；Condition needed for shaking table culture
For：120r/min, 28 DEG C, cultivate 10-12d under dark condition；The volume of one-level shaking flask strain access is second-level shake flask fermentation institute
With the 5% of wheat bran-dextrose broth volume.
4. a kind of two spore intends the extracting method of Aode mushroom SU41 bacterial strain Thick many candies, comprise the following steps：
(1) two spores described made from claim any one of 2-3 are intended into the centrifugation of Aode mushroom SU41 strain liquids zymotic fluid, discarded
Supernatant, precipitation obtain pure mycelium with distillation water washing, are dried to constant weight；
(2) first mycelium is crushed, adds distillation flooding, collected after centrifugation supernatant；
(3) merging supernatant and the concentrate that is concentrated under reduced pressure to obtain, add 95% ethanol of 3 times of volumes of concentrate, alcohol analyses 24h at 4 DEG C,
Abandoning supernatant, add after standing 30min with the isometric absolute ethyl alcohol of initial concentration liquid, abandoning supernatant, washing of precipitate 3
It is secondary, freeze-drying, produce two spores and intend Aode mushroom SU41 Thick many candies.
5. two spore as claimed in claim 4 intends the extracting method of Aode mushroom SU41 bacterial strain Thick many candies, it is characterised in that：The step
Suddenly in (1), described precipitation uses distillation water washing 2-4 times.
6. two spore as claimed in claim 4 intends the extracting method of Aode mushroom SU41 bacterial strain Thick many candies, it is characterised in that：The step
Suddenly in (2), the quality of the distilled water of addition is 50 times of mycelia weight, repeats extraction 3 times；Temperature is controlled in leaching process
50-70 DEG C, extract 2-4h.
7. two spore as claimed in claim 4 intends the extracting method of Aode mushroom SU41 bacterial strain Thick many candies, it is characterised in that：The step
Suddenly it is concentrated under reduced pressure into the 1/300 of supernatant volume in (3)；Washing precipitation reagent used is followed successively by absolute ethyl alcohol, acetone and second
Ether.
8. two spores as described in any one of claim 4 to 7 intend two spores made from the extracting method of Aode mushroom SU41 Thick many candies and intend Austria
Application of the moral mushroom SU41 Thick many candies in terms of antioxidant is prepared.
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| CN106258999B (en) * | 2016-08-11 | 2018-08-31 | 广东省微生物研究所 | A kind of thickness pleat Aode mushroom strain, cultural method and application thereof |
| CN110982704B (en) * | 2019-12-17 | 2021-06-25 | 华南农业大学 | Tricholoma sporotrichum strain and breeding method thereof |
| CN112674199B (en) * | 2020-12-09 | 2021-07-06 | 广州市金龟寿药品有限公司 | Polypeptide food preservative and preparation method thereof |
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