CN106350456A - Manufacture and detection method of lucid ganoderma liquid strains - Google Patents
Manufacture and detection method of lucid ganoderma liquid strains Download PDFInfo
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- 239000007788 liquid Substances 0.000 title claims abstract description 44
- 241000222336 Ganoderma Species 0.000 title claims abstract description 19
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 19
- 238000001514 detection method Methods 0.000 title claims abstract description 13
- 238000000855 fermentation Methods 0.000 claims abstract description 33
- 230000004151 fermentation Effects 0.000 claims abstract description 33
- 239000001963 growth medium Substances 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 20
- 238000000746 purification Methods 0.000 claims abstract description 14
- 238000009630 liquid culture Methods 0.000 claims abstract description 9
- 239000000463 material Substances 0.000 claims abstract description 9
- 240000008397 Ganoderma lucidum Species 0.000 claims description 27
- 235000001637 Ganoderma lucidum Nutrition 0.000 claims description 27
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 22
- 239000008103 glucose Substances 0.000 claims description 21
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 19
- 241000894007 species Species 0.000 claims description 18
- 238000012360 testing method Methods 0.000 claims description 17
- 239000002609 medium Substances 0.000 claims description 16
- 239000007787 solid Substances 0.000 claims description 14
- 210000000582 semen Anatomy 0.000 claims description 13
- 230000001954 sterilising effect Effects 0.000 claims description 12
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 229920001817 Agar Polymers 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 7
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 5
- 229920000742 Cotton Polymers 0.000 claims description 3
- 241000973497 Siphonognathus argyrophanes Species 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
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- 229920002678 cellulose Polymers 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 3
- 235000013312 flour Nutrition 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
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- 238000002360 preparation method Methods 0.000 claims description 3
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- 238000003756 stirring Methods 0.000 claims description 3
- 239000002023 wood Substances 0.000 claims description 3
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- 244000000231 Sesamum indicum Species 0.000 claims 1
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- 241000233866 Fungi Species 0.000 description 1
- 208000007443 Neurasthenia Diseases 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 206010033557 Palpitations Diseases 0.000 description 1
- 241001558929 Sclerotium <basidiomycota> Species 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
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- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000004140 cleaning Methods 0.000 description 1
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- 208000002173 dizziness Diseases 0.000 description 1
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- 238000012807 shake-flask culturing Methods 0.000 description 1
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Abstract
The invention relates to a manufacture and a detection method of lucid ganoderma liquid strains. The method comprises the following steps: firstly, purifying a lucid ganoderma mother strain in a mother strain purification culture medium, inoculating the purified mother strain into a shake flask to be cultured in a liquid culture medium, taking out the cultured liquid culture medium, and inoculating the cultured liquid culture medium into a fermentation tank for continuous culture, and using the obtained liquid strain after the obtained liquid strain is qualified through detection. The mother strain purification culture medium, the liquid culture medium and a fermentation tank culture medium all have various formulas and can be flexibly selected according to situations. The liquid strain prepared from the method has the characteristics of consistent cell age, high permeability, short production period, early sprouting and high material absorbing speed.
Description
Technical field
The present invention relates to fungus growing technique field is and in particular to a kind of making of Liquid Strain of Ganoderma Lucidum, detection method.
Background technology
Ganoderma (ganoderma lucidum) is commonly called as Ganoderma lucidum seu Japonicum, is the sporophore of On Polyporaceae Ganoderma.Ganoderma has
Invigorating QI and tranquilization, effect of relieving cough and asthma, for diseases such as dizziness sleeplessness, shortness of breath and palpitation, neurasthenia, asthenia cough with asthma, have to human body
Health-care effect, has prevention, treatment or inhibitory action to disease.
The high temperatures mushroom of Ganoderma, is mainly grown in the place relatively moistening, and is required in its whole growth and development process
Ozone and the larger growing environment of humidity.Ganoderma has been realized in artificial greenhouseization plantation at present, and traditional field is adopted
The sporophore (or sclerotium) that collection or artificial culture produce is subject to that wild resource is rare, sporophore cultivation and production cycle length (4～5
Month), produce the condition such as climate and control and the restriction of bioactive substance content low factors, a large amount of productions to Ganoderma are made
Become obstruction, also limit the industrialization of Ganoderma.
The liquid production of hybrid seeds is with respect to traditional solid production of hybrid seeds advantageously: the liquid production of hybrid seeds utilizes biofermentation principle, is mycelia
Growth provides optimal nutrition, acid-base value, temperature, oxygen content, cleaning condition, so that mycelia fast-growth is bred rapidly, relatively
A fermentation period is completed in the short time.
Content of the invention
It is an object of the invention to solving the problems referred to above present in existing cultivation of glossy ganoderma, provide a kind of Liquid Strain of Ganoderma Lucidum
Making and detection method, the liquid spawn that the method produces has that cell age is consistent, permeability is strong, growth cycle is short, sprouting is early,
Material feeding waits many advantages soon, has the prospect of large-scale promotion application.Technical scheme is as follows.
A kind of making of Liquid Strain of Ganoderma Lucidum and detection method, comprise the steps: a volume fraction be 75% medical
Alcohol disinfecting test tube, the parent species preparing pure medium is loaded and sterilizes again after test tube, cut above alcohol burner flame
0.3cm2Mycelia is put in test tube and is stoppered test tube mouth, cultivates to obtain purification of ganoderma lucidum parent species under optimum conditions；B prepares liquid culture
Base sterilization, inoculation lucid ganoderma stock culture after purification, shaking table is cultivated；C disappears to fermentation tank and filtration system
Poison；D prepares fermentation tank culture medium sterilization；The Liquid Strain of Ganoderma Lucidum that step b makes is inoculated in fermentation tank culture medium e
Cultivated.
In such scheme, in step a, every 1l parent species pure medium formula includes Semen Tritici aestivi or bean sprout 50g, Rhizoma Solani tuber osi 200g, Portugal
Grape sugar 20g, peptone 2g, agar 18-20g, kh2po41.5g and mgso4Semen Tritici aestivi is boiled during preparation or soil by 0.75g first
Bean boils shortcake, and bean sprout and agar are shredded, and adds distilled water constant volume, conventional sterilant 40min with other dispensings after mixing.
In such scheme, in step b every 1l liquid culture based formulas include Rhizoma Solani tuber osi 200g, Semen Maydis powder 50g, glucose 30g,
Peptone 2g, kh2po41g、mgso40.5g, or Rhizoma Solani tuber osi 200g, Semen Glyciness 5g, glucose 30g, kh2po41.5g、mgso4
0.5g, or Rhizoma Solani tuber osi 200g, defatted soya milk 8g, glucose 30g, kh2po41g、mgso40.5g.
In such scheme, step b specifically includes following steps: the fluid medium preparing is distributed into shaking flask, will shake
Autoclave put into by bottle, is incubated 40min, then naturally cools to room temperature under 121 DEG C of steam, will be pure in inoculating hood after sterilization
Lucid ganoderma stock culture after change is inoculated in shaking flask, and postvaccinal shaker flask stand is placed on stirring training on 23-25 DEG C of shaking table after cultivating 4 days
Support.
In such scheme, the sterilization of step c, particularly as follows: closing bottom valve of fermentation tank, adds water to total measurement (volume) into fermentation tank
70%, close fermentation tank air intake valve, turn on the power switch, temperature be set to 121-125 DEG C, after reaching design temperature, close
Close air total valve, open fermentation tank steam valve, allow steam enter filter, the drain tap of filter bottom is opened,
Discharge part steam, sterilizes to filtration system.
In such scheme, in step d, fermentation tank culture medium includes the raw material of the following meter of fraction by weight: 0.5% starch,
0.8% Semen Glyciness, 1% glucose, 2% sucrose, 0.15% kh2po4And 0.05% mgso4Or 0.5% glucose, 2%
Sucrose, 0.1% yeast extract, 0.1% peptone, 0.15% kh2po4And 0.05% mgso4, surplus is distilled water.
In such scheme, step e Liquid Strain of Ganoderma Lucidum is cultivated under the conditions of 26-28 DEG C, 0.03-0.04mpa.
Ganderma lucidum strain cultured in fermentation tank is specially inoculated into by a kind of detection method of Liquid Strain of Ganoderma Lucidum respectively
3 pieces of solid culture primary surfaces, are cultivated at 20-25 DEG C, are all turned white with 3 media surface after 10h, after 24h, mycelia starts to eat
Material, after 48h, material feeding depth is more than 0.2cm, and after 72h, no living contaminantses are the standard using.
Described solid culture based formulas are calculated as according to mass fraction: corncob cellulose 40%, Testa Tritici 30%, wood flour 20%, cotton seed hullss
8.5%, glucose 1.5%.
The inventive method has the advantages that (1) adopts the Liquid Strain of Ganoderma Lucidum cell age of the inventive method culture relatively
For consistent, mycelia permeability is strong, growth is vibrant；(2) life of Liquid Strain of Ganoderma Lucidum can be greatly shortened using the inventive method
The product cycle was to 5-7 days, and adopted existing solid state cultivation strain technology to need 30 days about；(3) Ganderma lucidum that the present invention produces
Kind sprout early, material feeding fast, more ripe than solid spawn shifts to an earlier date 10 ~ 20d；(4) liquid spawn has mobility, each mycelium pellet and mycelia
Segment can be scattered in different position rudiments, develops many, inside and outside long together up and down, the 6 ~ 12h mycelium germination of point, and 15 ~ 20d can grow
Full cultivating bag；(5) liquid spawn yield height, raw material are cheap, and the cost of comprehensively measuring and calculating liquid spawn is less than solid spawn cost
1/3, eliminate bottling, choose and abandon pollution bottle, inoculate, dig numerous and diverse work such as bottle, save a large amount of labours, energy consumption and space.
Specific embodiment
For making those skilled in the art fully understand technical solution of the present invention and beneficial effect, below in conjunction with specific embodiment
Absolutely proved.
The raw material of culture medium of the present invention all by conventional route from market purchasing, the test tube being used, inoculating hood, shaking flask,
Shaking table and fermentation tank etc. are commonly commercially available.
The first step: lucid ganoderma stock culture purification.
The formula of parent species pure medium has a following two kinds: formula a:1l parent species pure medium formula include Semen Tritici aestivi 50g,
Rhizoma Solani tuber osi 200g, glucose 20g, peptone 2g, agar 18-20g, kh2po41.5g and mgso40.75g.Formula b:1l parent species are pure
Change culture medium prescription and include bean sprout 50g, Rhizoma Solani tuber osi 200g, glucose 20g, peptone 2g, agar 18-20g, kh2po41.5g and
mgso40.75g.
First Semen Tritici aestivi is boiled Rhizoma Solani tuber osi during preparation and boil shortcake, bean sprout and agar shred, and add distilled water together with other dispensings
Middle constant volume, conventional sterilant 40min.Lucid ganoderma stock culture and test tube are put into sterilizing 20min in inoculating hood.It is 75% with volume fraction
Medical alcohol cleans sterilized test tube, the parent species preparing pure medium is loaded and sterilizes again after test tube, in alcohol burner flame
Top cuts 0.3cm2Mycelia is put in test tube and is stoppered test tube mouth, cultivates to obtain purification of ganoderma lucidum parent species under optimum conditions.
Second step: lucid ganoderma stock culture shake-flask culture after purification.
Fluid medium during shaking flask has three kinds of different formula, calculates its component according to 1l liquid culture as follows:
Formula c: Rhizoma Solani tuber osi 200g, Semen Maydis powder 50g, glucose 30g, peptone 2g, kh2po41g、mgso40.5g.
Formula d: Rhizoma Solani tuber osi 200g, Semen Glyciness 5g, glucose 30g, kh2po41.5g、mgso40.5g.
Formula e: Rhizoma Solani tuber osi 200g, defatted soya milk 8g, glucose 30g, kh2po41g、mgso40.5g.
The manufacture method of fluid medium is similar to parent species pure medium manufacture method.By the fluid medium preparing
It is distributed in shaking flask, is put in autoclave and seals pot mouth and open vent valve, close vent valve, pot interior temperature when a large amount of steam are discharged
Degree is incubated 40min after rising to 121 DEG C, makes it naturally cool to room temperature afterwards, is dried tampon using preheating.By shaking flask and purification
Parent species afterwards put into inoculating hood together, remove moistureproof paper and back-out tampon after sterilizing, carry out inoculation operation above alcohol burner flame.
Picking 0.3cm2Truffle rapidly by mycelia access shaking flask in, cover tampon at once, make mycelia be evenly distributed on media surface.
Then shaking flask is placed under preference temperature and rests 4 days about, backlight does not observe and during any miscellaneous bacteria, shaking flask is placed in 23-25 DEG C and shakes
Stir culture on bed.
3rd step: fermentation tank and filtration system sterilization.
Close bottom valve of fermentation tank, add water to the 70% of total measurement (volume) into fermentation tank, close fermentation tank air intake valve, beat
Switch power supply switchs, and temperature is set to 121 DEG C -- and 125 DEG C, after reaching design temperature, close air total valve, open fermentation tank
Steam valve, allows steam enter filter, the drain tap of filter bottom is opened, discharge part steam, to filtration system
Sterilized.
4th step: fermentor cultivation.
The formula of fermentation tank culture medium also has two kinds, the starch of formula f:0.5%, 0.8% Semen Glyciness, 1% glucose, 2%
Sucrose, 0.15% kh2po4And 0.05% mgso4, the glucose of formula g:0.5%, 2% sucrose, 0.1% yeast extract,
0.1% peptone, 0.15% kh2po4And 0.05% mgso4.Above formula is fraction meter by weight, and surplus is
Distilled water.The compound method of fermentation tank culture medium is similar with aforementioned.After the completion of fermentation tank culture medium is prepared, second step is made
Liquid Strain of Ganoderma Lucidum is inoculated in fermentation tank culture medium and is cultivated.Pressure in fermentation tank needs stability contorting in 0.03-
0.04mpa, temperature controller temperature is set in 26-28 DEG C.
Fermentor cultivation liquid spawn out also needs to be detected, detection method is as follows before use: will cultivate
Liquid spawn be inoculated into 3 pieces of solid culture primary surfaces respectively, at 20-25 DEG C cultivate, after 10h, 3 media surface are all sent out
In vain, after 24h, mycelia all starts material feeding, and after 48h, material feeding depth is all higher than 0.2cm, and after 72h, no living contaminantses can use.Described
Solid culture based formulas are calculated as according to mass fraction: corncob cellulose 40%, Testa Tritici 30%, wood flour 20%, cotton seed hullss 8.5%, glucose
1.5%, each raw material of culture medium using before need to pulverize mixing.Need to carry out Bacteria Detection in whole incubation to grasp bacterium at any time
The situation of kind, Bacteria Detection is specific as follows: aseptically accesses sample liquid equipped with the test tube of culture medium, at 28-30 DEG C
If no any abnormal phenomena after culture 24h, meet the requirements.
Embodiment 1.
According to parent species pure medium described in formula a, purification is carried out to lucid ganoderma stock culture, using fluid medium described in formula c
Shaking table is cultivated to purification parent species, according to formula f, fermentor cultivation is carried out to the liquid spawn of previous step and obtain lucidum liquid
Body strain, qualified after testing after can use.
Embodiment 2.
The parent species pure medium of embodiment 1 is replaced by formula b, changing liquid cultivation matrix is formula d, by fermentation tank
Culture medium is replaced by formula g, and other conditions are constant.
Embodiment 3.
The parent species pure medium of embodiment 1 is replaced by formula b, changing liquid cultivation matrix is formula e, other conditions
Constant.
Embodiment 1-3 averagely only needs 5-7 days to prepare the Liquid Strain of Ganoderma Lucidum that cell age is consistent, permeability is strong, only sends out
The bacterium time just decreases more than 1/2 than solid spawn, and the production cycle of strain averagely shortens 15-20 days.Using system of the present invention
When standby liquid spawn comes amplification culture original seed or cultigen as parent species or original seed, more faster than using solid spawn.Control
In the case of other conditions identical, it is ripe that general liquid spawn shifts to an earlier date 10 ~ 20d than solid kind.Further, since liquid spawn has stream
The position rudiment that dynamic property, each mycelium pellet and mycelia segment can be scattered different, growth point is many, inside and outside long together up and down, 6 ~
12h mycelia can sprout, and 15 ~ 20d can cover with cultivating bag.Calculate by analysis, liquid bacteria is produced using triangular flask or fermentation tank
Kind of cost less than the 1/3 of solid spawn, simultaneously because with short production cycle, do not use seed bottle, eliminate bottling, choose and abandon pollution
Bottle, inoculate, dig numerous and diverse technique such as bottle, save a large amount of labours, energy consumption and space.The inventive method is applied in Xiaogan City
Technical research institute tests successfully, achieves preferable economic and social benefit, has preferable application and popularization value.
Claims (9)
1. a kind of manufacture method of Liquid Strain of Ganoderma Lucidum is it is characterised in that comprise the steps: the doctor that a volume fraction is 75%
Disinfect test tube in alcohol, the parent species preparing pure medium is loaded and sterilizes again after test tube, cut above alcohol burner flame
Take 0.3cm2Mycelia put in test tube and be stoppered test tube mouth, cultivate to obtain purification of ganoderma lucidum parent species under optimum conditions；B prepares liquid
Culture medium sterilization, inoculation lucid ganoderma stock culture after purification, shaking table is cultivated；C enters to fermentation tank and filtration system
Row sterilization；D prepares fermentation tank culture medium sterilization；The Liquid Strain of Ganoderma Lucidum that step b makes is inoculated into fermentor cultivation by e
Cultivated in base.
2. Liquid Strain of Ganoderma Lucidum as claimed in claim 1 manufacture method it is characterised in that: every 1l parent species purification in step a
Culture medium prescription includes Semen Tritici aestivi or bean sprout 50g, Rhizoma Solani tuber osi 200g, glucose 20g, peptone 2g, agar 18-20g, kh2po41.5g
And mgso4Semen Tritici aestivi is boiled during preparation or Rhizoma Solani tuber osi is boiled shortcake, bean sprout and agar shredded, is mixed with other dispensings by 0.75g first
Add distilled water constant volume, conventional sterilant 40min afterwards.
3. Liquid Strain of Ganoderma Lucidum as claimed in claim 1 manufacture method it is characterised in that: every 1l liquid culture in step b
Based formulas include Rhizoma Solani tuber osi 200g, Semen Maydis powder 50g, glucose 30g, peptone 2g, kh2po41g、mgso40.5g, or Rhizoma Solani tuber osi
200g, Semen Glyciness 5g, glucose 30g, kh2po41.5g、mgso40.5g, or Rhizoma Solani tuber osi 200g, defatted soya milk 8g, glucose
30g、kh2po41g、mgso40.5g.
4. the manufacture method of Liquid Strain of Ganoderma Lucidum as claimed in claim 1 is it is characterised in that step b specifically includes following step
Rapid: the fluid medium preparing to be distributed into shaking flask, shaking flask is put into autoclave, be incubated 40min under 121 DEG C of steam, connect
And naturally cool to room temperature, in inoculating hood after sterilization, the lucid ganoderma stock culture of purification is inoculated in shaking flask, postvaccinal shaking flask
Quiescent culture is placed on stir culture on 23-25 DEG C of shaking table after 4 days.
5. Liquid Strain of Ganoderma Lucidum as claimed in claim 1 manufacture method it is characterised in that: step c sterilization particularly as follows: close
Bottom valve of fermentation tank, adds water to the 70% of total measurement (volume) into fermentation tank, closes fermentation tank air intake valve, turns on the power switch, will
Temperature is set to 121-125 DEG C, after reaching design temperature, closes air total valve, opens fermentation tank steam valve, allow steam to enter
Enter filter, the drain tap of filter bottom is opened, discharge part steam, filtration system is sterilized.
6. Liquid Strain of Ganoderma Lucidum as claimed in claim 1 manufacture method it is characterised in that: fermentation tank culture medium in step d
Raw material including following fraction meter by weight: 0.5% starch, 0.8% Semen Glyciness, 1% glucose, 2% sucrose, 0.15%
kh2po4And 0.05% mgso4, or 0.5% glucose, 2% sucrose, 0.1% yeast extract, 0.1% peptone,
0.15% kh2po4And 0.05% mgso4, surplus is distilled water.
7. Liquid Strain of Ganoderma Lucidum as claimed in claim 1 manufacture method it is characterised in that: step e Liquid Strain of Ganoderma Lucidum exists
26-28 DEG C, cultivated under the conditions of 0.03-0.04mpa.
8. a kind of detection method of Liquid Strain of Ganoderma Lucidum is it is characterised in that comprise the following steps: by spirit cultured in fermentation tank
Sesame strain is inoculated into 3 pieces of solid culture primary surfaces respectively, cultivates, all turned white with 3 media surface after 10h at 20-25 DEG C,
After 24h, mycelia starts material feeding, and after 48h, material feeding depth is more than 0.2cm, and after 72h, no living contaminantses are the standard using.
9. Liquid Strain of Ganoderma Lucidum as claimed in claim 8 detection method it is characterised in that: described solid culture based formulas are pressed
It is calculated as according to mass fraction: corncob cellulose 40%, Testa Tritici 30%, wood flour 20%, cotton seed hullss 8.5%, glucose 1.5%.
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