CN105624200B - A kind of recoverying and utilizing method of Hirsutella sinensis ferment filtrate - Google Patents
A kind of recoverying and utilizing method of Hirsutella sinensis ferment filtrate Download PDFInfo
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- CN105624200B CN105624200B CN201610082312.0A CN201610082312A CN105624200B CN 105624200 B CN105624200 B CN 105624200B CN 201610082312 A CN201610082312 A CN 201610082312A CN 105624200 B CN105624200 B CN 105624200B
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- water
- hirsutella sinensis
- ferment filtrate
- filtrate
- electrodialysis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
Abstract
The present invention provides a kind of recoverying and utilizing methods of Hirsutella sinensis ferment filtrate, the method are as follows: the filtrate after the filtering fermentation liquor obtained with the fermented culture of Hirsutella sinensis is water inlet, using homogeneous ion-exchange membrane, electrodialysis process is carried out under conditions of flow of inlet water is 200-400L/h, working voltage is 20-30V, collect electrodialysis efflux, as Hirsutella sinensis ferment filtrate recycle-water recycles；The recycling is that Hirsutella sinensis ferment filtrate recycle-water is used for preparing microorganism culture medium；The selected electrodialysis plant of the present invention and parameter are not only cheap compared with import, and under corresponding parameter setting salt rejection rate up to 95% or more.After electrodialysis, the yield of water is about 90%.1000L ferment filtrate is about 1.88 yuan per ton with electroosmose process processing energy consumption cost, and about 0.5 yuan of equipment cost per ton.Therefore, the production cost of enterprise can be substantially reduced in culture medium water by being handled ferment filtrate using electroosmose process and being recycled.
Description
(1) technical field
The present invention relates to the recoverying and utilizing method of the ferment filtrate of cordyceps sinensis production bacterium Hirsutella sinensis and electrodialysis
The cycle applications of filtrate afterwards.
(2) background technique
Cordyceps sinensis (Cordyceps sinensis (Berk.) Sacc.) is that aweto colonizes in Lepidoptera
(Lepidoptera) stroma and larva corpse on Hepialidae insect (Hepialus armoricanus Oberthur) larva
Complex (including stroma and polypide) on body.Cordyceps sinensis is a kind of precious traditional fungi herb resource, and there is metabolism to produce
The characteristics of object and diverse biological activities, shows huge application and development prospect in biomedicine field.
Last century the eighties, the Zhongmei Huadong Pharmaceutical Co., Ltd. Hangzhou of one of the present inventor complete artificial production winter worm
The producing strains Hirsutella sinensis of cordyceps sinensis is carried out microbial liquid fermented and cultured, and utilizes mycelia by the research of summer grass
Drug is made through drying in body --- and " hundred enable " capsule (authentication code: national drug standard Z10910036) can be substituted partly wild
The function of cordyceps sinensis, clinically using extensively and obtaining the curative effect of affirmative.
It is in the field of business, it is contemplated that production cost, to the ferment filtrate of Hirsutella sinensis and wherein, effective component is not ground
Study carefully, ferment filtrate is generally treated as wastewater treatment.Ring is be easy to cause due to containing a small amount of heavy metal ion etc. in ferment filtrate
The substance of border pollution, if directly when discharge of wastewater can cause certain harm to environment, it is therefore necessary to reach by wastewater treatment
Direct emission after industry wastewater discharge standard.Wastewater treatment will generate high expense, handle one ton of Hirsutella sinensis at present
Ferment filtrate, only energy consumption cost is just more than 5 yuan, and the industrial cost of 1 ton of tap water is also above 6 yuan；Fermentation filter is wasted simultaneously
The a large amount of useful resources contained in liquid, such as microelement.
The processing method of ferment filtrate generally has physical treatment process, method of chemical treatment and biological treatment.Physical treatment process
Using the methods of precipitating, filtering, centrifuge separation, air bearing, evaporative crystallization, reverse osmosis, method of chemical treatment has neutralization, coagulation, oxidation
Reduction, extraction, stripping, stripping, absorption, ion exchange and electro-osmosis etc., and the active sludge of biological treatment, biological filter
Pond and oxidation pond etc., physics and method of chemical treatment have the shortcomings that complex steps and at high cost, using biological treatment not only at
This is low and environmentally friendly.
Electrodialysis belongs to physical method, is generally used for desalination, material concentration of seawater etc..Industrially utilize electrodialysis desalination
Principle handle simultaneously reuse to production waste water.Yu-Mei Chao etc. to electrodialytic technique for industrial effluent reusing handle into
Technology and feasibility study (Chao Y M, Liang T M.A feasibility study of economically are gone
industrial wastewater recovery using electrodialysis reversal[J]
.Desalination.2008,221(1):433–439.)(ref).T.Sirivedhin etc. utilizes electrodialysis removing production waste water
In salinity, try hard to reach drinking water, irrigation water and livestock drinking water standard (Sirivedhin T, Mccue J,
Dallbauman L.Reclaiming produced water for beneficial use:salt removal by
electrodialysis[J].J.Membrane.Sci.2004,243(s 1–2):335-343.).But never document report
Lead the recycling (ref) that ferment filtrate is carried out using electrodialytic technique.
It is well known that some fermented products are included in ferment filtrate, and some fermented products are then included in thallus, and
And the ferment filtrate after filtering off degerming filament might not contain a certain amount of active constituent, be more containing harmful
Secondary metabolite not necessarily has the value recycled.Therefore pharmacy corporation is all directly worked as ferment filtrate at present
It is handled at waste water.In addition, the ferment filtrate before electrodialysis, which pre-processes (filtering etc.), needs to spend certain expense, and concentration
The reason of difference is easy to make electrodialysis plant fouling and occurs reverse osmosis, causes desalting efficiency undesirable, therefore, electrodialysis seldom quilt
Pharmacy corporation is utilized.
(3) summary of the invention
Present invention aims at for the deficiency present on, a kind of " hundred enable " production bacterium cordyceps sinensis China coat is provided
The recycling method of spore submerged fermentation filtrate.
The present invention provides a kind of recoverying and utilizing method of Hirsutella sinensis ferment filtrate, the methods are as follows: with Chinese quilt
Filtrate after the filtering fermentation liquor that the fermented culture of hair spore obtains is water inlet, using homogeneous ion-exchange membrane, is in flow of inlet water
200-400L/h, working voltage carry out electrodialysis process under conditions of being 20-30V, collect electrodialysis efflux, as Chinese quilt
Hair spore ferment filtrate recycle-water, recycles.
The wherein electrodialysis process, specifically, film used is amberplex, preferably homogeneous ion in electrodialysis
Exchange membrane, more preferable polyethylene homogeneous ion-exchange membrane can use domestic film.If selecting other films, as different-phase ion exchanges
Film, salt rejection rate are lower.Such as the I type heterogeneous ion-exchange membrane for selecting Zhejiang Saite membrane technology Co., Ltd to provide, feed liquid flow
When for 300L/h, working voltage being 30V, the processing time is about 30 minutes, salt rejection rate 85%.Select Zhejiang Saite membrane technology
The II type heterogeneous ion-exchange membrane that Co., Ltd provides, when feed liquid flow 400L/h, working voltage are 30V, the processing time is about
20 minutes, salt rejection rate 70%.
The wherein electrodialysis condition are as follows: flow of inlet water 200-400L/h, working voltage 20-30V, processing time
It is 18-35 minutes.It is preferred that the processing time is about 20 minutes when flow of inlet water 300L/h, working voltage are 30V.
Further, described recycle is that Hirsutella sinensis ferment filtrate recycle-water is used for preparing microorganism culture medium,
Substitute distilled water, tap water；The microorganism is fungi, bacterium or actinomyces, and the preferably described microorganism is Hirsutella sinensis
In L0106, e. coli jm109, e. coli bl21, pichia pastoris X-33, Pichia pastoris GS115 or actinoplanes utahensis
One kind.
Hirsutella sinensis ferment filtrate of the present invention, can be obtained with prior art preparation.Such as: using Chinese quilt
Hair spore (Hirsutella sinensis) L0106, is preserved in China typical culture collection center, address: China, and Wuhan is military
Chinese university, 430072, deposit number CCTCC No:M 2011278, preservation date on August 12nd, 2011.By Hirsutella sinensis
L0106 liquid medium within top fermentation obtains mycelium, and the hair containing fermented supernatant fluid and one of the various metabolites
Ferment product；By tunning by being separated by solid-liquid separation, such as using the methods of plate-frame filtering, fermentation filter is obtained after isolating mycelium
Liquid.This filtrate can later use electrodialysis plant progress desalting processing.The fermented cultural method of Hirsutella sinensis are as follows: will
Hirsutella sinensis L0106 is inoculated in slant medium, and inclined-plane culture is carried out under the conditions of 12~16 DEG C, then inclined-plane bacterium is taken to be inoculated in
In seed culture medium, seed culture is carried out under the conditions of 15-18 DEG C, and seed liquor is then taken to connect by the inoculum concentration of volumetric concentration 10%
Enter fermentation medium, fermented and cultured is carried out under the conditions of 15-18 DEG C and obtains fermentation liquid；The slant medium quality composition
Are as follows: (murphy juice refers to 200g/L potato by glucose 1.8-2.2%, corn flour 0.8-1.2%, murphy juice 0.4-0.6%
Be brewed into paste liquid with water, the filtrate after eight layers of filtered through gauze), dextrin 0.4-0.6%, yeast powder 0.4-0.6%, wheat bran 0.8-
1.2%, dried silkworm chrysalis meal 1.8-2.2%, peptone 0.8-1.2%, magnesium sulfate 0.04-0.06%, potassium dihydrogen phosphate 0.04-
0.06%, agar powder 0.8-1.2%, solvent are water, and pH value is naturally, it is preferred that slant medium quality group becomes: glucose
2.0%, corn flour 1.0%, murphy juice 0.5%, dextrin 0.5%, yeast powder 0.5%, wheat bran 1.0%, dried silkworm chrysalis meal 2.0%, egg
White peptone 1.0%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.05%, agar powder 1.0%, solvent are water, and pH value is natural；Described
Seed culture medium and fermentation medium are fluid nutrient medium, and the fluid nutrient medium quality group becomes: glucose 0.8-
1.2%, molasses 0.8-1.2%, dried silkworm chrysalis meal 0.4-0.6%, soybean cake powder 0.8-1.2%, yeast extract 0.4-0.6%, magnesium sulfate
0.01-0.015%, potassium dihydrogen phosphate 0.015-0.025%, solvent are water, and pH value is natural；It is preferred that the Liquid Culture matrix
Amount composition are as follows: glucose 1.0%, molasses 1.0%, dried silkworm chrysalis meal 0.5%, soybean cake powder 1.0%, yeast extract 0.5%, magnesium sulfate
0.01%, potassium dihydrogen phosphate 0.02%, solvent are water, and pH value is natural.
The specific fermented cultural method of Hirsutella sinensis are as follows: Hirsutella sinensis L0106 is inoculated in slant medium,
It is cultivated 25 days at 12~16 DEG C, obtains inclined-plane thalline；Slant medium quality group becomes: glucose 1.8-2.2%, corn flour
0.8-1.2%, murphy juice 0.4-0.6%, dextrin 0.4-0.6%, yeast powder 0.4-0.6%, wheat bran 0.8-1.2%, dried silkworm chrysalis meal
1.8-2.2%, peptone 0.8-1.2%, magnesium sulfate 0.04-0.06%, potassium dihydrogen phosphate 0.04-0.06%, agar powder 0.8-
1.2%, solvent is water, and pH value is naturally, it is preferred that inclined-plane culture liquid quality group becomes: glucose 2.0%, corn flour 1.0%, potato
Juice 0.5%, dextrin 0.5%, yeast powder 0.5%, wheat bran 1.0%, dried silkworm chrysalis meal 2.0%, peptone 1.0%, magnesium sulfate 0.05%,
Potassium dihydrogen phosphate 0.05%, agar powder 1.0%, solvent are water, and pH value is natural；Then inclined-plane thalline is seeded to Liquid Culture
Base shaken cultivation 30 days, obtains seed liquor under the conditions of 100rmp, 15-18 DEG C；Seed liquor is vibrated broken mycelium to be made
Seed suspension liquid cultivates 5- then by the inoculum concentration access first order seed fermentor of volumetric concentration 10% under the conditions of 15-18 DEG C
15 days, then it is pressed into second order fermentation tank by the inoculum concentration of volumetric concentration 10% by first class seed pot, 5-15 is cultivated under the conditions of 15-18 DEG C
Tank is put after being pressed into the culture of three grade fermemtation tank 40 days by the inoculum concentration of volumetric concentration 10% again after it, is obtained tunning, will be fermented
Product separation, obtains filtrate；The first order seed fermentor, second order fermentation tank in three grade fermemtation tank are fluid nutrient medium,
Fluid nutrient medium quality group becomes: glucose 0.8-1.2%, molasses 0.8-1.2%, dried silkworm chrysalis meal 0.4-0.6%, soybean cake powder
0.8-1.2%, yeast extract 0.4-0.6%, magnesium sulfate 0.01-0.015%, potassium dihydrogen phosphate 0.015-0.025%, solvent are
Water, pH value is naturally, preferred liquid culture medium quality forms are as follows: glucose 1.0%, molasses 1.0%, dried silkworm chrysalis meal 0.5%, soyabean cake
Powder 1.0%, yeast extract 0.5%, magnesium sulfate 0.01%, potassium dihydrogen phosphate 0.02%, solvent are water, and pH value is natural；The fermentation
Liquid separating method is to be filtered using plate and frame filter press or filtered using head pressure, collects filtrate.It is described to utilize head pressure mistake
Filter is squeezed into fermentation liquid in high-order storage tank, using potential difference pressure and by control valve by fermentation liquid with 10 ton/hours
Speed squeezes into rotary drum, is separated by solid-liquid separation fermentation liquid using rotary drum, obtains filtrate.
The further high speed centrifugation of filtrate that the present invention can also obtain Hirsutella sinensis fermentation, some of which is miscellaneous
The removal of matter particle, the Active Components for next step；Fractions of active ingredient needs to need by efficient liquid phase chromatographic analysis
It is carried out after being separated by filtration again using the film of 0.25 μ m diameter.
It is well known that some fermented products are included in ferment filtrate, and some fermented products are then included in thallus, and
And the ferment filtrate after filtering off degerming filament might not contain a certain amount of active constituent, be more containing harmful
Secondary metabolite not necessarily has the value recycled.Such as the fermentation being separated by solid-liquid separation after culturing gene engineering bacteria
Filtrate, wherein containing harmful substances such as a large amount of antibiotic, since these antibiotic are difficult from ferment filtrate effectively to divide
From, if recycled to these ferment filtrates, the antibiotic concentration in ferment filtrate can be made to accumulate, increase genetic engineering
The antibiotic-resistant activity of bacterium, to have the risk of Resident Evil.
The present invention has carried out composition detection analysis by the ferment filtrate to Hirsutella sinensis, and determination has wherein remained one
Point available active constituent, but the content of these active constituents is lower, one by one the cost of these active constituents of separation and Extraction compared with
Height, but it is very suitable as the accumulation that culture medium water promotes thallus.
Using the Hirsutella sinensis ferment filtrate after electrodialysis process as culture medium water, LB culture medium, investigation pair are prepared
The influence of e. coli jm109 and e. coli bl21 growth (referring to Fig. 3 and Fig. 4)；With the quilt containing China after electrodialysis process
Hair spore ferment filtrate prepares YPD culture medium as culture medium water, investigates raw to pichia pastoris X-33 and Pichia pastoris GS115
Long influence (referring to figs. 5 and 6)；, as culture medium water, to match containing the Hirsutella sinensis ferment filtrate after electrodialysis process
Hirsutella sinensis and acarbose producing strains actinoplanes utahensis liquid fermentation medium processed, investigation is to Hirsutella sinensis and still
The influence (referring to figs. 7 and 8) of his actinoplanes fermentation.Since Escherichia coli are mode bacterium, Pichia pastoris is that mode is true
Bacterium, the present invention also can be shown that the ferment filtrate after electrodialysis process can be used for the culture of most of bacterium and fungi.
Specifically, the application carries out as follows:
1, as culture medium water, to prepare LB culture medium, training containing the Hirsutella sinensis ferment filtrate after electrodialysis process
Supporting based component is peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, pH 7.0, and investigation is to e. coli jm109 and greatly
The influence of enterobacteria BL21 growth.
2, as culture medium water, to prepare YPD culture medium, training containing the Hirsutella sinensis ferment filtrate after electrodialysis process
Supporting based component is glucose 20g/L, peptone 10g/L, yeast powder 5g/L, pH 7.0, investigates to pichia pastoris X-33 and finishes red
The influence of yeast GS115 growth.
3, as culture medium water, to prepare Chinese quilt containing the Hirsutella sinensis ferment filtrate recycle-water after electrodialysis process
Hair spore liquid fermentation medium, culture medium prescription be glucose 0.8-1.2%, molasses 0.8-1.2%, dried silkworm chrysalis meal 0.4-0.6%,
Soybean cake powder 0.8-1.2%, yeast extract 0.4-0.6%, magnesium sulfate 0.01-0.015%, potassium dihydrogen phosphate 0.015-0.025%,
PH value naturally, it is preferred that are as follows: glucose 1.5%, corn flour 1.0%, dextrin 0.5%, wheat bran 1.5%, yeast powder 0.5%, silkworm chrysalis
Powder 1.5%, peptone 1.0%, MgSO4, 0.01%, KH2PO40.02%, pH value is natural.During preparation, first by silkworm chrysalis
Powder, corn flour and wheat bran are added in Hirsutella sinensis ferment filtrate appropriate and liquefy, and sterilizing takes supernatant, is then redissolved it
His nutrient media components, then sterilize, with the inoculum concentration of volumetric concentration 10%, investigate the influence fermented to Hirsutella sinensis.
4, as culture medium water, to prepare Utah trip containing the Hirsutella sinensis ferment filtrate recycle-water after electrodialysis process
Dynamic actinomycete fermentation culture medium, culture medium prescription are as follows: maltose 43-46g/L, glucose 39-41g/L, soybean cake powder 16-18g/
L, monosodium glutamate 2.5-3.5g/L, CaCO32.4-2.6g/L, CaCl22.4-2.6g/L, FeCl30.4-0.6g/L,
K2HPO40.9-1.1g/L, glycerol 0.9-1.1g/L, pH7.5, pH value naturally, it is preferred that are as follows: maltose 45g/L, glucose 40g/L,
Soybean cake powder 17g/L, monosodium glutamate 3g/L, CaCO32.5g/L, CaCl22.5g/L, FeCl30.5g/L, K2HPO41.0g/L
Glycerol 1.0g/L, pH7.5, pH value are boiled naturally, each component is miscible in suitable Hirsutella sinensis ferment filtrate and distilled water
It uniformly dispenses after boiling into the triangle shake bottle of 500mL, every bottle of packing 50mL, it is stand-by after the 30min that sterilizes under the conditions of 115 DEG C.With
10% inoculum concentration investigates the influence fermented to acarbose producing strains A.utahensis ZJB-08196.
The beneficial effects are mainly reflected as follows: from principle to the effective active in Hirsutella sinensis ferment filtrate at
It point is studied in detail, provides " hundred enable " and produce containing for effective component in bacterium cordyceps sinensis Hirsutella sinensis ferment filtrate
Amount removes micro harmful mineral element therein and certain density metal ion by electrodialytic method, assigns China
The circulation utilizability of coat spore ferment filtrate, and the culture medium water for cultivating other microorganisms, to expand Chinese quilt
The biologic applications of hair spore ferment filtrate provide effective way.Water resource effectively has been saved, effluent pressure has been reduced, reduces
The possibility of pollution, it is environmentally protective.The microbial fermentation carried out with ferment filtrate substitutive medium with water, fermentation unit are better than
Distilled water or tap water.It not only can solve cost caused by subsequent wastewater treatment, water used in culture medium can also be saved
Cost.The selected electrodialysis plant of the present invention and parameter are not only cheap compared with import, but also under corresponding parameter setting
Salt rejection rate is up to 95% or more.After electrodialysis, the yield of water is about 90%.1000L ferment filtrate, is handled with electroosmose process
Energy consumption cost is about 1.88 yuan per ton, and about 0.5 yuan of equipment cost per ton.Therefore, simultaneously using electroosmose process processing ferment filtrate
The production cost of enterprise can be substantially reduced in culture medium water by recycling.
The present invention develops and utilizes Hirsutella sinensis ferment filtrate, and the effective use and environmental protection to resource will have
Very positive meaning.
(4) Detailed description of the invention
Fig. 1 is electrodialysis plant and working principle diagram；
Fig. 2 is electric current and the variation diagram of conductivity during electrodialysis process；
Fig. 3 is that Hirsutella sinensis ferment filtrate recycle-water and distilled water are raw as the e. coli jm109 of culture medium water
Long curve；
Fig. 4 is that Hirsutella sinensis ferment filtrate recycle-water and distilled water are grown as the e. coli bl21 of culture medium water
Curve；
Fig. 5 is that Hirsutella sinensis ferment filtrate recycle-water and distilled water are grown as the pichia pastoris X-33 of culture medium water
Curve；
Fig. 6 is that Hirsutella sinensis ferment filtrate recycle-water and distilled water are raw as the Pichia pastoris GS115 of culture medium water
Long curve；
Fig. 7 is that Hirsutella sinensis ferment filtrate recycle-water and distilled water grow Hirsutella sinensis as culture medium water
It influences；
Fig. 8 is that Hirsutella sinensis ferment filtrate recycle-water and distilled water are raw to actinoplanes utahensis as culture medium water
Long influence.
(5) specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
Embodiment 1: the culture of " hundred enable " production bacterium cordyceps sinensis Hirsutella sinensis
Bacterium source: acquiring natural cordyceps from Qinghai first, and brings it back into Hangzhou and carry out separation screening, obtains
L0106 bacterial strain, and be Hirsutella sinensis (Hirsutella sinensis) through the strain idenfication bacterial strain, the culture presevation is in
State's Type Tissue Collection, deposit number is CCTCC No:M 2011278, in the patent previously applied
It is disclosed in CN102373190A.
The strain is inoculated in inclined-plane, (this is the liquid formulations before solidifying to culture medium prescription, is prepared in following ratios
Bevel again later) are as follows: glucose 2.0% (w/v contains 1g in 1% expression 100mL culture medium, similarly hereinafter), corn flour
1.0%, (murphy juice refers to is brewed into paste liquid with water with 200g/L potato to murphy juice 0.5%, after eight layers of filtered through gauze
Filtrate), dextrin 0.5%, yeast powder 0.5%, wheat bran 1.0%, dried silkworm chrysalis meal 2.0%, peptone 1.0%, magnesium sulfate 0.05%, phosphorus
Acid dihydride potassium 0.05%, agar powder 1.0%, solvent are water；It is cultivated 25 days at 12~16 DEG C；Then 4~5 are chosen from slant strains
Block soybean grain size contains mycelial culture medium, accesses 500ml triangle that is sterilized and filling 100ml fluid nutrient medium
In bottle, under the conditions of 15-18 DEG C, shaken cultivation 30 days (100rmp) on shaking table.By cultured shaking table strain, injection dress
It is acutely vibrated in the aseptic bottle for having small bead, seed suspension liquid is made to smash mycelium.Then dense by volume
In the inoculum concentration access first order seed fermentor of degree 10%, cultivated 10 days or so under the conditions of 100rmp, 15-18 DEG C, then by level-one
Seeding tank is pressed into second order fermentation tank by the inoculum concentration of volumetric concentration 10%, after cultivating 10 days or so under the conditions of 100rmp, 15-18 DEG C
Tank is put after being pressed into the culture of three grade fermemtation tank again 40 days, obtains fermentation liquid.The first order seed fermentor, second order fermentation tank, three
It is fluid nutrient medium in grade fermentor, fluid nutrient medium quality group becomes: glucose 1.0%, molasses 1.0%, dried silkworm chrysalis meal
0.5%, soybean cake powder 1.0%, yeast extract 0.5%, magnesium sulfate 0.01%, potassium dihydrogen phosphate 0.02%, solvent are water, and pH value is certainly
So.
Embodiment 2: the detection and analysis of active constituent in Hirsutella sinensis ferment filtrate
1, the preparation of Hirsutella sinensis ferment filtrate
After fermentation, fermentation liquid puts tank separation to embodiment 1, obtains hair of the invention after first separating using plate and frame filter press
Ferment filtrate.The ferment filtrate of acquisition is brown yellow transparent liquid.PH value is 6.0-7.0.
2, in ferment filtrate active constituent detection and analysis
Separated Hirsutella sinensis ferment filtrate 0.2mL is accurately drawn, ultrapure water is settled to 20mL, takes after mixing well
1mL potassium metaperiodate solution is added in clean tube in 1mL, and 25 DEG C of reaction 10min add 0.1wt%L- sandlwood syrup
4mL Fresh Nash solution is added in solution 2mL, shaken well, and 53 DEG C of colour developing 15min are rapidly cooled to room temperature.With ultrapure
Water does blank, and the reaction solution that is cooled to room temperature is in 96 hole elisa Plates after taking 200 μ L to develop the color, 3 multiple holes of every kind of reaction solution.?
It is scanned in the wave-length coverage of 380nm-450nm, determines maximum absorption wavelength.After testing, Hirsutella sinensis ferment filtrate
In cordycepic acid content be 3.16mg/mL.
Hirsutella sinensis ferment filtrate passes through 0.25 μm of micro-pore-film filtration, and filtrate uses high performance liquid chromatography detection.Condition
It is as follows, chromatographic column: Yi Lite C18 column (4.6mm × 250mm, 5 μm)；Column temperature: 35 DEG C；Sampling volume: 20 μ L；Flow velocity 1mL/
min；Detection wavelength 260nm；Mobile phase: A: ultrapure water；B: methanol.Gradient: 0min, 15%B keep 3min；3.0-
3.5min, 15-24%B；3.5min, 24% keeps 5min；8.5-9.0min, 24-35%B；35%B keeps 6min；15.0-
16.0min 35-85%B；85%B keeps 6min；22.0-22.5min 85%-15%B；15%B keeps 5min.By inspection
It surveys, the content of cordycepin is 0.56 μ g/ml in Hirsutella sinensis ferment filtrate, and the content of uridine is 16.4 μ g/ml, and guanosine contains
Amount is 7.9 μ g/ml, and the content of adenosine is 0.069 μ g/ml, and the content of thymidine is 4.0 μ g/ml.
The measurement of protein content uses Kjeldahl's method, after testing, Chinese coat in Hirsutella sinensis ferment filtrate
Crude protein content in spore ferment filtrate is 3.1mg/mL.3mL Hirsutella sinensis ferment filtrate is taken, the 95% (v/ of 4 times of volumes is added
V) ethanol water precipitating extracts polysaccharide, and 4 DEG C stand 1 hour, and 12000rpm is centrifuged 20min and removes supernatant, and removal as far as possible is dry
Only, distilled water and the concussion that 15mL is added into precipitating obtain Thick many candies solution so that precipitating is completely dissolved, for filter of fermenting
The measurement of exocellular polysaccharide in liquid.The measurement of exocellular polysaccharide is carried out using phend-sulphuric acid, through detecting, Hirsutella sinensis fermentation filter
Total sugar content in liquid is 11.2mg/mL, and the content of exocellular polysaccharide is 2.8mg/mL.
Through detecting, the aspartate content in Hirsutella sinensis ferment filtrate is 0.97 μ g/mL, threonine content 2.04
μ g/mL, serine content are 1.43 μ g/mL, and Glycine Levels are 1.55 μ g/mL, and alanine content is 1.075 μ g/mL, half Guang
Histidine content is 6.495 μ g/mL, and the content of isoleucine is 5.295 μ g/mL, and the content of leucine is 7.94 μ g/mL, phenylpropyl alcohol
The content of propylhomoserin is 74.255 μ g/mL, and the content of histidine is 90.855 μ g/mL, and the content of lysine is 28.58 μ g/mL, essence
The content of propylhomoserin is 24.69, and the total content of amino acid is 245.18 μ g/mL.
Hirsutella sinensis ferment filtrate and each metallic element titer distinguish sample introduction, Flame Atomic Absorption Spectrometry Determination its
Absorbance, through detecting, the content of Na element is 399.05 μ g/mL in Hirsutella sinensis ferment filtrate, and the content of K element is
The content of 172.02 μ g/mL, Mg elements is that the content that the content of 0.822 μ g/mL, Ca element is 41.22 μ g/mL, Fe elements is
The content of 1.15 μ g/mL, Zn elements is that the content that the content of 0.029 μ g/mL, Cu element is 0.014 μ g/mL, Ni element is
0.12 μ g/mL, does not have found Mn, Pb, Co element.
Through analyzing, cordycepic acid, nucleosides material, crude protein and amino acid isoreactivity in Hirsutella sinensis ferment filtrate at
Divide the growth to thallus not have to negatively affect or there is positive influence, and the mineral element accumulated in ferment filtrate may
It has a certain impact to the thallus osmotic pressure tool cultivated, the negative effect of the mineral element of accumulation need to be excluded.
Embodiment 3: the preparation of Hirsutella sinensis ferment filtrate
The cultured 78 tons of cordyceps sinensis Hirsutella sinensis fermentation liquid of 1 method of embodiment is squeezed into 6 tons of high-order storage tank,
Using the pressure of potential difference and fermentation liquid is squeezed into 10 ton/hours of speed by rotary drum by control valve, will be fermented using rotary drum
Liquid is separated by solid-liquid separation, and about 75 tons of ferment filtrates are obtained.During the experiment, it is de- that ferment filtrate stoste, active carbon are collected respectively
Centrifuge separation gained ferment filtrate and electrodialysis post-fermentation filtrate fresh water (recycle effect for comparing it, take distillation after color
Water is as experiment contrast).
Embodiment 4: the electrodialysis process of Hirsutella sinensis ferment filtrate
Electrodialysis plant (electrodialysis plant schematic diagram is as shown in Figure 1), film be polyethylene homogeneous ion-exchange membrane, anode membrane and
The model of cavity block is respectively LE-HeM-CM01 and LE-HeM-AM01, is produced by Zhejiang Qianqiu Environmental Water Treatment Co., Ltd., with
75 tons of ferment filtrates prepared by embodiment 3 are water inlet, optimal electrodialysis condition are as follows: feed liquid flow is 300L/h, working voltage is
When 30V, the processing time is about 20 minutes, and salt rejection rate is up to 95%；After processing, 75 tons of ferment filtrates can get the water 67 recycled
Ton (i.e. ferment filtrate recycle-water).If selecting other electrodialysis conditions, salt rejection rate and the water rate of recovery are lower.For example select material flow
When amount is 200L/h, working voltage is 20V, the processing time is about 80 minutes, salt rejection rate 92%；After processing, 75 tons of hairs
Ferment filtrate can get 64 tons of water recycled.When selection feed liquid flow 400L/h, working voltage are 30V, the processing time is about 18
Minute, salt rejection rate 88%；After processing, 75 tons of ferment filtrates can get 65 tons of water recycled.
The variation of electric current and conductivity is as shown in Figure 2 during electrodialysis process under the conditions of pressure stabilizing.It can be seen by Fig. 2
Out: under pressure stabilizing state, the electric current in ferment filtrate is gradually decreased, and conductivity is gradually lowered, after being disposed, conductivity
Basic and distilled water conductivity approaches, and most of metal ion in the ferment filtrate that illustrates that treated is removed.
Under similarity condition, polyethylene homogeneous ion-exchange membrane is changed to I type that Zhejiang Saite membrane technology Co., Ltd provides
Heterogeneous ion-exchange membrane, when feed liquid flow is 300L/h, working voltage is 30V, the processing time is about 30 minutes, and salt rejection rate is
85%.The II type heterogeneous ion-exchange membrane for selecting Zhejiang Saite membrane technology Co., Ltd to provide, feed liquid flow 400L/h, operation electricity
When pressure is 30V, the processing time is about 20 minutes, salt rejection rate 70%.
Embodiment 5: cycle applications of the Hirsutella sinensis ferment filtrate as culture medium water in Escherichia coli culture
Using the Hirsutella sinensis ferment filtrate recycle-water after 4 electrodialysis process of embodiment as culture medium water, examine respectively
Examined its influence grown to e. coli jm109 and e. coli bl21, and with using distilled water as culture medium water into
Comparison is gone.
, as culture medium water, to prepare LB culture containing the Hirsutella sinensis ferment filtrate recycle-water after electrodialysis process
Base, medium component are as follows: peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, pH 7.0 are inoculated with Escherichia coli respectively
JM109 and e. coli bl21 are cultivated 11 hours at 37 DEG C, and investigation grows e. coli jm109 and e. coli bl21
It influences, experimental result is as shown in Figure 3 and Figure 4, it can be seen that Hirsutella sinensis ferment filtrate recycle-water and distilled water are as culture
The influence that base water grows e. coli jm109 and e. coli bl21 is essentially identical, and growth curve is substantially similar.Therefore,
Hirsutella sinensis can be used as e. coli jm109 and the culture medium of e. coli bl21 is subject to cycle applications with water.Due to big
Enterobacteria is mode bacterium, and the conclusion of the present embodiment also can be shown that the ferment filtrate recycle-water after electrodialysis process can be used for
The culture of most of bacterium.
Embodiment 6: circulation of the Hirsutella sinensis ferment filtrate recycle-water as culture medium water in Pichia pastoris culture
Using
Using the Hirsutella sinensis ferment filtrate recycle-water after 4 electrodialysis process of embodiment as culture medium water, examine respectively
Examined its influence grown to pichia pastoris X-33 and Pichia pastoris GS115, and with using distilled water as culture medium water into
Comparison is gone.
, as culture medium water, to prepare YPD culture containing the Hirsutella sinensis ferment filtrate recycle-water after electrodialysis process
Base, medium component are as follows: glucose 20g/L, peptone 10g/L, yeast powder 5g/L, pH 7.0 are investigated to pichia pastoris X-33
With the influence of Pichia pastoris GS115 growth, condition of culture is 30 DEG C and cultivates 27 hours.Experimental result is as shown in Figure 5 and Figure 6, can
To find out Hirsutella sinensis ferment filtrate recycle-water and distilled water as culture medium water to pichia pastoris X-33 and Pichia pastoris
The influence of GS115 growth is essentially identical, and growth curve is substantially similar.Therefore, Hirsutella sinensis ferment filtrate recycle-water can be made
Culture medium for pichia pastoris X-33 and Pichia pastoris GS115 is subject to cycle applications with water.Since Pichia pastoris is mode fungi,
The conclusion of the present embodiment can be shown that the ferment filtrate recycle-water after electrodialysis process can be used for the culture of most of fungi.
Embodiment 7: Hirsutella sinensis ferment filtrate recycle-water following in Hirsutella sinensis culture as culture medium water
Ring application
Using the Hirsutella sinensis ferment filtrate recycle-water after 4 electrodialysis process of embodiment as culture medium water, investigate
Its influence to Hirsutella sinensis L0106 growth, and compared with using distilled water as culture medium with water.
, as culture medium water, to prepare Chinese coat containing the Hirsutella sinensis ferment filtrate recycle-water after electrodialysis process
Spore liquid fermentation medium, culture medium prescription are as follows: glucose 1.5%, corn flour 1.0%, dextrin 0.5%, wheat bran 1.5%, ferment
Female powder 0.5%, dried silkworm chrysalis meal 1.5%, peptone 1.0%, MgSO4, 0.01%, KH2PO40.02%, pH value is natural.In preparation
In the process, first dried silkworm chrysalis meal, corn flour and wheat bran are added in Hirsutella sinensis ferment filtrate recycle-water appropriate and are liquefied, gone out
Bacterium takes supernatant, is then redissolved other compositions, then sterilize.Inclined-plane culture based formulas be liquid medium on the basis of plus quality
The agar powder of concentration 2%.Picking about 2cm together is shoveled from slant medium with the inoculation of sterilizing2Fungus block is cultivated together with part
Base is into seed fluid nutrient mediums of saccharomycete, 16 DEG C of cultures, 30 days (100rpm).With the inoculum concentration of volumetric concentration 10%, it is inoculated with seed liquid
Culture medium is into shake flask fermentation fluid nutrient medium, about 10 days (100rpm) of 15-18 DEG C of culture, and investigates to Hirsutella sinensis
The influence of L0106 fermentation.Experimental result is as shown in Figure 7, it can be seen that Hirsutella sinensis ferment filtrate recycle-water and distilled water are made
Influence for culture medium water to mycelium dry weight after Hirsutella sinensis fermentation is essentially identical, after fermentation in 7 days, control group
Hirsutella sinensis mycelium dry weight be 18g/L, and using the Hirsutella sinensis ferment filtrate recycle-water after electrodialysis process as
The Hirsutella sinensis mycelium dry weight of culture medium culture is 27.1g/L.Therefore, during Hirsutella sinensis ferment filtrate recycle-water is used as
The culture medium of state's coat spore water has the function of raising Hirsutella sinensis mycelium amount, can be subject to cycle applications.
Embodiment 8: Hirsutella sinensis ferment filtrate recycle-water following on actinoplanes utahensis as culture medium water
Ring application
Using the Hirsutella sinensis ferment filtrate recycle-water after 4 electrodialysis process of embodiment as culture medium water, investigate
It is to acarbose producing strains Actinoplanes utahensis ZJB-08196 (referring to Chinese patent 201010605912.3
Denomination of invention: alpha-amylase inhibitor producing strain and its screening technique) growth influence, and with using distilled water as culture medium
It is compared with water.
Using containing after electrodialysis process Hirsutella sinensis ferment filtrate recycle-water and distilled water as culture medium water, respectively
Prepare A.utahensis ZJB-08196 liquid fermentation medium, culture medium prescription are as follows: maltose 45g/L, glucose 40g/L,
Soybean cake powder 17g/L, monosodium glutamate 3g/L, CaCO32.5g/L, CaCl22.5g/L, FeCl30.5g/L, K2HPO41.0g/L
Glycerol 1.0g/L, pH7.5, each component is miscible in suitable Hirsutella sinensis ferment filtrate recycle-water and distilled water, it boils
Uniformly packing is into the triangle shake bottle of 500mL afterwards, every bottle of packing 50mL, after the 30min that sterilizes under the conditions of 115 DEG C for use.Inclined-plane training
Support based formulas be liquid medium on the basis of plus mass concentration 2% agar powder.It is shoveled with the inoculation of sterilizing from slant medium
On picking about 2cm together2Fungus block together with partial medium into seed fluid nutrient mediums of saccharomycete, 26 DEG C of cultures, 15 days (100rpm).
With the inoculum concentration of volumetric concentration 10%, seed fluid nutrient mediums of saccharomycete is inoculated with into shake flask fermentation fluid nutrient medium, 26 DEG C of cultures about 15
Its (100rpm), and investigate the influence fermented to actinoplanes utahensis ZJB-08196.As a result as shown in Figure 8, it can be seen that in
The influence that state's coat spore ferment filtrate and distilled water grow actinoplanes utahensis as culture medium water is essentially identical, strain
Growth course it is substantially similar.Therefore, Hirsutella sinensis ferment filtrate can be used as the culture medium water of actinoplanes utahensis
It is subject to cycle applications.
Claims (9)
1. a kind of recoverying and utilizing method of Hirsutella sinensis ferment filtrate, it is characterised in that the method are as follows: with Hirsutella sinensis
Filtrate after the filtering fermentation liquor that fermented culture obtains is water inlet, is 200- in flow of inlet water using homogeneous ion-exchange membrane
400 L/h, working voltage carry out electrodialysis process under conditions of being 20-30 V, collect electrodialysis efflux, as Chinese coat
Spore ferment filtrate recycle-water, recycle-water are recycled for preparing microorganism culture medium.
2. the recoverying and utilizing method of Hirsutella sinensis ferment filtrate as described in claim 1, it is characterised in that the homogeneous ion
Exchange membrane is polyethylene homogeneous ion-exchange membrane.
3. the recoverying and utilizing method of Hirsutella sinensis ferment filtrate as described in claim 1, it is characterised in that at the electrodialysis
Managing the time is 18-35 minutes.
4. the recoverying and utilizing method of Hirsutella sinensis ferment filtrate as described in claim 1, it is characterised in that the electrodialysis item
Part are as follows: flow of inlet water is 300 L/h, working voltage is 30 V, and the electrodialysis process time is 20 minutes.
5. the recoverying and utilizing method of Hirsutella sinensis ferment filtrate as described in claim 1, it is characterised in that the microorganism is
Fungi, bacterium or actinomyces.
6. the recoverying and utilizing method of Hirsutella sinensis ferment filtrate as claimed in claim 5, it is characterised in that the microorganism is
Hirsutella sinensis L0106, e. coli jm109, e. coli bl21, pichia pastoris X-33, Pichia pastoris GS115 or Utah trip
One of dynamic actinomyces.
7. the recoverying and utilizing method of Hirsutella sinensis ferment filtrate as described in claim 1, it is characterised in that China's coat
The fermented cultural method of spore are as follows: Hirsutella sinensis L0106 is inoculated in slant medium, inclined-plane is carried out under the conditions of 12 ~ 16 DEG C
Culture；It takes inclined-plane bacterium to be inoculated in seed culture medium again, seed culture is carried out under the conditions of 15-18 DEG C, then takes seed liquor by body
The inoculum concentration of product concentration 10% accesses fermentation medium, and fermented and cultured is carried out under the conditions of 15-18 DEG C, obtains fermentation liquid；Described
Slant medium quality group becomes: glucose 1.8-2.2%, corn flour 0.8-1.2%, murphy juice 0.4-0.6%, dextrin 0.4-
0.6%, yeast powder 0.4-0.6%, wheat bran 0.8-1.2%, dried silkworm chrysalis meal 1.8-2.2%, peptone 0.8-1.2%, magnesium sulfate 0.04-
0.06%, potassium dihydrogen phosphate 0.04-0.06%, agar powder 0.8-1.2%, solvent are water, and pH value is natural；The seed culture medium
It is fluid nutrient medium with fermentation medium, the fluid nutrient medium quality group becomes: glucose 0.8-1.2%, molasses 0.8-
1.2%, dried silkworm chrysalis meal 0.4-0.6%, soybean cake powder 0.8-1.2%, yeast extract 0.4-0.6%, magnesium sulfate 0.01-0.015%, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.015-0.025%, solvent are water, and pH value is natural.
8. the recoverying and utilizing method of Hirsutella sinensis ferment filtrate as claimed in claim 7, it is characterised in that China's coat
The fermented cultural method of spore are as follows: Hirsutella sinensis L0106 is inoculated in slant medium, is cultivated 25 days at 12 ~ 16 DEG C, is obtained oblique
Face thallus；Slant medium quality group becomes: glucose 1.8-2.2%, corn flour 0.8-1.2%, murphy juice 0.4-0.6%, dextrin
0.4-0.6%, yeast powder 0.4-0.6%, wheat bran 0.8-1.2%, dried silkworm chrysalis meal 1.8-2.2%, peptone 0.8-1.2%, magnesium sulfate
0.04-0.06%, potassium dihydrogen phosphate 0.04-0.06%, agar powder 0.8-1.2%, solvent are water, and pH value is natural；Then by inclined-plane bacterium
Body is seeded to fluid nutrient medium, under the conditions of 100rmp, 15-18 DEG C, shaken cultivation 30 days, obtains seed liquor；Seed liquor is shaken
It swings broken mycelium and seed suspension liquid is made, then accessed in first order seed fermentor by the inoculum concentration of volumetric concentration 10%, 15-
It is cultivated 5-15 days under the conditions of 18 DEG C, then is pressed into second order fermentation tank, 15-18 by the inoculum concentration of volumetric concentration 10% by first class seed pot
Tank is put after being pressed into the culture of three grade fermemtation tank 40 days by the inoculum concentration of volumetric concentration 10% again after cultivating 5-15 days under the conditions of DEG C, is obtained
Separation of fermentative broth is obtained filtrate by fermentation liquid；The first order seed fermentor, second order fermentation tank are in three grade fermemtation tank
Fluid nutrient medium, fluid nutrient medium quality group become: glucose 0.8-1.2%, molasses 0.8-1.2%, dried silkworm chrysalis meal 0.4-0.6%, Huang
Beancake powder 0.8-1.2%, yeast extract 0.4-0.6%, magnesium sulfate 0.01-0.015%, potassium dihydrogen phosphate 0.015-0.025%, solvent are
Water, pH value are natural.
9. the recoverying and utilizing method of Hirsutella sinensis ferment filtrate as claimed in claim 7, it is characterised in that the fermentation liquid point
It is to be filtered using plate and frame filter press or filtered using head pressure from method, collects filtrate；Described is filtered using head pressure
To squeeze into fermentation liquid in high-order storage tank, using potential difference pressure and by control valve by fermentation liquid with 10 ton/hours of speed
Degree squeezes into rotary drum, is separated by solid-liquid separation fermentation liquid using rotary drum, obtains filtrate.
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| CN109082382B (en) * | 2018-06-15 | 2021-06-08 | 浙江工业大学 | Cordyceps sinensis hirsutella sinensis ZJB18002 and application thereof |
| CN110342991A (en) * | 2019-05-10 | 2019-10-18 | 宁夏西麒麟生物科技有限公司 | From the organic fertilizer of fermentation cordyceps sinensis |
| TWI768224B (en) * | 2019-07-25 | 2022-06-21 | 生展生物科技股份有限公司 | Use of Cordyceps sinensis CS-100 strain for promoting the growth of nerve cells |
| CN112126592B (en) * | 2020-08-06 | 2022-06-14 | 浙江工业大学 | Hirsutella sinensis mutant strain and application thereof in nucleoside production |
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