CN105624200A - Recycling method of hirsutella sinensis fermentation filtrate - Google Patents
Recycling method of hirsutella sinensis fermentation filtrate Download PDFInfo
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Abstract
The invention provides a recycling method of hirsutella sinensis fermentation filtrate. The method includes: using the filtrate obtained by filtering the fermentation broth acquired by the fermentation culture of hirsutella sinensis as inlet water, performing electrodialysis treatment under a condition that water inlet flow is 200-400L/h and the operation voltage is 20-30V, collecting electrodialysis effluent liquid, namely the recycled water of the hirsutella sinensis fermentation filtrate, and using the recycled water to prepare microorganism culture media. The recycling method has the advantages that the electrodialysis equipment selected by the method is cheaper than imported equipment, and the salt rejection rate of the electrodialysis equipment can reach more than 95% under corresponding parameter setting; the water yield after the electrodialysis is about 90%; when the electrodialysis method is used to treat 1000L of fermentation filtrate, the energy consumption cost of the electrodialysis method is about 1.88 yuan per ton, and the equipment cost is about 0.5 yuan per ton; enterprise production cost can be evidently lowered when the electrodialysis method is used to treat the fermentation filtrate and the recycled water is used for preparing the culture media.
Description
(1) technical field
The present invention relates to Cordyceps and produce the cycle applications of filtrate after the recoverying and utilizing method of ferment filtrate of bacterium China pilose spore and electrodialysis.
(2) background technology
Cordyceps (Cordycepssinensis (Berk.) Sacc.) is that Cordyceps fungus colonizes in the Stroma on Lepidoptera (Lepidoptera) Hepialidae insecticide (HepialusarmoricanusOberthur) larva and the complex on larva corpse (including Stroma and polypide). Cordyceps is traditional fungus herb resource that a class is precious, has the feature of metabolite and diverse biological activities, shows huge application and development prospect at biomedicine field.
Last century the eighties, the Zhongmei Huadong Pharmaceutical Co., Ltd. Hangzhou of one of the present inventor completes manually to produce the research of Cordyceps, microbial liquid fermentation culture is carried out by the producing strains China pilose spore of Cordyceps, and utilize mycelium drying to make medicine " hundred make " capsule (authentication code: the quasi-word Z10910036 of traditional Chinese medicines), can partly substitute the function of wild cordyceps, use extensively and obtained the curative effect of affirmative clinically.
In the field of business, it is contemplated that production cost, it does not have to the ferment filtrate of China pilose spore and wherein effective ingredient study, ferment filtrate is generally treated as wastewater treatment. Owing to ferment filtrate easily causing the material of environmental pollution containing a small amount of heavy metal ion etc., if directly when environment can be caused certain harm by discharge of wastewater, it is therefore necessary to reach directly to discharge after industry wastewater discharge standard through wastewater treatment. Wastewater treatment will produce high expense, processes the ferment filtrate of one ton of China pilose spore at present, and 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; Waste the substantial amounts of useful resources contained in ferment filtrate, such as trace element etc. simultaneously.
The processing method of ferment filtrate generally has physical treatment process, method of chemical treatment and biological treatment. Physical treatment process adopts the methods such as precipitation, filtration, centrifugation, air supporting, evaporative crystallization, reverse osmosis, method of chemical treatment has neutralization, coagulation, oxidoreduction, extraction, stripping, stripping, absorption, ion exchange and electro-osmosis etc., and biological treatment has activated sludge process, biofilter and oxidation pond etc., physics and method of chemical treatment have complex steps and the high shortcoming of cost, utilize biological treatment not only cost low but also environmental protection.
Electrodialysis belongs to physical method, is generally used for the desalination of sea water, material concentration etc. Industrial utilize electrodialysis desalination principle, carry out processing and reuse to producing waste water. Electrodialytic technique has been carried out technology and feasibility study (ChaoYM economically for industrial effluent reusing process by Yu-MeiChao etc., LiangTM.Afeasibilitystudyofindustrialwastewaterrecoveryu singelectrodialysisreversal [J] .Desalination.2008,221 (1): 433 439.) (ref). T.Sirivedhin etc. utilize electrodialysis elimination to produce the salinity in waste water, try hard to reach drinking water, irrigation water and domestic animal drinking water standard (SirivedhinT, MccueJ, DallbaumanL.Reclaimingproducedwaterforbeneficialuse:salt removalbyelectrodialysis [J] .J.Membrane.Sci.2004,243 (s1 2): 335-343.). But never reported in literature adopts electrodialytic technique to carry out the recycling (ref) of ferment filtrate.
It is known that, some fermented products are included in ferment filtrate, some fermented products are then included in thalline, and the ferment filtrate after the degerming filament of elimination might not contain a certain amount of active component, it is more the secondary metabolite containing harm, it is not necessary to there is the value recycled. Therefore at present ferment filtrate is all directly treated as waste water and is processed by pharmacy corporation. Additionally, ferment filtrate pretreatment (filtration etc.) before electrodialysis needs the expense that cost is certain, and the reason of concentration difference easily makes electrodialysis plant fouling and reverse osmosis occurs, and causes that desalting efficiency is undesirable, therefore, electrodialysis is seldom utilized by pharmacy corporation.
(3) summary of the invention
Present invention aim at for the above deficiency existed, it is provided that a kind of " hundred make " produces the recycling method of bacterium Cordyceps China pilose spore submerged fermentation filtrate.
The invention provides the recoverying and utilizing method of a kind of China pilose spore ferment filtrate, described method is: with the filtrate after the fermented filtering fermentation liquor cultivating acquisition of China pilose spore for water inlet, adopt homogeneous ion-exchange membrane, it is that 200-400L/h, working voltage carry out electrodialysis process when being 20-30V at flow of inlet water, collect electrodialysis effluent, it is China pilose spore ferment filtrate recycle-water, recycles.
Wherein said electrodialysis process, specifically, in electrodialysis, film used is ion exchange membrane, it is preferable that homogeneous ion-exchange membrane, more preferably polyethylene homogeneous ion-exchange membrane, it is possible to adopt domestic film. If selecting other films, such as heterogeneous ion-exchange membrane, salt rejection rate is relatively low. Such as selecting the I type heterogeneous ion-exchange membrane that Zhejiang Saite membrane technology company limited provides, feed liquid flow is 300L/h, working voltage when being 30V, and the process time is about 30 minutes, and its salt rejection rate is 85%. Selecting the II type heterogeneous ion-exchange membrane that Zhejiang Saite membrane technology company limited provides, when feed liquid flow 400L/h, working voltage are 30V, the process time is about 20 minutes, and its salt rejection rate is 70%.
Wherein said electrodialysis condition is: flow of inlet water is 200-400L/h, working voltage be 20-30V, process time is 18-35 minute. When preferred flow of inlet water 300L/h, working voltage are 30V, the process time is about 20 minutes.
Further, described recycling is that China pilose spore ferment filtrate recycle-water is used for preparing microorganism culture medium, substitutes distilled water, tap water; Described microorganism is fungus, antibacterial or actinomycetes, it is preferable that described microorganism is the one in China pilose spore L0106, e. coli jm109, e. coli bl21, pichia pastoris X-33, Pichia pastoris GS115 or actinoplanes utahensis.
China pilose spore ferment filtrate of the present invention, it is possible to prior art prepares. such as: adopt China pilose spore (Hirsutellasinensis) L0106, be preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, 430072, deposit number CCTCCNo:M2011278, preservation date on August 12nd, 2011. by China pilose spore L0106 liquid medium within top fermentation, it is thus achieved that mycelium, and the tunning containing fermented supernatant fluid and various metabolite therein, by tunning through solid-liquid separation, as adopted the methods such as plate-and-frame filtration, after isolating mycelium, obtain ferment filtrate. this filtrate can carry out desalting processing by later use electrodialysis plant. the fermented cultural method of described China pilose spore is: China pilose spore L0106 is inoculated in slant medium, slant culture is carried out under 12��16 DEG C of conditions, take inclined-plane bacterium again and be inoculated in seed culture medium, seed culture is carried out under 15-18 DEG C of condition, the inoculum concentration then taking seed liquor by volume concentration 10% accesses fermentation medium, carries out fermentation culture and obtain fermentation liquid under 15-18 DEG C of condition, described slant medium quality group becomes: glucose 1.8-2.2%, Semen Maydis powder 0.8-1.2%, (described murphy juice refers to and is brewed into paste liquid with 200g/L Rhizoma Solani tuber osi with water murphy juice 0.4-0.6%, 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 is water, pH value is natural, preferred slant medium quality group becomes: glucose 2.0%, Semen Maydis powder 1.0%, murphy 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 is water, pH value is natural, described seed culture medium and fermentation medium are fluid medium, described fluid 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 is water, and pH value is natural, preferably described fluid 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%, and solvent is water, and pH value is natural.
The concrete fermented cultural method of described China pilose spore is: China pilose spore L0106 is inoculated in slant medium, cultivates 25 days at 12��16 DEG C, it is thus achieved that inclined-plane thalline, slant medium quality group becomes: glucose 1.8-2.2%, Semen Maydis powder 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, pH value is natural, preferred slant culture liquid quality group becomes: glucose 2.0%, Semen Maydis powder 1.0%, murphy 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 is water, pH value is natural, then inclined-plane thalline is seeded to fluid medium, under 100rmp, 15-18 DEG C of condition, shaken cultivation 30 days, it is thus achieved that seed liquor, broken for seed liquor vibration mycelium is made seed suspension liquid, then by volume the inoculum concentration of concentration 10% accesses in first order seed fermentation tank, cultivate 5-15 days under 15-18 DEG C of condition, it is pressed into second order fermentation tank again by the inoculum concentration of first class seed pot by volume concentration 10%, after cultivating 5-15 days under 15-18 DEG C of condition, by volume the inoculum concentration press-in three grade fermemtation tank of concentration 10% puts tank after cultivating 40 days again, obtain tunning, tunning is separated, it is thus achieved that filtrate, described first order seed fermentation tank, second order fermentation tank, it is fluid medium in three grade fermemtation tank, fluid 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 is water, pH value is natural, preferred liquid culture medium quality consists of: 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 is water, pH value is natural, described separation of fermentative broth method, for adopting filter press filter or utilize head pressure to filter, collects filtrate. described utilize head pressure to filter to be squeezed into by fermentation liquid in high-order storage tank, utilize the pressure of potential difference and by control valve, with the speed of 10 ton hour, fermentation liquid squeezed into rotary drum, utilizing rotary drum that fermentation liquid is carried out solid-liquid separation, it is thus achieved that filtrate.
The further high speed centrifugation of filtrate that China pilose spore fermentation can also be obtained by the present invention, removes some of which contaminant particles, for next step Active Components; Fractions of active ingredient needs through efficient liquid phase chromatographic analysis, it is necessary to carry out again with after the membrane filtration separation of 0.25 �� m diameter.
It is known that, some fermented products are included in ferment filtrate, some fermented products are then included in thalline, and the ferment filtrate after the degerming filament of elimination might not contain a certain amount of active component, it is more the secondary metabolite containing harm, it is not necessary to there is the value recycled. Such as, carry out the ferment filtrate of solid-liquid separation after culturing gene engineering bacteria; contain the harmful substances such as substantial amounts of antibiotic wherein; owing to these antibiotic are difficult to efficiently separate from ferment filtrate; if these ferment filtrates are circulated utilization; the antibiotic concentration in ferment filtrate can be made to accumulate; increase the antibiotic-resistant activity of genetic engineering bacterium, thus having the risk of Resident Evil.
The present invention is by having carried out composition detection analysis to the ferment filtrate of China pilose spore, determine and wherein remained a part of available active component, but the content of these active component is relatively low, these active component of separation and Extraction is relatively costly one by one, but is very suitable as culture medium water and promotes the accumulation of thalline.
Using the China pilose spore ferment filtrate after electrodialysis process as culture medium water, prepare LB culture medium, investigate the impact (referring to Fig. 3 and Fig. 4) that e. coli jm109 and e. coli bl21 are grown; Using after electrodialysis process containing China pilose spore ferment filtrate as culture medium water, prepare YPD culture medium, investigate the impact (referring to Fig. 5 and Fig. 6) that pichia pastoris X-33 and Pichia pastoris GS115 are grown; Using containing the China pilose spore ferment filtrate after electrodialysis process as culture medium water, preparation China pilose spore and acarbose producing strains actinoplanes utahensis liquid fermentation medium, investigate the impact (referring to Fig. 7 and Fig. 8) that China pilose spore and actinoplanes utahensis are fermented. Owing to escherichia coli are pattern antibacterial, Pichia sp. is pattern fungus, the present invention also can be shown that electrodialysis process after ferment filtrate can be used for major part antibacterial and the cultivation of fungus.
Concrete, described application carries out as follows:
1, using containing the China pilose spore ferment filtrate after electrodialysis process as culture medium water, prepare LB culture medium, medium component is peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, pH are 7.0, investigate the impact that e. coli jm109 and e. coli bl21 are grown.
2, using containing the China pilose spore ferment filtrate after electrodialysis process as culture medium water, prepare YPD culture medium, medium component is glucose 20g/L, peptone 10g/L, yeast powder 5g/L, pH are 7.0, investigate the impact that pichia pastoris X-33 and Pichia pastoris GS115 are grown.
3, using containing the China pilose spore ferment filtrate recycle-water after electrodialysis process as culture medium water, preparation China pilose spore liquid fermentation medium, culture medium prescription is 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 is natural, it is preferably: glucose 1.5%, Semen Maydis powder 1.0%, dextrin 0.5%, wheat bran 1.5%, yeast powder 0.5%, dried silkworm chrysalis meal 1.5%, peptone 1.0%, MgSO4, 0.01%, KH2PO40.02%, pH value is natural. In the process of preparation, first dried silkworm chrysalis meal, Semen Maydis powder and wheat bran are joined in suitable China pilose spore ferment filtrate and liquefy, sterilizing, take supernatant, then other nutrient media componentses of re-dissolved, then sterilizing, with the inoculum concentration of volumetric concentration 10%, investigate the impact on China pilose spore fermentation.
4, using containing the China pilose spore ferment filtrate recycle-water after electrodialysis process as culture medium water, prepare actinoplanes utahensis fermentation medium, culture medium prescription is: 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 is natural, it is preferred to: 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 is natural, and by each component miscibility in appropriate China pilose spore ferment filtrate and distilled water, after boiling, uniform subpackage is to the triangle shaking flask of 500mL, and every bottle of subpackage 50mL is stand-by after sterilizing 30min under 115 DEG C of conditions. With the inoculum concentration of 10%, investigate the impact on acarbose producing strains A.utahensisZJB-08196 fermentation.
The beneficial effects are mainly as follows: principle, the effective active composition in China pilose spore ferment filtrate is studied in detail, provide " hundred make " and produce the content of effective ingredient in bacterium Cordyceps China pilose spore ferment filtrate, the harmful mineral element of trace therein and certain density metal ion is removed by electrodialytic method, give the circulation utilizability of China pilose spore ferment filtrate, and for cultivating the culture medium water of other microorganisms, the biologic applications for expanding China pilose spore ferment filtrate provides effective way. Effectively save water resource, reduced effluent pressure, decrease the possibility of pollution, environmental protection. The fermentable carried out with ferment filtrate substitutive medium water, its fermentation unit is better than distilled water or tap water. It is possible not only to solve cost produced by follow-up wastewater treatment, it is also possible to save the cost of water used by culture medium. Electrodialysis plant selected by the present invention and parameter not only relatively import cheap, and in corresponding parameter, lower salt rejection rate is set up to more than 95%. After electrodialysis, the yield of water is about 90%. 1000L ferment filtrate, with electroosmose process process energy consumption cost be about 1.88 yuan per ton, equipment cost about 0.5 yuan is per ton. Therefore, electroosmose process is adopted to process ferment filtrate and recycle the production cost that can substantially reduce enterprise in culture medium water.
China pilose spore ferment filtrate is developed and utilized by the present invention, and effectively utilization and the environmental conservation of resource will be had very positive meaning.
(4) accompanying drawing explanation
Fig. 1 is electrodialysis plant and fundamental diagram;
Fig. 2 is the variation diagram of electric current and electrical conductivity in electrodialysis process process;
Fig. 3 is China pilose spore ferment filtrate recycle-water and the distilled water e. coli jm109 growth curve as culture medium water;
Fig. 4 is China pilose spore ferment filtrate recycle-water and the distilled water e. coli bl21 growth curve as culture medium water;
Fig. 5 is China pilose spore ferment filtrate recycle-water and the distilled water pichia pastoris X-33 growth curve as culture medium water;
Fig. 6 is China pilose spore ferment filtrate recycle-water and the distilled water Pichia pastoris GS115 growth curve as culture medium water;
The impact that Fig. 7 is China pilose spore ferment filtrate recycle-water and China pilose spore is grown by distilled water as culture medium water;
The impact that Fig. 8 is China pilose spore ferment filtrate recycle-water and actinoplanes utahensis is grown by distilled water as culture medium water.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited to that:
Embodiment 1: " hundred make " produces the cultivation of bacterium Cordyceps China pilose spore
Bacterium source: first gather natural cordyceps from Qinghai, and bring it back into Hangzhou and be easily separated screening, obtain L0106 bacterial strain, and through this bacterial strain of strain identification be China pilose spore (Hirsutellasinensis), this culture presevation is in China typical culture collection center, deposit number is CCTCCNo:M2011278, discloses in the patent CN102373190A previously applied for.
This strain is inoculated in inclined-plane, (this is the liquid formulations before solidifying to culture medium prescription, by following proportions well after bevel again) be: glucose 2.0% (w/v, 1% represents in 100mL culture medium containing 1g, lower same), Semen Maydis powder 1.0%, (described murphy juice refers to and is brewed into paste liquid with 200g/L Rhizoma Solani tuber osi with water murphy juice 0.5%, filtrate after eight layers of filtered through gauze), 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 is water, cultivate 25 days at 12��16 DEG C, that then chooses 4��5 pieces of soybean grain sizes from slant strains contains mycelial culture medium, accesses sterilized and fills in the 500ml triangular flask of 100ml fluid medium, under 15-18 DEG C of condition, and shaken cultivation 30 days (100rmp) on shaking table. by cultured shaking table strain, inject equipped with the aseptic bottle of little bead carries out thermal agitation, in order to mycelium is smashed and makes seed suspension liquid. then by volume the inoculum concentration of concentration 10% accesses in first order seed fermentation tank, cultivate about 10 days under 100rmp, 15-18 DEG C of condition, it is pressed into second order fermentation tank again by the inoculum concentration of first class seed pot by volume concentration 10%, it is pressed into again after cultivating about 10 days under 100rmp, 15-18 DEG C of condition after three grade fermemtation tank is cultivated 40 days and puts tank, it is thus achieved that fermentation liquid. it is fluid medium in described first order seed fermentation tank, second order fermentation tank, three grade fermemtation tank, fluid 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 is water, and pH value is natural.
Embodiment 2: the detection analysis of active component in China pilose spore ferment filtrate
1, the preparation of China pilose spore ferment filtrate
After embodiment 1 fermentation ends, fermentation liquid is put tank and is separated, and first adopts filter press to obtain the ferment filtrate of the present invention after separating. The ferment filtrate obtained is brown yellow transparent liquid. PH value is 6.0-7.0.
2, the detection analysis of active component in ferment filtrate
Accurately draw separated China pilose spore ferment filtrate 0.2mL, ultra-pure water is settled to 20mL, fully take 1mL in clean tube after mixing, it is simultaneously introduced 1mL Potassium metaperiodate. solution, 25 DEG C of reaction 10min, add 0.1wt%L-rhamnose aqueous solution 2mL, shaken well, add 4mL Fresh Nash solution, 53 DEG C of colour developing 15min, it is quickly cooled to room temperature. Do blank with ultra-pure water, be cooled to the reactant liquor of room temperature after taking 200 �� L colour developings in 96 hole ELISA Plate, 3 multiple holes of every kind of reactant liquor. It is scanned in the wave-length coverage of 380nm-450nm, it is determined that maximum absorption wavelength. Through detection, the content of the cordycepic acid in China pilose spore ferment filtrate is 3.16mg/mL.
China pilose spore ferment filtrate is through 0.25 ��m of micro-pore-film filtration, and filtrate adopts high performance liquid chromatography detection. Condition is as follows, chromatographic column: Yi Lite C18 post (4.6mm �� 250mm, 5 ��m); Column temperature: 35 DEG C; Sampling volume: 20 �� L; Flow velocity 1mL/min; Detection wavelength 260nm; Mobile phase: A: ultra-pure water; B: methanol. Gradient: 0min, 15%B, keeps 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. Through detection, in China pilose spore ferment filtrate, the content of cordycepin is 0.56 �� g/ml, and the content of uridnine is 16.4 �� g/ml, and the content of guanosine is 7.9 �� g/ml, and the content of adenosine is 0.069 �� g/ml, and the content of thymidine is 4.0 �� g/ml.
In China pilose spore ferment filtrate, the mensuration of protein content adopts Kjeldahl's method, and through detection, the crude protein content in China pilose spore ferment filtrate is 3.1mg/mL. Take 3mL China pilose spore ferment filtrate, the ethanol water precipitation of 95% (v/v) adding 4 times of volumes extracts polysaccharide, 4 DEG C stand 1 hour, the centrifugal 20min of 12000rpm removes supernatant, remove clean as far as possible, in precipitation, add the distilled water of 15mL and shake so that precipitation is completely dissolved, obtain crude polysaccharides solution, for the mensuration of extracellular polysaccharide in ferment filtrate. The mensuration of extracellular polysaccharide adopts phend-sulphuric acid to carry out, and after testing, the total sugar content in China pilose spore ferment filtrate is 11.2mg/mL, and the content of extracellular polysaccharide is 2.8mg/mL.
After testing, aspartate content in China pilose spore ferment filtrate is 0.97 �� g/mL, threonine content is 2.04 �� g/mL, serine content is 1.43 �� g/mL, Glycine Levels is 1.55 �� g/mL, alanine content is 1.075 �� g/mL, cysteine content is 6.495 �� g/mL, the content of isoleucine is 5.295 �� g/mL, leucic content is 7.94 �� g/mL, the content of phenylalanine is 74.255 �� g/mL, the content of histidine is 90.855 �� g/mL, the content of lysine is 28.58 �� g/mL, arginic content is 24.69, amino acid whose total content is 245.18 �� g/mL.
China pilose spore ferment filtrate and each metallic element titer sample introduction respectively, its absorbance of Flame Atomic Absorption Spectrometry Determination, after testing, in China pilose spore ferment filtrate, the content of Na element is 399.05 �� g/mL, the content of K element is 172.02 �� g/mL, the content of Mg element is 0.822 �� g/mL, the content of Ca element is 41.22 �� g/mL, the content of Fe element is 1.15 �� g/mL, the content of Zn element is 0.029 �� g/mL, the content that the content of Cu element is 0.014 �� g/mL, Ni element is 0.12 �� g/mL, does not find Mn, Pb, Co element.
By analysis, the growth of thalline is not had negative effect or has positive impact by cordycepic acid in China pilose spore ferment filtrate, nucleosides material, crude protein and aminoacid isoreactivity composition, and the thalline osmotic pressure tool cultivated may be had a certain impact by the mineral element of accumulation in ferment filtrate, the negative effect of the mineral element of accumulation need to be got rid of.
Embodiment 3: the preparation of China pilose spore ferment filtrate
Cultured for embodiment 1 method 78 tons of Cordyceps China pilose spore fermentation liquids are squeezed in the high-order storage tank of 6 tons, utilize the pressure of potential difference and by controlling valve, fermentation liquid squeezed into rotary drum with the speed of 10 ton hour, utilize rotary drum that fermentation liquid is carried out solid-liquid separation, it is thus achieved that about 75 tons of ferment filtrates. In experimentation, centrifugation gained ferment filtrate and electrodialysis after fermentation filtrate fresh water (be used for contrasting it and recycle effect, take distilled water as experiment contrast) after collection ferment filtrate stock solution, activated carbon decolorizing respectively.
Embodiment 4: the electrodialysis process of China pilose spore ferment filtrate
Electrodialysis plant (electrodialysis plant schematic diagram is as shown in Figure 1), film is polyethylene homogeneous ion-exchange membrane, the model of anode membrane and cavity block respectively LE-HeM-CM01 and LE-HeM-AM01, produced by Zhejiang Qianqiu Environmental Water Treatment Co., Ltd., with 75 tons of ferment filtrates of embodiment 3 preparation for water inlet, optimum electrodialysis condition is: feed liquid flow is 300L/h, working voltage when being 30V, and the process time is about 20 minutes, and salt rejection rate reaches 95%; After treatment, 75 tons of ferment filtrates can obtain 67 tons of the water (i.e. ferment filtrate recycle-water) of recycling. If selecting other electrodialysis conditions, salt rejection rate and Water Sproading rate relatively low. Such as to select feed liquid flow be 200L/h, working voltage when being 20V, and the process time is about 80 minutes, and its salt rejection rate is 92%; After treatment, 75 tons of ferment filtrates can obtain 64 tons of the water of recycling. When selection feed liquid flow 400L/h, working voltage are 30V, the process time is about 18 minutes, and its salt rejection rate is 88%; After treatment, 75 tons of ferment filtrates can obtain 65 tons of the water of recycling.
When voltage stabilizing, in electrodialysis process process, the change of electric current and electrical conductivity is as shown in Figure 2. As seen in Figure 2: under voltage stabilizing state, electric current in ferment filtrate is gradually lowered, and electrical conductivity is gradually lowered, after being disposed, its electrical conductivity is basic and distillation electrical conductivity of water is close, and the most of metal ion in ferment filtrate after explanation process is removed.
Under similarity condition, polyethylene homogeneous ion-exchange membrane changing the I type heterogeneous ion-exchange membrane that Zhejiang Saite membrane technology company limited provides into, feed liquid flow is 300L/h, working voltage when being 30V, and the process time is about 30 minutes, and its salt rejection rate is 85%. Selecting the II type heterogeneous ion-exchange membrane that Zhejiang Saite membrane technology company limited provides, when feed liquid flow 400L/h, working voltage are 30V, the process time is about 20 minutes, and its salt rejection rate is 70%.
Embodiment 5: China pilose spore ferment filtrate as culture medium water escherichia coli cultivate on cycle applications
Using the China pilose spore ferment filtrate recycle-water after embodiment 4 electrodialysis process as culture medium water, investigate its impact that e. coli jm109 and e. coli bl21 are grown respectively, and contrasted as culture medium water with using distilled water.
Using containing the China pilose spore ferment filtrate recycle-water after electrodialysis process as culture medium water, preparation LB culture medium, medium component is: peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, pH is 7.0, inoculation e. coli jm109 and e. coli bl21 respectively, cultivate 11 hours at 37 DEG C, investigate the impact that e. coli jm109 and e. coli bl21 are grown, experimental result is as shown in Figure 3 and Figure 4, can be seen that the impact that e. coli jm109 and e. coli bl21 are grown by China pilose spore ferment filtrate recycle-water and distilled water as culture medium water is essentially identical, growth curve basic simlarity. therefore, China pilose spore can as the culture medium water of e. coli jm109 and e. coli bl21 cycle applications in addition. owing to escherichia coli are pattern antibacterial, the conclusion of the present embodiment also can be shown that electrodialysis process after ferment filtrate recycle-water can be used for major part antibacterial cultivation.
Embodiment 6: China pilose spore ferment filtrate recycle-water as culture medium water Pichia sp. cultivate on cycle applications
Using the China pilose spore ferment filtrate recycle-water after embodiment 4 electrodialysis process as culture medium water, investigate its impact that pichia pastoris X-33 and Pichia pastoris GS115 are grown respectively, and contrasted as culture medium water with using distilled water.
Using containing the China pilose spore ferment filtrate recycle-water after electrodialysis process as culture medium water, preparation YPD culture medium, medium component is: glucose 20g/L, peptone 10g/L, yeast powder 5g/L, pH is 7.0, investigates the impact that pichia pastoris X-33 and Pichia pastoris GS115 are grown, and condition of culture is 30 DEG C and cultivates 27 hours. Experimental result is as shown in Figure 5 and Figure 6, it can be seen that the impact that pichia pastoris X-33 and Pichia pastoris GS115 are grown by China pilose spore ferment filtrate recycle-water and distilled water as culture medium water is essentially identical, growth curve basic simlarity. Therefore, China pilose spore ferment filtrate recycle-water can as the culture medium water of pichia pastoris X-33 and Pichia pastoris GS115 cycle applications in addition. Owing to Pichia sp. is pattern fungus, the conclusion of the present embodiment can be shown that electrodialysis process after ferment filtrate recycle-water can be used for major part fungus cultivation.
Embodiment 7: China pilose spore ferment filtrate recycle-water as culture medium water China pilose spore cultivate on cycle applications
Using the China pilose spore ferment filtrate recycle-water after embodiment 4 electrodialysis process as culture medium water, investigate its impact on China pilose spore L0106 growth, and contrasted as culture medium water with using distilled water.
Using containing the China pilose spore ferment filtrate recycle-water after electrodialysis process as culture medium water, preparation China pilose spore liquid fermentation medium, culture medium prescription is: glucose 1.5%, Semen Maydis powder 1.0%, dextrin 0.5%, wheat bran 1.5%, yeast powder 0.5%, dried silkworm chrysalis meal 1.5%, peptone 1.0%, MgSO4, 0.01%, KH2PO40.02%, pH value is natural. In the process of preparation, first dried silkworm chrysalis meal, Semen Maydis powder and wheat bran are joined in suitable China pilose spore ferment filtrate recycle-water and liquefy, sterilizing, take supernatant, then other compositions of re-dissolved, then sterilizing. Slant culture based formulas is add the agar powder of mass concentration 2% on liquid medium within basis. The about 2cm of picking in the lump is shoveled from slant medium with the inoculation of sterilizing2Truffle is together with, in partial medium to seed fluid medium, cultivating 30 days (100rpm) for 16 DEG C. With the inoculum concentration of volumetric concentration 10%, inoculation seed fluid medium, to shake flask fermentation fluid medium, is cultivated about 10 days (100rpm), and is investigated the impact on China pilose spore L0106 fermentation for 15-18 DEG C. Experimental result is as shown in Figure 7, can be seen that China pilose spore ferment filtrate recycle-water and distilled water are essentially identical on the impact of mycelium dry weight after China pilose spore fermentation as culture medium water, after the fermentation of 7 days, the Hirsutella sinensis mycelium dry weight of matched group is 18g/L, and using the China pilose spore ferment filtrate recycle-water after electrodialysis process as the Hirsutella sinensis mycelium dry weight of culture medium culturing for 27.1g/L. Therefore, China pilose spore ferment filtrate recycle-water is improved the effect of Hirsutella sinensis mycelium amount as the culture medium glassware for drinking water of China pilose spore, it is possible to cycle applications in addition.
Embodiment 8: China pilose spore ferment filtrate recycle-water is as culture medium water cycle applications on actinoplanes utahensis
Using the China pilose spore ferment filtrate recycle-water after embodiment 4 electrodialysis process as culture medium water, investigate it to acarbose producing strains ActinoplanesutahensisZJB-08196 (referring to Chinese patent 201010605912.3 denomination of invention: alpha-amylase inhibitor producing strain and the screening technique thereof) impact grown, and contrasted as culture medium water with using distilled water.
Using containing the China pilose spore ferment filtrate recycle-water after electrodialysis process and distilled water as culture medium water, preparation A.utahensisZJB-08196 liquid fermentation medium respectively, culture medium prescription is: 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, by each component miscibility in appropriate China pilose spore ferment filtrate recycle-water and distilled water, after boiling, uniform subpackage is to the triangle shaking flask of 500mL, and every bottle of subpackage 50mL is stand-by after sterilizing 30min under 115 DEG C of conditions. Slant culture based formulas is add the agar powder of mass concentration 2% on liquid medium within basis. The about 2cm of picking in the lump is shoveled from slant medium with the inoculation of sterilizing2Truffle is together with, in partial medium to seed fluid medium, cultivating 15 days (100rpm) for 26 DEG C. With the inoculum concentration of volumetric concentration 10%, inoculation seed fluid medium, to shake flask fermentation fluid medium, is cultivated about 15 days (100rpm), and is investigated the impact on actinoplanes utahensis ZJB-08196 fermentation for 26 DEG C. Result is as shown in Figure 8, it can be seen that the impact that actinoplanes utahensis is grown by China pilose spore ferment filtrate and distilled water as culture medium water is essentially identical, the growth course basic simlarity of strain. Therefore, China pilose spore ferment filtrate can as the culture medium water of actinoplanes utahensis cycle applications in addition.
Claims (10)
1. the recoverying and utilizing method of a China pilose spore ferment filtrate, it is characterized in that described method is: with the filtrate after the fermented filtering fermentation liquor cultivating acquisition of China pilose spore for water inlet, adopt homogeneous ion-exchange membrane, it is that 200-400L/h, working voltage carry out electrodialysis process when being 20-30V at flow of inlet water, collect electrodialysis effluent, it is China pilose spore ferment filtrate recycle-water, recycles.
2. the recoverying and utilizing method of China pilose spore ferment filtrate as claimed in claim 1, it is characterised in that described homogeneous ion-exchange membrane is polyethylene homogeneous ion-exchange membrane.
3. the recoverying and utilizing method of China pilose spore ferment filtrate as claimed in claim 1, it is characterised in that the described electrodialysis process time is 18-35 minute.
4. the recoverying and utilizing method of as claimed in claim 1 China pilose spore ferment filtrate, it is characterised in that described electrodialysis condition is: flow of inlet water is 300L/h, working voltage is 30V, and the electrodialysis process time is 20 minutes.
5. the recoverying and utilizing method of China pilose spore ferment filtrate as claimed in claim 1, it is characterised in that described recycling is that China pilose spore ferment filtrate recycle-water is used for preparing microorganism culture medium.
6. the recoverying and utilizing method of China pilose spore ferment filtrate as claimed in claim 5, it is characterised in that described microorganism is fungus, antibacterial or actinomycetes.
7. the recoverying and utilizing method of China pilose spore ferment filtrate as claimed in claim 5, it is characterised in that described microorganism is the one in China pilose spore L0106, e. coli jm109, e. coli bl21, pichia pastoris X-33, Pichia pastoris GS115 or actinoplanes utahensis.
8. the recoverying and utilizing method of China pilose spore ferment filtrate as claimed in claim 1, it is characterised in that the fermented cultural method of described China pilose spore is: China pilose spore L0106 is inoculated in slant medium, carries out slant culture under 12��16 DEG C of conditions; Taking inclined-plane bacterium again and be inoculated in seed culture medium, carry out seed culture under 15-18 DEG C of condition, the inoculum concentration then taking seed liquor by volume concentration 10% accesses fermentation medium, carries out fermentation culture, it is thus achieved that fermentation liquid under 15-18 DEG C of condition; Described slant medium quality group becomes: glucose 1.8-2.2%, Semen Maydis powder 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 natural; Described seed culture medium and fermentation medium are fluid medium, described fluid 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 is water, and pH value is natural.
9. the recoverying and utilizing method of China pilose spore ferment filtrate as claimed in claim 8, it is characterised in that the fermented cultural method of described China pilose spore is: China pilose spore L0106 is inoculated in slant medium, cultivates 25 days at 12��16 DEG C, it is thus achieved that inclined-plane thalline; Slant medium quality group becomes: glucose 1.8-2.2%, Semen Maydis powder 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 natural; Then inclined-plane thalline is seeded to fluid medium, under 100rmp, 15-18 DEG C of condition, shaken cultivation 30 days, it is thus achieved that seed liquor; Broken for seed liquor vibration mycelium is made seed suspension liquid, then by volume the inoculum concentration of concentration 10% accesses in first order seed fermentation tank, cultivate 5-15 days under 15-18 DEG C of condition, it is pressed into second order fermentation tank again by the inoculum concentration of first class seed pot by volume concentration 10%, after cultivating 5-15 days under 15-18 DEG C of condition, by volume the inoculum concentration press-in three grade fermemtation tank of concentration 10% puts tank after cultivating 40 days again, obtain fermentation liquid, by separation of fermentative broth, it is thus achieved that filtrate; It is fluid medium in described first order seed fermentation tank, second order fermentation tank, three grade fermemtation tank, fluid 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 is water, and pH value is natural.
10. the recoverying and utilizing method of China pilose spore ferment filtrate as claimed in claim 8, it is characterised in that described separation of fermentative broth method, for adopting filter press filter or utilize head pressure to filter, collects filtrate; The described head pressure filtration that utilizes is to be squeezed into by fermentation liquid in high-order storage tank, utilizes the pressure of potential difference and by controlling valve, with the speed of 10 ton hour, fermentation liquid is squeezed into rotary drum, utilizing rotary drum that fermentation liquid is carried out solid-liquid separation, it is thus achieved that filtrate.
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