CN104774182A - Extraction and purification method of ergothioneine - Google Patents

Extraction and purification method of ergothioneine Download PDF

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CN104774182A
CN104774182A CN201410008812.0A CN201410008812A CN104774182A CN 104774182 A CN104774182 A CN 104774182A CN 201410008812 A CN201410008812 A CN 201410008812A CN 104774182 A CN104774182 A CN 104774182A
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thioneine
mycelium
extraction
water
ergothioneine
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CN104774182A8 (en
CN104774182B (en
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姜文侠
刘琦
王洪宇
李运华
刘伟
梅保良
张维亚
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Tianjin Institute of Industrial Biotechnology of CAS
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Tianjin Institute of Industrial Biotechnology of CAS
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D233/00Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/66Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D233/84Sulfur atoms

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

The invention relates to an extraction and purification method of ergothioneine. The extraction and purification method comprises following steps: (1) hot water is used for extraction of a mushroom mycelium fermentation broth so as to obtain a mycelium and ergothioneine aqueous solution; (2) the ergothioneine aqueous solution is subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff ranging from 1kDa to 30kDa, and an obtained filtrate is collected; (3) the filtrate is subjected to chromatography purification, wherein HILIC filling material is taken as a chromatographic packing material, mobile phase is composed of acetonitrile and water at a volume ratio of 80:20 to 88:12, and each 100g of the chromatographic packing material is used for purifying 0.01 to 0.323g sample amount of ergothioneine. The extraction and purification method is simple; operation is convenient; cost is low; large amounts of ergothioneine products with different purities can be prepared rapidly; and sample purity can be as high as 99.0%.

Description

The extraction of thioneine and purification process
Technical field
The invention belongs to biochemical product, food and medical art, relate to a kind of separation and refining method of thioneine, particularly relate to a kind of method of extraction and purifying thioneine from mycelium.
Background technology
Thioneine (L-Ergothioneine; EGT) be a kind of rare natural chiral amino acid; it is physiologically active substance important in organism; there is scavenging free radicals, removing toxic substances, the biosynthesizing of maintenance DNA, the normal growth of cell, cellular immunization, radioprotective, whitening and anti-ageing function of waiting for a long time; be identified a kind of distinctive, multi-functional cell physiological protective material; especially play an important role in anti-oxidant and capacity control etc., therefore thioneine has broad application prospects in industries such as food, makeup and biological medicines.
Whole world NI first executive officer Marvin S.Hausman MD points out: various diseases can be suppressed human body cell due to thioneine and organize the injury caused, what can look is a kind of important VITAMIN in human body, and can as a kind of composition (GrigatS of meals functional food, Harlfinger S, Pal S, et al.Probing the substrate specificity of theergothioneine transporter with methimazole, hercynine, and organic cations.BiochemPharmacol, 2007, 74:309-316.).
Experiment proves, humans and animals can not produce any toxic side effect after taking in thioneine, is generally recognized as safe (generally recognized as safe, GRAS).The biological function special based on thioneine and pharmacologically active, OXIS international corporation of the U.S. is proposed a dietary supplements ErgoFlex made with thioneine, there is relieve chronic arthralgia, promote cartilage health, reduce inflammation, improve effect (the Benson KF of physique and supplementing energy etc., Ager DM, Landes B, et al.Improvement of joint range of motion (ROM) andreduction of chronic pain after consumption of an ergothioneine-containingnutritional supplement.Prev Med, 2012, 54:S83-S89.).
Comparing with natural biological extraction method with chemical synthesis, gill fungus deep-fermentation biosynthesizing thioneine has that output is high, cost is low, easily accomplish scale production and the advantage such as product safety, is the technological development direction of synthesis thioneine.The biosynthetic thioneine overwhelming majority of gill fungus bacterium accumulates in mycelium, therefore, prepares thioneine product with gill fungus deep-fermentation, must possess the technology of Isolation and purification thioneine from mycelium.
The Mitsubishi dried powder of acetone or alcohol solution extraction Clitocybe sporophore, recycling column chromatography, thin-layer chromatography, ion exchange chromatography and paper electrophoresis technology are further purified thioneine (Mitsubishi, Chem Ind.JP58057365-A), Harada E utilizes ethyl acetate lixiviate tea plant mushroom fruit body, again by Zeo-karb and the further separation and Extraction of chromatographed on silica gel, after lyophilize, highly purified thioneine (Univ Nagoya is obtained through dextran gel filtration and reverse-phase HPLC purifying, Iwade Kingaku Kenkyusho Kk, DaicelChem.Method for producing ergothioneine [P] .JP:JP2009161498A), Nukina M etc. extracts thioneine from tame mushroom fruitbody, first by mushroom drying and crushing, hot water extraction, dissolve with ethanol is used again after filtration, use Zeo-karb, silica gel thin-layer chromatography separation and Extraction, high purity thioneine (Nukina M.Preparation of ergothioneine useful in pharmaceuticals is prepared by high pressure liquid chromatography and lyophilize, by fractionating mushroom extract containing ergothioneine, with solvent, separating the ergothioneine, and purifying the fraction liquid containingthe ergothioneine [P] .JP:JP2008110988-A), the domestic purifying had no about thioneine is studied, only there is report Zhou Nianbo etc. from bisporous mushroom mushroom handle, extract thioneine, first mushroom handle is pulverized, utilize the NaOH solution lixiviate of heat, centrifugal segregation solid substance, supernatant liquor concentrating under reduced pressure, re-uses alumina column chromatography separation and Extraction, the thioneine rate of recovery is 91.2%, and purity reaches 25.6%.(Zhou Nianbo, Li Yiqun, solicitous red, alumina column chromatography extracts thioneine [J] from bisporous mushroom mushroom handle. Agriculture of Anhui science, 2010,38 (27): 14842 ~ 14843.).
Above-mentioned purification process step is many, and program is complicated; Extraction solvent mostly adopts organic solvent or adds chemical reagent, must remove organic solvent or other reagent of extra interpolation when being further purified; Although adopt the normal pressure such as ion exchange chromatography, alumina column chromatography chromatography can realize extensive preparation, separation efficiency and product purity low, complicated operation, is not especially suitable for the separation of the more complicated sample of composition; Utilize high performance liquid chromatography (HPLC method) although can obtain highly purified thioneine by purifying, cost is high, and preparation amount is little, only can obtain milligram level product, is only limited to laboratory study application.
Middle pressure preparative chromatography is natural product, biological products purifying and refining novel separation and purification system, and have sepn process constant pressure, middle compacting is standby, and flow velocity is fast, and treatment capacity is large, and post effect is high, and product purity is high, is suitable for the advantages such as suitability for industrialized production.There are no the report of the standby chromatogram purification thioneine of compacting in employing in prior art.
Summary of the invention
The object of the invention is to the deficiency overcoming prior art existence; a kind of method of rapid, high volume extraction and purifying thioneine from mycelium is provided; thus provide the thioneine product of required purity for industries such as food, health care, medicine; the method is simple, is particularly useful for the large-scale production of thioneine.
Object of the present invention is achieved through the following technical solutions.
1, hot water extraction: after the submerged fermentation of gill fungus bacterium mycelium terminates, mycelium fermentation broth, through solid-liquid separation, collects mycelium, by wet mycelium weight: water (g: mL) adds the water of 1: 0.5 ~ 1: 40, makes mycelial aqueous suspensions.Mycelium aqueous suspensions is warming up to 70 ~ 100 DEG C, and with 0 ~ 600rpm stirring and leaching, 1 ~ 60min, thioneine is leached into the extracellular aqueous solution in mycelial cell.Solid-liquid separation, collect mycelium and the thioneine aqueous solution respectively, mycelium can repeat lixiviate as stated above, for extracted many times, merge the thioneine aqueous solution after lixiviate.
Described solid-liquid separating method can be centrifugal, filter or natural subsidence.
2, ultrafiltration: the thioneine aqueous solution of step 1 gained is adopted ultrafiltration membrance filter, removes the macromole impurity of albumen, polysaccharide etc.Collect ultrafiltration permeate, carry out drying or concentrated according to actual needs, obtain dry crude product or concentrated solution.Thioneine can dryly be solid crude product or be concentrated into content higher than 0.1g/L.
Ultrafiltration ultra-filtration membrane used can be the film of ceramic membrane or macromolecular material.The molecular weight cut-off of ultra-filtration membrane selects 1kDa ~ 30kDa, preferred 4kDa ~ 6kDa.
Can first micro-filtration before ultrafiltration.Microfiltration membrane can be the film of ceramic membrane, metallic membrane or macromolecular material.0.01 ~ 5 μm, the aperture of microfiltration membrane, preferably 0.1 ~ 0.5 μm.
Described drying means can be but be not limited to spraying dry, vacuum-drying or lyophilize, obtains the thick product of thioneine after drying.
Described concentration method can be but be not limited to evaporation or vacuum concentration.
3, chromatogram purification: the acetonitrile thick product of step 2 gained or concentrated solution being added 0.5 ~ 5 times of volume dissolves, supersound process 1 ~ 30min, through the micro-filtrate membrane filtration in 0.1 ~ 0.5 μm of aperture, be concentrated into the sample introduction reaching preparing chromatography system and require laggard row chromatogram purification, the concentration of such as thioneine is at 6g/L ~ 48g/L.
Described purifying chromatographic column can be but be not limited to Flash chromatographic column, dynamic axial compression column etc., and chromatograph packing material is HILIC (hydrophilic Interaction Chromatography) filler.Chromatographic column specification is determined according to actual treatment amount.
Described moving phase consists of acetonitrile-water, adopts isocratic elution, mobile phase volume than selection 80: 20 ~ 88: 12, preferably 88: 12 ~ 85: 15.Described chromatogram determined wavelength is 280nm, and collection wavelength is 254nm; Thioneine sample size is every 100g filler 0.01g ~ 0.323g, preferred 0.113g ~ 0.225g, and flow velocity is determined according to the specification of chromatographic column.
According to ultraviolet detection spectrogram, collect, utilize high pressure liquid chromatography (HPLC) to detect to the counterpart of each chromatographic peak be separated, the concentrated and dry component containing thioneine, the thioneine purity of gained sample reaches 96.6 ~ 99.0%.
Described concentration method can be but be not limited to evaporation or vacuum concentration.
Described drying means can be but be not limited to spraying dry, vacuum-drying or lyophilize, obtains highly purified thioneine product after drying.
The detection method of thioneine:
High pressure lipuid chromatography (HPLC) (HPLC), chromatographic column is HILIC analytical column (250mm × 4.6mm, 5 μm), moving phase is acetonitrile-water, and volume ratio is 85: 15, uses 20mmol/L acetic acid-ammonium acetate buffer to regulate the pH to 6.0 of moving phase, determined wavelength 254nm, column temperature 40 DEG C, flow velocity 1mL/min, sample size 10 μ L.
Advantage of the present invention is:
(1) step is few, and consuming time short, easy and simple to handle, yield is high, and the purity of product is high, and according to different application needs, can prepare the thioneine product of multiple purity.
(2) in mycelium, the extracting technology of thioneine is only extraction solvent with water, instead of lower boiling organic extraction solution, does not add any inorganic chemical and petrochemical complex class reagent in leaching process; The cost of digestion agent is minimum, avoids the danger that with an organic solvent may bring in a large number in operating process; Do not increase impurity, subsequent technique need not be considered to remove the artificial impurity added.
(3) adopt ultra-filtration technique to achieve being separated of thioneine and macromole impurity, obtain the product that thioneine purity reaches 25.2% ~ 34.7%, be suitable for the application that purity requirement is lower.Avoid the harm of macromole impurity to the chromatograph packing material of follow-up use, ultrafiltration technology does not have phase transformation, simple to operate, and energy consumption is low, and cost is low simultaneously, does not add chemistry examination, pollution-free.
(4) be further purified in employing and press chromatogram, compared to normal pressure column chromatography for separation, thioneine purity prepared by present method is high, technique and quality product easy to control, be suitable for the separation and purification of the thioneine in complicated component sample.Compared with high-pressure liquid chromatography, the present invention can prepare highly purified thioneine on a large scale, and output is high, and facility investment is little, and running cost is low, is suitable for suitability for industrialized production.
Accompanying drawing explanation
Fig. 1 is the middle pressure chromatographic separation collection of illustrative plates in embodiment 2;
Fig. 2 is that the thioneine HPLC in embodiment 2 after chromatogram purification detects collection of illustrative plates;
Fig. 3 is the middle pressure chromatographic separation collection of illustrative plates in embodiment 3;
Fig. 4 is that the thioneine HPLC in embodiment 3 after chromatogram purification detects collection of illustrative plates;
Fig. 5 is the middle pressure chromatographic separation collection of illustrative plates in embodiment 4;
Fig. 6 is that the thioneine HPLC in embodiment 4 after chromatogram purification detects collection of illustrative plates.
Embodiment
Following examples of the present invention are only used for explanation and realize the specific embodiment of the present invention, and these embodiments can not be interpreted as it is limitation of the present invention.On the contrary, the present invention not only will comprise these embodiments, but also to contain other any do not deviate from do under spirit of the present invention and principle change, modification, substitute, combine, simplify.
Laboratory apparatus, material and reagent
Middle pressure preparing chromatography system (Dr Flash S type, sharp fringe science and technology (Suzhou) company limited); High pressure liquid chromatograph (Agela LC-10F type), Flash chromatographic column (HILIC, 20 ~ 45 μm, 80g) be Tianjin Bonaaijieer Technology Co., Ltd with HILIC analytical column (250mm × 4.6mm, 5 μm, VH510525BXN0068) to produce; Hollow fiber ultrafiltration membrane (Tianjin Ai Sheng membrane filtration technique company limited); The millipore filtration (Tianjin Bonaaijieer Technology Co., Ltd) in 0.22 μm of aperture; Rotary Evaporators (Hei-VAPPrecsion ML/G6 type, Heidolph company); Freeze drier (FreeZone, Labconco company); Water-bath (TW20 type, Julabo company); Digital heating magnetic stirring apparatus (MIX Control20 type, WIGGENS company); High speed desktop refrigerated centrifuge (TGL-16M type, Xiang Yi whizzer Instrument Ltd.); Ultrasonic machine (SB-5200D type, NingBo XinZhi Biology Science Co., Ltd).
Main agents: thioneine reference substance (purity >=98%, Biomol InternationalInc.); The reagent such as acetonitrile, acetic acid, ammonium acetate are commercially available chromatographically pure.Chemical reagent KH 2pO 4, MgSO 47H 2o etc., purchased from Chemical Reagent Co., Ltd., Sinopharm Group.Semen Maydis powder is purchased from meter Ye limited liability company of Meihekou City Xingda, bean cake powder is purchased from Zhongmianziguang Biological Science and Technology Co., Ltd., Beijing, α-amylase is purchased from Beijing Suo Laibao Science and Technology Ltd., glycerine is purchased from Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd., and casein peptone is purchased from various schools of thinkers Trade Co., Ltd. of Yanshi City.
Embodiment 1: the preparation of oyster cap fungus (Pleurotus ostreatus) CGMCC No.6232 mycelium fermentation broth
Slant medium: PDA substratum (Becton, Dickinson and Company).
Liquid seed culture medium: Semen Maydis powder 30g/L, bean cake powder 15g/L, α-amylase 54U/L, KH 2pO 43g/L, MgSO 47H 2o1.5g/L, all the other are water, and in 121 DEG C of sterilizing 20min, the liquid amount in 500mL triangular flask is 150mL.
Liquid fermentation medium: glycerine 50g/L, casein peptone 35g/L, KH 2pO 43g/L, MgSO 47H 2o1.5g/L, all the other are water, 121 DEG C of sterilizing 20min, and in 500mL triangular flask, liquid amount is 150mL.
The lawn access seed culture medium of picking slant medium bacterial classification CGMCC No.6232,25 DEG C of shaking table 150rpm cultivate 4 days, obtain seed liquor.By seed liquor by volume 5% inoculum size access fermention medium, 25 DEG C of shaking table 150rpm cultivate 15 days, obtain mycelium fermentation broth.
Embodiment 2: the extraction of thioneine and purifying
The mycelium fermentation broth of oyster cap fungus (Pleurotusostreatus) CGMCC No.6232, through centrifugal, collects 50g mycelium, by wet mycelium weight: water (g: mL) adds the water of 1: 40, makes mycelial aqueous suspensions.Mycelium aqueous suspensions is placed in 100 DEG C of water-baths, without stirring and leaching 5min, thioneine is leached in the aqueous solution.Collect mycelium and filtrate respectively after filtration, obtain the 1700mL thioneine aqueous solution.
By the hollow fiber ultrafiltration membrane ultrafiltration that above-mentioned thioneine aqueous solution employing aperture is 4kDa, obtain 1000mL permeate, the purity that high-pressure liquid phase measures thioneine reaches 31.6%.Permeate in 50 DEG C of rotary evaporations to thioneine concentration be the concentrated solution of 1.72g/L or spray-dried the thick product of thioneine.
In above-mentioned 300mL concentrated solution, add the acetonitrile of 0.5 times of volume, supersound process 20min, through the filtering with microporous membrane in 0.22 μm of aperture, filtrate, in 55 DEG C of rotary evaporations to 15mL, is concentrated by rotary evaporation liquid.
Concentrated by rotary evaporation liquid presses preparing chromatography system to be further purified in utilizing, two Flash Coupled columns use, adopt isocratic elution, moving phase is acetonitrile-water, volume ratio 88: 12, determined wavelength and collection wavelength are respectively 280nm and 254nrn, and the sample size of flow velocity 40mL/min, 100g filler thioneine is 0.323g.
Collect the corresponding component of each chromatographic peak, the component that high-pressure liquid phase detects 27.9 ~ 28.3min is thioneine, and lyophilize obtains the thioneine that purity is 98.93%, and the rate of recovery reaches 37.5%.
Embodiment 3: the extraction of thioneine and purifying
After the submerged fermentation of oyster cap fungus (Pleurotusostreatus) CGMCC No.6232 mycelium terminates, mycelium fermentation broth is through centrifugal, collect 600g mycelium, by wet mycelium weight: water (g: mL) adds the water of 1: 0.5, makes mycelial aqueous suspensions.Mycelium aqueous suspensions is placed in 70 DEG C of water-baths, with 600rpm stirring and leaching 60min, thioneine is leached in the aqueous solution.Collect mycelium and filtrate respectively after filtration, mycelium repeats lixiviate 5 times as stated above, merges the filtrate after lixiviate, obtains the 1100mL thioneine aqueous solution.
By the hollow fiber ultrafiltration membrane ultrafiltration that above-mentioned thioneine aqueous solution employing aperture is 6kDa, obtain 600mL permeate, the purity that high-pressure liquid phase measures thioneine reaches 25.2%.Permeate in 50 DEG C of rotary evaporations to thioneine concentration be the concentrated solution of 0.82g/L or spray-dried the thick product of thioneine.
In above-mentioned 20mL concentrated solution, add the acetonitrile of 5 times of volumes, supersound process 20min, through the filtering with microporous membrane in 0.22 μm of aperture, filtrate is in 40 DEG C of rotary evaporations to 15mL concentrated by rotary evaporation liquid.
Preparing chromatography system is pressed to be further purified in being utilized by concentrated by rotary evaporation liquid, two Flash Coupled columns use, adopt isocratic elution, moving phase is acetonitrile-water, volume ratio 80: 20, determined wavelength and collection wavelength are respectively 280nm and 254nm, and the sample size of flow velocity 60mL/min, 100g filler thioneine is 0.01g.
Collect the corresponding component of each chromatographic peak, the component that high-pressure liquid phase detects 15.7 ~ 16.2min is thioneine, and lyophilize obtains the thioneine that purity is 96.62%, and the rate of recovery reaches 28.9%.
Embodiment 4: the extraction of thioneine and purifying
After the submerged fermentation of oyster cap fungus (Pleurotuso streatus) CGMCC No.6232 mycelium terminates, mycelium fermentation broth after filtration, collect 500g mycelium, by wet mycelium weight: water (g: mL) adds the water of 1: 1, makes mycelial aqueous suspensions.Mycelium aqueous suspensions is placed in 90 DEG C of water-baths, with 200rpm stirring and leaching 10min, thioneine is leached in the aqueous solution.Collect mycelium and filtrate respectively after filtration, mycelium repeats lixiviate 3 times as stated above, merges the aqueous solution after lixiviate, obtains the 1200mL thioneine aqueous solution.
By the hollow fiber ultrafiltration membrane ultrafiltration that above-mentioned thioneine aqueous solution employing aperture is 4kDa, obtain 650mL permeate, the purity that high-pressure liquid phase measures thioneine reaches 34.7%.Permeate in 50 DEG C of rotary evaporations to thioneine concentration be the concentrated solution of 1.8g/L or spray-dried the thick product of thioneine.
In above-mentioned 100mL concentrated solution, add the acetonitrile of 2 times of volumes, supersound process 15min, through the filtering with microporous membrane in 0.22 μm of aperture, filtrate is in 40 DEG C of rotary evaporations to 15mL concentrated by rotary evaporation liquid.
Concentrated by rotary evaporation liquid presses preparing chromatography system to be further purified in utilizing, two Flash Coupled columns use, application isocratic elution mode, moving phase is acetonitrile-water, volume ratio 85: 15, determined wavelength and collection wavelength are respectively 280am and 254nm, and the sample size of flow velocity 50mL/min, 100g filler thioneine is 0.113g.
Collect the corresponding component of each chromatographic peak, the component that high-pressure liquid phase detects 22.8 ~ 23.3min is thioneine, and lyophilize obtains the thioneine that purity is 98.99%, and the rate of recovery reaches 47.6%.

Claims (6)

1. an Isolation and purification method for thioneine, comprises the following steps:
(1) with the step of hot water extraction gill fungus bacterium mycelium fermentation broth, comprise
By mycelium fermentation broth through solid-liquid separation, collect mycelium, by wet mycelium weight: water (g: mL) adds the water of 1: 0.5 ~ 1: 40, makes mycelial aqueous suspensions,
Mycelium aqueous suspensions is warming up to 70 ~ 100 DEG C, with 0 ~ 600rpm stirring and leaching 1 ~ 60 minute, and
Through solid-liquid separation, collect mycelium and the thioneine aqueous solution respectively;
(2) ultrafiltration is carried out to the ultra-filtration membrane that the described thioneine aqueous solution take molecular weight cut-off as 1kDa ~ 30kDa, and collect ultrafiltration permeate, carry out drying or the concentrated step obtaining dry crude product or concentrated solution, and
(3) described ultrafiltration permeate is carried out to the step of chromatogram purification, comprise
In described dry crude product or concentrated solution, add the acetonitrile of 0.5 ~ 5 times of volume, supersound process 1 ~ 30min, after filtration, be concentrated into the sample introduction requirement reaching preparing chromatography system, and
Carry out chromatogram purification, the condition of described chromatogram is: chromatograph packing material is HILIC filler, and moving phase consists of the acetonitrile-water of volume ratio 80: 20 ~ 88: 12, and thioneine sample size is every 100g filler 0.01g ~ 0.323g.
2. the method for claim 1, wherein said gill fungus bacterium is oyster cap fungus (Pleurotusostreatus) CGMCC No.6232.
3. the method for claim 1, wherein after described (1) step terminates, the described mycelium also comprised collecting repeats described (1) step, carry out extracted many times, and the thioneine aqueous solution that each lixiviate obtains is combined for (2) step.
4. the method according to any one of claims 1 to 3, the molecular weight cut-off of the ultra-filtration membrane adopted in wherein said (2) step is 4kDa ~ 6kDa.
5. the method according to any one of claims 1 to 3, in wherein said (3) step mule, moving phase consists of the acetonitrile-water of volume ratio 88: 12 ~ 85: 15.
6. the method according to any one of claims 1 to 3, in wherein said (3) step, thioneine sample size is every 100g filler 0.113g ~ 0.225g.
CN201410008812.0A 2014-01-09 2014-01-09 The extraction of erythrothioneine and purification process Active CN104774182B (en)

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