CN107778357A - A kind of extraction of Pneumocandin B0, purification process - Google Patents
A kind of extraction of Pneumocandin B0, purification process Download PDFInfo
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Abstract
The invention discloses a kind of extraction of Pneumocandin B0, purification process, including：1) zymotic fluid containing Pneumocandin B0 is adjusted into pH value, adds filter aid, be filtrated to get bacteria residue；2) bacteria residue is extracted with the aqueous solution of the high concentration methanol aqueous solution or ethanol；3) polymeric adsorbent resin adsorption leaching liquor is used, is eluted with methanol (ethanol) 80 90% (V/V) aqueous solution, collects the eluent rich in Pneumocandin B0；4) activated carbon is added in the eluent of Pneumocandin B0 and carries out decolorization；5) adsorbed with polymeric adsorbent, 80 100% (v/v) methanol (ethanol) aqueous solution elution resin, collect the eluent rich in Pneumocandin B0；6) it is concentrated in vacuo to obtain Pneumocandin B0 crude product.By the improvement of method for extraction and purification, decolouring impurity-eliminating effect is obvious, and product purity significantly improves, and production cost is low, and processing step is simple, is adapted to industrialized production.
Description
Technical field
The invention belongs to field of biological pharmacy, and in particular to a kind of method for extraction and purification of Pneumocandin B0.
Background technology
Caspofungin is the echinocandin of first listing in the whole world, and its mechanism of action is unique, using fungal cell wall as target position,
Specificity suppresses the synthesis of cell membrane β (1,3)-D- glucans, destroys the integrality of fungal cell wall, makes to permeate in fungal cell
Press it is unstable, ultimately result in fungal cell dissolving.And-β-(1,3)-glucan is free of in mammalian cell, without similar thin
Cell wall building-up process, therefore, Caspofungin are antimycotic simultaneously, will not destroy and influence human body cell.External pharmaceutical research
It has been shown that, Caspofungin is effective to a variety of candida albicans, region fungi, aspergillus and Pneumocystis carinii, thus increasingly by people
Concern.
Pneumocandin B0 (Pneumocandin B0) is the cometabolism as caused by fungi Zalerion arB0ricola
Product, it is the intermediate of synthetic antifunguses thing Caspofungin (CasPofungin).
As the intermediate of synthetic antifunguses thing Caspofungin (CasPofungin), Pneumocandin B0 is by fungi fermentation
Produce, can produce the accessory substance close with Pneumocandin B0 structure and substantial amounts of pigment in fermentation process, therefore knob not Kangding
B0 method for extraction and purification is more complicated.Difference according to substituent on proline in structure is broadly divided into three major types：A0 (32 hydroxyls
242 methylprolines), B0 (32 hydroxy-proline) and C0 (42 hydroxy-proline), in addition, according to substituent on the more skins of ring-type
Kangding A0 can not be divided into the group of AO, Al, AZ, A3, A4 five to different knobs again, and B0 can be divided into the group of B0, B2 two again.
In Pneumocandins From Zalerion arboricola I.Discovery And Isolation
(The Journal Of Antibiotics 45(12):1853-1866) in a text, extracting, being pure for Pneumocandin B0 is described
Change method.Its method is described as follows：
1) zymotic fluid pH is adjusted, mycelium is taken after high speed centrifugation, is soaked with the methanol of two volumes, after centrifugation, used again
80% methanol is soaked to extract Pneumocandin B0 and merge methanol extract；
2) after being diluted with water methanol extract liquid, with HP-207 resin columns on 40%-50% methanol, first with 65:35 first
Alcohol washes pillar, then is eluted Pneumocandin B0 with 100% methanol；
3) at 25 degree, isopropyl acetate precipitation is slowly added in Pneumocandin B0 solution, then by filtering and centrifuging steaming
It is about 67% to obtain Pneumocandin B0 purity after dry, and yield is about 84%；
4) purified again with silica gel column chromatography and HP20 resin adsorptions, with 85:10:5 EtOAC-MEOH-5%AcOH
Pillar is washed, HP20 is being dissolved with 50% methanol-water after collection, is first being washed with 50%, then rinsed with 100% methanol-water, 50%
Recycling；
5) plus nitrile precipitates Pneumocandin B0, refilters, and final purity is 85%.
6) in normal propyl alcohol, 50mg/ml concentration, 60 degree of heating, crystallizing at room temperature is cooled to after adding water.The main of this technique lacks
Point is that separating step is loaded down with trivial details, and the pigment in upstream fermentation liquid is difficult thoroughly to remove, it is maximum the shortcomings that be silicon in silica gel column chromatography
Glue is very unstable, it is difficult to be produced greatly.
United States Patent (USP) Purification Process (US6610822) also provide the extraction of Pneumocandin B0, purifying
Method, that is, repeatedly using extraction and stripping process, repeatedly extract and wash, this method will use substantial amounts of isobutanol,
The organic solvent such as methanol and heptane, operation is more complicated, finally precipitates obtained Pneumocandin B0 solid, and purity only has 80% left side
It is right.Not only purity is not high, and pigment impurity removes very incomplete.Not it is adapted to big production.
CN200910133118.0《A kind of method for preparing Pneumocandin B0》It is pure to describe a kind of new Pneumocandin B0
Change method, has main steps that：
1) zymotic fluid is centrifuged, takes mycelium, Pneumocandin B0 is extracted with methanol；
2) methanol extract is evaporated, n-butanol extraction Pneumocandin B0；
3) n-butanol leaching liquor is evaporated, then Pneumocandin B0 is extracted with 70-80% methanol, peracidity alumina column, received
Collect efflux；
4) after Pneumocandin B0 collection liquid is evaporated, dissolved with 60-70% methanol, upper HP20 polymeric adsorbents, use 85-95%
Methanol elutes, and collects eluent Pneumocandin B0 purity in 50-65%；
5) Pneumocandin B0 collection liquid is evaporated, be dissolved in 60-70% methanol, upper reversed-phase resin YPR- II, use 85-95%
Methanol elutes, and the Pneumocandin B0 purity of collection is more than 90%；
6) after Pneumocandin B0 collection liquid is evaporated, dissolved with methanol, a small amount of water is added dropwise and is allowed to supersaturation crystallization precipitation, system
Pneumocandin B0 is obtained, purity can reach 96%.
Increased although this purification process can improve Pneumocandin B0 product quality, eliminate a large amount of pigments and carry
High product purity, but whole processing step is complicated, and almost Pneumocandin B0 solution will be evaporated by each step, be only used for
Lab scale is studied, and is applied in production, and energy consumption, loss can be very big, and Pneumocandin B0 is easily destroyed under long-time heating.More
Importantly, the purity of knob not Kangding can not be effectively improved using so-called reversed-phase resin YPR- II, particularly it is very difficult to
Except the analog of Pneumocandin B0, purity reaches 96% technology and needs further to be verified, therefore, this method is not appropriate for Niu Mokang
Determine B0 industrialized production.
CN201110266790.4《A kind of method for extraction and purification of Pneumocandin B0》Describe a kind of new knob not Kangding
B0 extraction, purification process, have main steps that：
1) the zymotic fluid pH value containing Pneumocandin B0 is adjusted to 2.0-4.0, filtered, bacteria residue is extracted with low mass molecule alcohol, obtained
Leaching liquor；
2) determining alcohol and pH of leaching liquor are adjusted；
3) after adsorbing leaching liquor with polymeric adsorbent, resin is rinsed with water, the aqueous solution of acetone one successively, then with the acid of acetone one
Solution elutes resin, collects the eluent rich in Pneumocandin B0；
4) after Pneumocandin B0 eluent is diluted with water, adsorbed with polymeric adsorbent, with using low mass molecule alcohol after water rinse resin
Elution, collect the eluent rich in Pneumocandin B0；
5) Pneumocandin B0 is crystallized to obtain in conventional manner, and solid is light yellow or off-white color, and purity is more than 85%；Knob is not
Kangding B0 solid low molecule alcohols, such as methanol, ethanol, propyl alcohol and butanol dissolving, after adding a small amount of water and being allowed to supersaturation
Crystallization separates out, or uses other known method for crystallising, and the Pneumocandin B0 of purity more than 90% can be prepared, pass through extraction
The improvement of purification process, the extraction process step picture of Pneumocandin B0 are treated simply, and product purity can obtain improving relatively, but
Decolorizing effect unobvious during extraction purification.
The major defect of this technique is：
1) zymotic fluid regulation pH is too low, the impurity such as a large amount of pigments is all precipitated, adds downstream purification difficulty；
2) product purity is still not ideal enough, the difficulty of increase downstream synthesis extraction purification.
Therefore find more effective discoloration method and formulate simple method for extraction and purification, be possible to realize knob not Kangding
B0 industrialized production.
The content of the invention
It is difficult to remove the pigment in zymotic fluid, and whole stream to solve to prepare the technique of Pneumocandin B0 in the prior art
The relatively complicated technical problem of journey, the present invention provide a kind of straightforward procedure for preparing Pneumocandin B0, and this method can not only be very
The good pigment removed in Pneumocandin B0, and its purity can be brought up to more than 93%.Therefore, the present invention provides one kind and prepared
The method of Pneumocandin B0, this method comprise the following steps：
1) by the zymotic fluid containing Pneumocandin B0 in the state of stirring, pH value 4.2-6 is adjusted, adds filter aid, is mixed
It is even, filtering, obtain solid bacteria residue；
2) the low molecule alcohol-water solution extraction of bacteria residue high concentration, obtains leaching liquor；The determining alcohol of leaching liquor is adjusted to 20-
30% (V/V)；
3) after adsorbing leaching liquor with polymeric adsorbent, rinsed successively with purified water, 30-50% (V/V) low molecules alcohol-water solution
Resin, resin then is eluted with 80-90% (V/V) low molecules alcohol-water solution, collect the eluent rich in Pneumocandin B0；
4) 0.5-5% activated carbons are added in eluent, are stirred under the conditions of 20-50 DEG C 30-60 minutes, filtering is decolourized；
5) Pneumocandin B0 destainer is diluted with water the concentration of alcohol to 20-30% (V/V), with suction after ultra-filtration filters
Attached resin adsorption, resin is rinsed with containing low mass molecule alcohol 30-50% (V/V) solution, then with 80-100% containing low mass molecule alcohol (V/V)
Solution elutes, and collects the eluent rich in Pneumocandin B0；
6) the Pneumocandin B0 eluent that will be collected into, is concentrated in vacuo under the conditions of 30-50 DEG C, obtains off-white color crude product；
Crude product purity is more than 96.5%.
In the inventive method step 1), due to the integrated application of regulation pH and filter aid, that is, avoid that pH is too low to be caused to sink
The impurity in shallow lake is excessive, improves filtrate filter efficiency, enabling knob again, Kangding completely as thalline settles down together, does not improve
Yield, reduce post-processing difficulty.The pH value of Pneumocandin B0 zymotic fluid is wherein adjusted to 4.2-6, preferable ph with phosphoric acid
4.5。
In the inventive method step 1), add filter aid amount be zymotic fluid quality 0.5-5%, preferably 2%, drainage
Agent is diatomite, the one or more in perlite, cellulose, magnesia, gypsum, Emathlite, the preferred perlite of filter aid；
In the inventive method step 2), the aqueous solution of the low mass molecule alcohol of high concentration used in extraction, wherein low mass molecule alcohol are：First
One kind in alcohol, ethanol, propyl alcohol；Concentration is 90-98% (V/V), preferably 95% ethanol water；Leaching liquor addition is bacterium
2-5 times of slag amount, preferably 3 times amounts；
In the inventive method step 3), polymeric adsorbent model skp-10-4300, D101, HPD300, HPD700, BS-5,
Preferably skp-10-4300；Pure water is added to make the concentration of alcohol in leaching liquor be 20-30% (V/V), leaching liquor determining alcohol is preferably
30%；It is preferably 45% (V/V) that 30-50% (V/V) methanol (ethanol)-aqueous solution, which rinses resin and rinses resin column determining alcohol, is washed
De- liquid determining alcohol preferably 80% (V/V).
Because the described polymeric adsorbent material larger to polarity does not have suction-operated, the impurity slightly stronger to polarity is inhaled
Attached effect is also very weak, therefore is rinsed by loading and purified water, can substantially remove the polar impurity in solution, while
Most of water colo(u)r can be removed；Resin is eluted using 80-90% (V/V) low molecules alcohol-water solution so that Pneumocandin B0
It can relatively centrally elute, while the extremely weak pigment of polarity and other impurities is remained on pillar.Pass through
These means, the purity of Pneumocandin B0 are significantly improved.
In the inventive method in step 4), activated carbon, decolorizing effect highly significant are used in high concentration alcoholic solution so that
The color of Pneumocandin B0 solution is changed into extremely light yellow from light brown, and causes the Pneumocandin B0 damage control within 5%,
The addition of activated carbon is preferably 2%, and bleaching temperature is preferably 40 DEG C.
In the inventive method step 5), the polymeric adsorbent of selection is skp-40-4300, and the resin is by lipophilicity divinyl
The macroporous copolymer that two kinds of monomers of benzene and hydrophily NVP aggregate into by a certain percentage.Its retention mechanism is anti-
Phase, good water logging lubricant nature is provided come the reservation increased to polar substances by one " special polarity captures group ".Utilize
The characteristics of this polymer, while being adsorbed Pneumocandin B0, the slightly stronger impurity of polarity can be made to be rushed in chromatographic column
It can be eluted when washing, Pneumocandin B0 is further purified, purity is further improved, and pigment is substantially removed
Totally, meanwhile, the polymer has obvious enrichment to target product, is advantageous to the vacuum concentration of next step.By vacuum
After concentration, Pneumocandin B0 is essentially colourless.
Resin is preferably skp-40-4300, and the concentration adjustment of upper prop liquid alcohol is preferably 30% (V/V), cleaning solution it is pure and strong
Degree is preferably 50% (V/V), and the determining alcohol of eluent is preferably 90% (V/V).
The present invention 6) step in, due to inventive step 5) in enrichment so that the effluent volume received is little
And determining alcohol is higher, be suitable for being concentrated in vacuo, still using the method being concentrated in vacuo, target product do not lose substantially.
Large-molecular peptides substance crystallization technique unmanageable (the cooling crystallization time is very long), yield are low, and purification effect is bad.Pass through
A large amount of contrast tests, the method for vacuum concentration are more much better than traditional method for crystallising.In an experiment, Pneumocandin B0 96%
Purity above can meet the needs of Caspofungin synthesis, if it is desired to further improving purity, can use and be published in 1992
An entitled Pneumocandin from Zalerion on June 15, the journal of antibiotics magazines
Method prepared by the silica gel in arboricloa article, collects the efflux rich in Pneumocandin B0, purity can be further
Bring up to more than 95%.Certainly, we have also been developed the preparation method of maturation, and can be further purified makes Pneumocandin B0 pure
Degree reaches more than 98%.
Relative to prior art, advantages of the present invention is it is obvious that be easily achieved industrialized production.Carried simultaneously using the invention
The technique of confession, the pigment in zymotic fluid and impurity can be removed substantially so that the purity of Pneumocandin B0, which reaches, to be more than
96.5% level.
Embodiment
Beneficial effects of the present invention now are further described by following examples, embodiment is only used for the purpose of illustration,
Do not limit the scope of the invention, while the obvious change and modification that those of ordinary skill in the art are made according to the present invention
It is also contained within the scope of the invention.
Embodiment
Pneumocandin B0 HPLC testing conditions of the present invention are as follows：Chromatographic column：The C18 of ODS mono- (5um)；
Detection wavelength：210nm；
Mobile phase：Water：Acetonitrile (55:45)；Flow velocity：1.0ml/min
Column temperature：40℃.
Pneumocandin B0 fermentation condition of the present invention is as follows：
Strain：ATCC 20957
Inclined-plane culture
Culture medium：PDA
Condition：25 DEG C, 12 days
Seed culture
Culture medium：Glucose 1.0%, mannitol 0.5%, glycerine 1.0%, peptone 0.5%, cotton seed meal 2.5%,
KH2P040.5%, pH is 6.8 1 7.0 before disappearing
Condition：25 DEG C, 4 days, shaking speed：250rpm
Fermented and cultured
Culture medium：Mannitol 8.0%, the cotton seed meal 3.0% of glucose 2.0%, dusty yeast 0.5%, KHZPO4 0.5%, disappear
Preceding regulation pH7.0.
121 DEG C of sterilizing 30min, inoculum concentration 10%, shaking flask loading amount 200ml/1000ml, are placed in 25 DEG C of thermostatic chambers rotary
Shaking table 250rpm shake flask fermentations 12 days, obtain Pneumocandin B0 zymotic fluid.
Embodiment 1
1) by the zymotic fluid 1000ml containing Pneumocandin B0, purity 2.6%, fermentation titer 675mg/L, in stirring
Under state, with phosphorus acid for adjusting pH value 4.2,2% filter aid diatomite is added, is mixed, stands 0.5 hour, filtering, obtains solid
Bacteria residue；
2) 2 times of 95% alcohol steep of bacteria residue, filters off except mycelia, obtains leaching liquor, color is dark red；Adjust the alcohol of leaching liquor
For concentration to 20 (V/V), this step yield is 81%；
3) a height 25cm is filled, diameter about 4cm D101 posts, sample solution is flowed through into D101 posts with 5ml/min speed,
Resin column is rinsed with purified water, 30% (V/V) ethanol-water solution successively, is then eluted and set with 80% (V/V) ethanol-water solution
Fat post, the eluent rich in Pneumocandin B0 is collected, be 46% through high pressure liquid phase detection purity, color substantially shoals, this step
Yield is 90%；
4) 0.5% activated carbon is added in eluent, is stirred 30 minutes under the conditions of 20 DEG C, filtering is decolourized, decolorizing effect
Preferably, color is in light yellow that this step yield is 96%；
5) Pneumocandin B0 destainer is diluted with water to 30% (V/V) after ultra-filtration filters.A height 25cm is filled,
Diameter about 4cm KB-SPE-HLB posts, sample solution is flowed through into KB-SPE-HLB posts with 5ml/min speed, with containing ethanol 30%
(V/V) solution rinses resin column, then is eluted with ethanol 80% (V/V) solution, collects the eluent rich in Pneumocandin B0, liquid phase
It is 96.5% to detect purity, and it is 91% that color, which substantially shoals as light yellow, this step yield,；
6) the Pneumocandin B0 eluent that will be collected into, it is concentrated in vacuo under the conditions of 30 DEG C, obtains Pneumocandin B0 crude product；
Crude product purity 96.5%.
Above step total recovery 64%, Pneumocandin B0 purity reach 96.5%.
Embodiment 2
1) by the zymotic fluid 1000ml containing Pneumocandin B0, purity 2.7%, fermentation titer 701mg/L, in stirring
Under state, with phosphorus acid for adjusting pH value 6,5% filter aid perlite is added, is mixed, stands 0.5 hour, filtering, obtains solid bacterium
Slag, bacteria residue color are shallower；
2) 5 times of 95% alcohol steep of bacteria residue, filters off except mycelia, obtains leaching liquor, color is dark red；Adjust the alcohol of leaching liquor
Concentration is to 30% (V/V), this step yield 87%；
3) a height 25cm is filled, diameter about 4cm HPD300 posts, sample solution is flowed through with 5ml/min speed
HPD300 posts, resin column is rinsed with purified water, 40% (V/V) ethanol-water solution successively, it is then molten with 90% (V/V) alcohol-water
Liquid elutes resin column, collects the eluent rich in Pneumocandin B0, is 41% through high pressure liquid phase detection purity, color is red, bright
Aobvious to shoal, this step yield is 93%；
4) 5% activated carbon is added in eluent, is stirred 60 minutes under the conditions of 50 DEG C, filtering decolourize, decolorizing effect compared with
Good, color is in light yellow, and this step yield is 88%；
5) Pneumocandin B0 destainer is diluted with water to 20% (V/V) after ultra-filtration filters.A height 25cm is filled,
Diameter about 4cm skp-40-4300 posts, sample solution is flowed through into skp-40-4300 posts with 5ml/min speed, with containing ethanol
50% (V/V) solution rinses resin column, then is eluted with containing ethanol 90% (V/V) solution, collects the elution rich in Pneumocandin B0
Liquid, liquid phase detection purity is 97.9%, and color substantially shoals, and this step yield is 94%；
6) the Pneumocandin B0 eluent that will be collected into, it is concentrated in vacuo under the conditions of 50 DEG C, obtains Pneumocandin B0 crude product；
Crude product purity 97.9%.
Above step total recovery 67%, Pneumocandin B0 purity reach 97.9%.
Embodiment 3
1) by the zymotic fluid 1000ml containing Pneumocandin B0, purity 2.2%, fermentation titer 694mg/L, in stirring
Under state, with phosphorus acid for adjusting pH value 5,2% filter aid Emathlite is added, is mixed, stands 0.5 hour, filtering, obtains solid
Bacteria residue, bacteria residue color are shallower；
2) 3 times of 95% alcohol steep of bacteria residue, filters off except mycelia, obtains leaching liquor, color is dark red；Adjust the alcohol of leaching liquor
Concentration is to 30% (V/V), this step yield 85%；
3) a height 25cm is filled, diameter about 4cm skp-10-4300 posts, sample solution is flowed through with 5ml/min speed
Skp-10-4300 posts, resin column is rinsed with purified water, 50% (V/V) ethanol-water solution successively, then uses the (V/ containing ethanol 80%
V) solution elution resin column, collects the eluent rich in Pneumocandin B0, is 39.8% through high pressure liquid phase detection purity, color is red
Color, hence it is evident that shoal, this step yield is 95%；
4) 3% activated carbon is added in eluent, is stirred 40 minutes under the conditions of 40 DEG C, filtering decolourize, decolorizing effect compared with
Good, color is in light yellow, and this step yield is 92%；
5) Pneumocandin B0 destainer is diluted with water to 30% (V/V) after ultra-filtration filters.A height 25cm is filled,
Diameter about 4cm Waters Oasis HLB posts, sample solution is flowed through into Waters Oasis HLB posts with 5ml/min speed,
Resin column is rinsed with containing ethanol 45% (V/V) solution, then is eluted with ethanol solution, collects the elution rich in Pneumocandin B0
Liquid, liquid phase detection purity is 97.1%, and color substantially shoals, and this step yield is 95%；
6) the Pneumocandin B0 eluent that will be collected into, it is concentrated in vacuo under the conditions of 40 DEG C, obtains Pneumocandin B0 crude product；
Crude product purity 97.1%.
Above step total recovery 70%, Pneumocandin B0 purity reach 97.1%.
Embodiment 4
1) by the zymotic fluid 1000ml containing Pneumocandin B0, purity 2.8%, fermentation titer 639mg/L, in stirring
Under state, with phosphorus acid for adjusting pH value 4.2,2% filter aid perlite is added, is mixed, stands 0.5 hour, filtering, obtains solid
Bacteria residue, bacteria residue color are shallower；
2) bacteria residue is extracted with 3 times of absolute methanols, is filtered off except mycelia, obtains leaching liquor, and color is dark red；Adjust the alcohol of leaching liquor
Concentration is to 30% (V/V), this step yield about 80%；
3) a height 25cm is filled, diameter about 4cm skp-10-4300 posts, sample solution is flowed through with 5ml/min speed
Skp-10-4300 posts, resin column is rinsed with purified water, 50% (V/V) methanol-water solution successively, then uses the (V/ containing methanol 80%
V) solution elution resin column, collects the eluent rich in Pneumocandin B0, is 44.9% through high pressure liquid phase detection purity, color is red
Color, hence it is evident that shoal, this step yield is 91%；
4) 3% activated carbon is added in eluent, is stirred 30 minutes under the conditions of 30 DEG C, filtering decolourize, decolorizing effect compared with
Good, color is in light yellow, and this step yield is 89%；
5) Pneumocandin B0 destainer is diluted with water to 30% (V/V) after ultra-filtration filters.A height 25cm is filled,
Diameter about 4cm skp-40-4300 posts, sample solution is flowed through into skp-40-4300 posts with 5ml/min speed, with containing methanol
45% (V/V) solution rinses resin column, then is eluted with absolute methanol solution, collects the eluent rich in Pneumocandin B0, liquid phase
It is 97.8% to detect purity, and color substantially shoals, and this step yield is 96%；
6) the Pneumocandin B0 eluent that will be collected into, it is concentrated in vacuo under the conditions of 30 DEG C, obtains Pneumocandin B0 crude product；
Crude product purity 97.8%.
Above step total recovery 62%, Pneumocandin B0 purity reach 97.8%.
Embodiment 5
1) by the zymotic fluid 1000ml containing Pneumocandin B0, purity 2.9%, fermentation titer 679mg/L, in stirring
Under state, with phosphorus acid for adjusting pH value 4.5,2% filter aid perlite is added, is mixed, stands 0.5 hour, filtering, obtains solid
Bacteria residue, bacteria residue color are shallower；
2) 3 times of 95% alcohol steep of bacteria residue, filters off except mycelia, obtains leaching liquor, color is dark red；Adjust the alcohol of leaching liquor
Concentration is to 30% (V/V), this step yield 88%；
3) a height 25cm is filled, diameter about 4cm skp-10-4300 posts, sample solution is flowed through with 5ml/min speed
Skp-10-4300 posts, resin column is rinsed with purified water, 45% (V/V) ethanol-water solution successively, then uses the (V/ containing ethanol 80%
V) solution elution resin column, collects the eluent rich in Pneumocandin B0, is 48.7% through high pressure liquid phase detection purity, color is red
Color, hence it is evident that shoal, this step yield is 94%；
4) 2% activated carbon is added in eluent, is stirred 40 minutes under the conditions of 40 DEG C, filtering decolourize, decolorizing effect compared with
Good, color is in light yellow, and this step yield is 93%；
5) Pneumocandin B0 destainer is diluted with water to 30% (V/V) after ultra-filtration filters.A height 25cm is filled,
Diameter about 4cm skp-40-4300 posts, sample solution is flowed through into skp-40-4300 posts with 5ml/min speed, with containing ethanol
50% (V/V) solution rinses resin column, then is eluted with containing ethanol 80% (V/V) solution, collects the elution rich in Pneumocandin B0
Liquid, liquid phase detection purity is 99.2%, and color substantially shoals, and this step yield is 96%；
6) the Pneumocandin B0 eluent that will be collected into, it is concentrated in vacuo under the conditions of 40 DEG C, obtains Pneumocandin B0 crude product；
Crude product purity 99.2%.
Above step total recovery 74%, Pneumocandin B0 purity reach 99.2%.
Claims (8)
1. a kind of extraction of Pneumocandin B0, purification process, comprise the following steps：
1) by the zymotic fluid containing Pneumocandin B0 in the state of stirring, pH value 4.2-6 is adjusted, adds filter aid, is mixed, mistake
Filter, obtains solid bacteria residue；
2) the low molecule alcohol-water solution extraction of bacteria residue high concentration, filtering, obtains leaching liquor；Pure water is added to make the dense of alcohol in leaching liquor
Spend for 20-30% (V/V)；
3) after adsorbing leaching liquor with polymeric adsorbent, tree is rinsed with purified water, 30-50% (V/V) low mass molecule alcohol-aqueous solution successively
Fat, resin then is eluted with low mass molecule alcohol 80-90% (V/V)-aqueous solution, collect the eluent rich in Pneumocandin B0；
4) eluent quality 0.5-5% activated carbons are added in eluent, are stirred under the conditions of 20-50 DEG C 30-60 minutes, filtering
Decolourize；
5) Pneumocandin B0 destainer is diluted with water to the concentration 20-30% (V/V) of alcohol after ultra-filtration filters, is set with absorption
Fat adsorbs, and first washs resin with 30-50% (V/V) low mass molecule alcohol-aqueous solution, then with 80-100% (V/V) low mass molecule alcohol-water
Solution elutes, and collects the eluent rich in Pneumocandin B0；
6) the Pneumocandin B0 eluent that will be collected into, is concentrated in vacuo under the conditions of 30-50 DEG C, obtains off-white color crude product.
2. in accordance with the method for claim 1, it is characterised in that in described step 1), regulation pH value and addition filter aid are same
When use.
3. in accordance with the method for claim 1, it is characterised in that the drainage dosage that described step 1) adds is zymotic fluid matter
The 0.5-5% of amount.
4. in accordance with the method for claim 1, it is characterised in that the filter aid that described step 1) adds is diatomite, pearl
One or more in rock, cellulose, magnesia, gypsum, Emathlite.
5. in accordance with the method for claim 1, it is characterised in that described low mass molecule alcohol is：One in methanol, ethanol, propyl alcohol
Kind；Described high concentration is 90-98% (V/V) low mass molecule alcohol-aqueous solution.
6. in accordance with the method for claim 1, it is characterised in that in described step 2), leaching liquor addition is bacteria residue quality
2-5 times.
7. in accordance with the method for claim 1, it is characterised in that in described step 3), polymeric adsorbent model skp- used
10-4300、D101、HPD300、HPD700、BS-5。
8. in accordance with the method for claim 1, it is characterised in that in described step 5), polymeric adsorbent model used
skp-40-4300、KB-SPE-HLB、Waters Oasis HLB(30-60um)。
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| CN111925421A (en) * | 2020-07-02 | 2020-11-13 | 北大方正集团有限公司 | Method for extracting pneumocandin B0 from fermentation liquor and application |
| CN112321682A (en) * | 2020-11-24 | 2021-02-05 | 苏州纳微科技股份有限公司 | Purification method of pneumocandin B0 |
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| US20130030149A1 (en) * | 2010-03-29 | 2013-01-31 | Kushal Rastogi | Process for purification of pneumocandin |
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