CN109400458A - A method of the separation and Extraction Co-Q10 from microbial fermentation solution - Google Patents
A method of the separation and Extraction Co-Q10 from microbial fermentation solution Download PDFInfo
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- C07—ORGANIC CHEMISTRY
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- C07C46/00—Preparation of quinones
- C07C46/10—Separation; Purification; Stabilisation; Use of additives
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Abstract
The method of the invention discloses a kind of from microbial fermentation solution separation and Extraction Co-Q10.Include: to filter microbial fermentation solution, is dried to obtain bacterium powder；Recycle superfine communication technique that bacterium powder is ground into the Co-Q10 micro mist that partial size is 2-35 μm；It extracts and is separated by solid-liquid separation again, chromatograph, crystallization obtains Co-Q10 finished product.Method high income of the invention, good product quality, simple process, extraction time is short, and separation and Extraction is high-efficient, at low cost, is suitable for industrialized production.
Description
Technical field
The present invention relates to Co-Q10s to extract field, more particularly to one kind separation and Extraction Co-Q10 from microbial fermentation solution
Method.
Background technique
Co-Q10 (Coenzyme Q10, be abbreviated as CoQ10) also known as ubiquinone, are a kind of biostearin substances, in animals and plants
And it is widely present in microorganism.Co-Q10 is the cell metabolism activator and antioxidant of archebiosis synthesis, it can be acted on
Certain enzymes are allowed to that the variation of three-dimensional structure occurs, to influence its physiological activity.Past research and clinical test prove coenzyme
Q10, which has, increases immunity of organisms, prevents the effect of cerebrovascular sclerosis, to improvement hypertension, Congestive heart failure, nerveous system
The treatment of disease of uniting and tumour etc. is helpful.Currently, natural products of the Co-Q10 as a kind of preciousness, is commonly used to give birth to
In the production of chemical drug object, health-preserving food and cosmetics.
There are mainly three types of the preparation methods of Co-Q10: chemical synthesis, animal vegetable tissue extraction method and microbial fermentation
Method.And microbe fermentation method has the advantage that microbial resources are abundant compared to first two method, production cost is low, does not generate
Optical siomerism, bioactivity height etc..So microbe fermentation method is considered as a kind of production method most with prospects.
Due to microbial fermentation solution complicated component, when the separation and Extraction Co-Q10 from microorganism, a large amount of impurity also can be by
It extracts together, this directly affects the complexity of subsequent purification purification operations.The superiority and inferiority of extraction process also directly influences
The yield of extraction, the quality of product, cost of purification etc..
According to current document report, the method that Co-Q10 is extracted from coenzyme Q 10 fermentation broth mainly has the saponification of alkali alcohol
Method, alkalization saponification method, ultrasonic fragmentation, supercritical CO2Extraction etc..The Chinese patent of Publication No. CN104694613A is public
A kind of method that alkali alcohol saponification method extracts Co-Q10 has been opened, using coenzyme Q 10 fermentation broth as raw material, has passed through organic solvent extraction, alkali
Alcohol saponification, silica gel column chromatography, dehydrated alcohol crystallization, suction filtration, vacuum drying obtain Co-Q10 product, this technique can be in certain journey
The purity of Co-Q10 crude extract is improved on degree, but organic solvent consumption is big in technical process, extraction efficiency is not high, generates big
Saponification waste-water and washes are measured, environment is seriously polluted, is the non-friendly technique of environment.
The Chinese patent literature of Publication No. CN102557912A discloses a kind of saponification method and prepares Co-Q10 crude extract
Method.It is saponified Co-Q10 extracting solution with lye, the extracting solution after being saponified after organic phase washing, for further purifying
It refines, in the technique, does not generate single or double oxyethyl derivatives, but extraction process emulsification is serious, influence to extract yield and quality,
Extraction efficiency is low.
The Chinese patent literature of Publication No. CN103819326A discloses a kind of ultrasonication extraction, chromatographic purifying preparation
The method of Co-Q10.In the technique, extraction efficiency can be improved, but ultrasonication process is highly exothermic, frozen cooling need to be increased
Equipment, ultrasonic wave is fast with processing thickness degree increase energy attenuation, limits treating capacity, and ultrasonic equipment value is expensive, big life
It produces and applies high production cost, be not suitable for industrialized production.
A kind of supercritical CO disclosed in the Chinese patent of Publication No. CN101591685A2Under the conditions of microorganism conversion prepare
The method of Co-Q10 accesses schizosaccharomyces pombe bacterium in the culture medium containing Salanesol, temperature is 25-35 DEG C, pressure is
The supercritical CO of 5-20MPa2Environment in ferment, obtain Co-Q10, the technique can improve Co-Q10 synthesis speed
And yield, but the method operating pressure is high, and investment is big, and energy consumption is high, and equipment is unstable, is unfavorable for commercial introduction.
A kind of preparation method of Co-Q10 sterling disclosed in the Chinese patent of Publication No. CN107337593A, using pottery
Porcelain membrane microfiltration, spray drying process crush mycelia, then separate through acetone reflux extraction, purpose ceramic-film filter, and hyphal cell is broken
Wall, separation are enriched with Co-Q10, and this method can be improved extract yield 7% or so, but spray drying process crushes mycelia, equipment compared with
Complexity takes up a large area, and primary investment is big；Atomizer, bacterium powder reclaiming device price are higher；It needs air capacity more, increases air blast
The power consumption of machine and the capacity of recyclable device；The thermal efficiency is not high, and heat loss is big, and the mycelia partial size crushed is larger, and cell is broken
Wall is not thorough, and the more difficult Co-Q10 extracted is still remained in cell.Prior fermentation liquid uses microfiltration of ceramic membrane and separation of solid and liquid
The purpose ceramic-film filter that process uses is perishable in alkaline environment, and elasticity is small frangible, and difficult forming, filter membrane easily blocks, and is unfavorable for
Commercial introduction.
Superfine communication technique is a kind of materiel machining new technology derived from the end of the 20th century, main application be mineral drug,
Valuable medicine and Chinese medicine with special nature are current international advanced crushing technologies, can obtain material from conventional size reduction technique
To 75 μm of medium particle diameter hereinafter, being increased to 5 μm hereinafter, cell-wall breaking ratio >=95%, can make the effective component in cell direct
Be exposed, rather than it is traditional pass through cell wall and cell membrane release.
Yang Jing etc., which is reported, pulverizes polysaccharide process research " Yang Jing, Zhang Shuai, Wang Dong in combining ultrasonic method extraction safflower
Plum waits to pulverize combining ultrasonic method and extracts polysaccharide process research [J] Harbin University of Commerce journal (natural science in safflower
Version), 2014 (3): 354-356. ", by the comparative study to the recovery rate for pulverizing front and back safflower polysaccharide, determine Ultramicro-powder
It is broken to polysaccharide extract rate have significantly improve effect；Lv Ping reports ultramicro grinding combining ultrasonic auxiliary extraction and prepares the total soap of ginseng
Glycosides research " Lv Ping pulverizes combining ultrasonic auxiliary extraction and prepares research [J] food research and development of general ginsenoside,
2016,37 (16): 42-46. ", test result shows that Radix Ginseng micropowder's minimum average particle diameters after ultramicro grinding can reach 49.18 μ
M, only 1/3 to the 1/4 of coarse powder, and the extraction efficiency of total saposins in ginseng can be significantly improved.Polysaccharide is macromolecule carbon aquation
Object is closed, general ginsenoside is steroid compound, triterpenoid saponin, and Co-Q10 is a kind of biostearin substance, fat-soluble quinones
Compound, by superfine communication technique applied to there is not been reported for separation and Extraction Co-Q10 from microbial fermentation solution.
Summary of the invention
It is an object of the invention to overcome the shortage of prior art, a kind of high income is provided, good product quality, simple process,
Extraction time is short, and separation and Extraction is high-efficient, at low cost, is suitable for separation and Extraction coenzyme in the slave microbial fermentation solution of industrialized production
The method of Q10.
To achieve the above object, the present invention provides a kind of method of separation and Extraction Co-Q10 from microbial fermentation solution, packet
Include following steps:
The preparation of bacterium powder: microbial fermentation solution is filtered, bacterium powder is dried to obtain；
Bacterium powder crushes: bacterium powder being ground into the Co-Q10 micro mist that partial size is 2-35 μm using superfine communication technique；
It extracts: will be extracted after solvent 1 and Co-Q10 micro mist stirring；
It is separated by solid-liquid separation: the above-mentioned extract liquor 1 being obtained by extraction and bacteria residue being entered into cyclone separator, using cyclone separation process
Carry out isolated extract liquor 2；
Chromatography: extract liquor 2 carries out column chromatography, and chromatographic column therein is washed with eluant, eluent, elutes, Fractional Collections elution
Liquid, reduced pressure steam organic solvent, obtain Co-Q10 concentrate；
Crystallization: solvent 2 being added into the Co-Q10 concentrate, crystallizes, and filtering obtains Co-Q10 finished product.
Further, the microbial fermentation solution be produce Co-Q10 microbial fermentation solution, it is preferred that for hydrogenlike silicon ion,
Aspergillus fumigatus, Fermentation with Rhizobium radiobacter, capsula Rhodopseudomonas, mucilage binding red pseudomonas, false calico, which are strangled, plays spore yeast, the false silk ferment in the torrid zone
At least one of mother, Pseuomonas denitrifican, this paddy pseudomonad, Paracoccus denitrificans, the fermentation liquid of nucleoprotamine Bacillus.
Further, the bacterium powder pulverising step refers to is crushed with micronizer, and temperature when crushing is in 10-45
℃。
Further, in the extraction step, Co-Q10 micro mist is conveyed into extractor, solvent 1 is passed through and is stirred extraction
It takes, speed of agitator control stirs 0.5-3h in 15-20r/min；
Optional, the solvent 1 is acetone, chloroform, ether, n-hexane, ethyl alcohol or petroleum ether.
Optional, the feed liquid mass ratio in the Co-Q10 micro mist and solvent 1 is 1:4-1:20；
Optional, the Extracting temperature control is at 15-45 DEG C.
Further, in the solid-liquid separation step, extract liquor 1 extracted and bacteria residue is passed through cyclone separator and divided
It is 0.1-0.5MPa from, control cyclone separator feed pressure, the cyclone separator is made of or more primary cyclone
Grade cyclone separator is composed in series.
Further, in the chromatographic step, the filler of chromatographic column is aluminium oxide or silica gel；
Optional, eluant, eluent includes component A and component B, wherein component A be petroleum ether, ether, isopropyl ether, diisopropyl ether,
One kind or several of ethyl-butyl ether, n-hexane, normal heptane, normal octane, pentamethylene, methyl cyclopentane, hexamethylene, hexahydrotoluene
Kind；
Component B be acetone, butanone, methanol, ethyl alcohol, normal propyl alcohol, isopropanol, methyl formate, Ethyl formate, propyl formate,
The one or more of ethyl acetate, methyl acetate；
Optional, the volumetric concentration of component A and component B are than control at (97=30): (3=70)；
Optional, the eluent refers to eluent of the purity of Co-Q10 at 90%-98% sections.
Further, in the crystallisation step, the dosage of the solvent 2 is 2-15 times of concentrate quality；
Optional, the solvent 2 is acetone, butanone, methanol, ethyl alcohol, normal propyl alcohol, isopropanol, methyl formate, formic acid second
Ester, propyl formate, ethyl acetate, methyl acetate, oily ether, ether, isopropyl ether, diisopropyl ether, ethyl-butyl ether, n-hexane, just
One or more of heptane, normal octane；
Optional, the crystallization, filtering, drying are that heating is dissolved, and solution temperature controls after 40-65 DEG C, dissolution
It is stirred for cooling down, speed of agitator control is in 15=20r/min, and rate of temperature fall control is in 5-15 DEG C/h, decrease temperature crystalline, final temperature control
At 0-25 DEG C, centrifuge filtering is dry.
For the present invention on the basis of extracting method of existing Co-Q10, lower for extraction process efficiency, time-consuming, yield
Not high disadvantage introduces the Co-Q10 micro mist that bacterium powder is ground into 2-35 μm by superfine communication technique before extraction, significantly improves
Extract yield greatly improves extraction efficiency, shortens extraction time.
Applicant of the present invention has found that ultramicro grinding can effectively improve Co-Q10 in bacterium powder after further investigation
Dissolution, but proportionate relationship is not presented in the partial size and extraction efficiency that pulverize, is crushed in 75-35 μ m in bacterium powder
When, the variation of Co-Q10 dissolution rate is smaller, basically reaches dissolution balance；When bacterium powder is crushed in 35-2 μ m, powder cell
Tissue changes, and effective component is in abundant release conditions, and dissolution rate is accelerated, and the amount of dissolution increases suddenly within a certain period of time,
It discharges more complete；And when partial size, which continues, to be reduced, cell is almost all broken, but recovery rate decreases instead.Therefore really
When extracting Co-Q10 surely, the optimum grain-diameter range that bacterium powder pulverizes is 35-2 μm.Office will not be generated during ultramicro grinding
Portion's superheating phenomenon, or even can be carried out under low-temperature condition, crushing instantaneously can be completed, thus can retain powder to the maximum extent
Bioactive ingredients are conducive to that high quality of products is made.The setting of superfine communication technique hierarchy system both strictly limits big
Grain, in turn avoided broken, obtained the uniform superfine powder of particle diameter distribution, while largely increasing the specific surface area of micro mist,
Make smashed bacterium powder that there is more good dissolubility, dispersibility, adsorptivity, chemical reactivity etc..Ultramicro grinding is to seal
Progress in system is closed, had not only avoided micro mist pollution ambient enviroment, but also the dust polluted product in air can be prevented.Due to passing through
There is raw material after ultramicro grinding great specific surface to react the area of contact significantly in the reaction process such as biology, chemistry
It increases, thus the application of superfine communication technique can thoroughly mention the remaining Co-Q10 ingredient for being difficult to extract intracellular
It takes out.
During the extraction process, discovery thallus crushing is too thin, easily agglomerates by applicant of the present invention, glues wall, blocking causes
Subsequent process has that thallus is difficult to separate with extracting solution, therefore creative the introducing in separation of solid and liquid process of applicant is revolved
Flow separation technology makes bacteria residue and extract liquor under a certain pressure from sand filter import tangentially to enter eddy flow sand filter, in it
High speed rotation generates centrifugal field, is separated according to the effect of density variation and centrifugal force between object, can accomplish serialization,
And can be carried out plural serial stage, greatly improve separating effect.The subsequent method using crystallization is purified.The method has dirt
Dye is less, easy to operate, process is simple, extraction time is short, separation and Extraction efficiency and high income, it is at low cost the advantages that, have very high
Commercial introduction potentiality.
The beneficial effects of the present invention are:
Present invention process method introduces superfine communication technique before extraction and crushes to bacterium powder, during ultramicro grinding
Hot-spot phenomenon will not be generated, or even can be carried out under low-temperature condition, crushing instantaneously can be completed, thus can be to the maximum extent
The bioactive ingredients for retaining powder, are conducive to that high quality of products is made.The setting of superfine communication technique hierarchy system, both strictly
Bulky grain is limited, in turn avoided broken, obtains the uniform superfine powder of particle diameter distribution, while largely increasing micro mist
Specific surface area makes smashed bacterium powder have more good dissolubility, dispersibility, adsorptivity, chemical reactivity etc..Ultramicro-powder
Broken carried out in closed system, has not only avoided micro mist pollution ambient enviroment, but also can prevent the dust polluted product in air.
Due to the raw material after pulverizing, there is great specific surface to react contact in the reaction process such as biology, chemistry
Area considerably increases, and improves Co-Q10 dissolution rate, thus the application of superfine communication technique remaining can be difficult to intracellular
The Co-Q10 ingredient extracted thoroughly extracts, and significantly improves extract yield, greatly improves extraction efficiency, contracting
Short extraction time.
Present invention process method uses cyclone separation process in separation of solid and liquid process, makes bacteria residue and extract liquor in a level pressure
From sand filter import tangentially to enter eddy flow sand filter under power, high speed rotation in it generates centrifugal field, according to close between object
The effect for spending difference and centrifugal force, achievees the effect that separation, can accomplish serialization, and can be carried out series connection, greatly improves point
From effect.
Present invention process method is compared with the traditional method, have pollution less, easy to operate, process is simple, extraction time is short,
The advantages that separation and Extraction is high-efficient, at low cost finally extracts the purity > 99.5% of obtained Co-Q10 crystal, total recovery >
95%.
Microbial fermentation solution of the present invention is the fermentation liquid for having Co-Q10 production capacity, can generate micro- life of Co-Q10
The fermentation liquid of object is all suitable for.Such as hydrogenlike silicon ion (Rhodobacter sphaeroides), aspergillus fumigatus (Aspergillus
Fumigatus), Fermentation with Rhizobium radiobacter (Rhizobium radiobacter), capsula Rhodopseudomonas
(Rhodopseudomonas capsulata), mucilage binding red pseudomonas (Rhodopseudomonas gelatinosa), vacation are white
Bu Le bullet spore yeast (Bullera psedoalba), candida tropicalis (Candida tropicalis), Pseuomonas denitrifican
(Pseudomonas denitrificans), this paddy pseudomonad (Pseudomonas umoros), Paracoccus denitrificans
The fermentation liquid of (Paracoccus denitrificans), nucleoprotamine Bacillus (Protaminobacter) etc..
The embodiment of the present invention shows there was only 89.32% without the extraction of ultramicro grinding, total recovery.And use this
The total recovery of the embodiment 1-9 of inventive technique scheme is all larger than 95%, and purity is all larger than 99.5%.It sufficiently proves using the present invention
The total recovery of the embodiment 1-9 of technical solution is high, purity is high.
Specific embodiment
The embodiment of the present invention is described below in detail, the examples of the embodiments are intended to be used to explain the present invention, and cannot
It is interpreted as limitation of the present invention.In the examples where no specific technique or condition is specified, described according to the literature in the art
Technology or conditions or carried out according to product description.Reagents or instruments used without specified manufacturer is that can lead to
Cross the conventional products of commercially available acquisition.
Embodiment 1:
10L hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid (content 3150mg/L) is taken, Bu Shi is used
Funnel dries bacterium powder after filtering in a vacuum drying oven, and the bacterium powder after drying is crushed to 2 μm of partial size or so with micronizer,
It is placed in wide-mouth bottle, 5 times of quality n-hexanes is added in 30 DEG C or so stirrings and extract 1h, extract liquor and bacteria residue are passed through cyclone separator
It being separated, extract liquor crosses silicagel column, is washed with 4% ethyl alcohol-hexane solution, elutes, and it collects principal piece and is concentrated,
2 times of quality ethyl alcohol are added into concentrate for recycled solvent, after being heated to 60 DEG C of dissolutions, then with 5 DEG C/h rate are cooled to 5
DEG C crystallization, centrifugal filtration is dried to obtain Co-Q10 finished product 30.02g, and purity reaches 99.60%, total recovery 95.30%.
Embodiment 2:
10L hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid (content 3150mg/L) is taken, Bu Shi is used
Funnel dries bacterium powder after filtering in a vacuum drying oven, and the bacterium powder after drying is crushed to 5 μm of partial size or so with micronizer,
It is placed in wide-mouth bottle, 10 times of quality ether is added, extract 1.5h in 40 DEG C or so stirrings, extract liquor and bacteria residue are passed through cyclonic separation
Device is separated, and extract liquor crosses silicagel column, is washed with 10% Data for Methyl Acetate-Methanol solution, elution, and it is dense to collect principal piece progress
Contracting, recycled solvent, 4 times of quality acetone of addition into concentrate after heating for dissolving, then with 10 DEG C/h rate are cooled to 10 DEG C
Crystallization, centrifugal filtration are dried to obtain Co-Q10 finished product 30.53g, and purity reaches 99.61%, total recovery 96.92%.
Embodiment 3:
10L hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid (content 3150mg/L) is taken, Bu Shi is used
Funnel dries bacterium powder after filtering in a vacuum drying oven, and the bacterium powder after drying is crushed to 10 μm of left sides of partial size with micronizer
The right side is placed in wide-mouth bottle, and 15 times of quality petroleum ethers are added in 35 DEG C or so stirrings and extract 1.2h, extract liquor and bacteria residue are passed through eddy flow
Separator is separated, and extract liquor crosses silicagel column, is washed with 15% butanone-n-heptane solution, elution, collect principal piece into
8 times of quality ethyl alcohol are added into concentrate for row concentration, recycled solvent, after being heated to 55 DEG C of dissolutions, then with 12 DEG C/h rate
15 DEG C of crystallizations are cooled to, centrifugal filtration is dried to obtain Co-Q10 finished product 30.72g, and purity reaches 99.67%, total recovery
97.52%.
Embodiment 4:
1000L hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid (content 3150mg/L) is taken, plate is used
By bacterium powder pneumatic conveying drying after frame filtering, the bacterium powder after drying is crushed to 20 μm of partial size or so with micronizer, is placed in wide-mouth
In bottle, 20 times of quality hexamethylenes are added, extract 2h in 45 DEG C or so stirrings, extract liquor and bacteria residue are passed through cyclone separator and are divided
From, extract liquor crosses silicagel column, is washed with 30% methanol-normal octane solution, elutes, and it collects principal piece and is concentrated, solvent
It recycles, 10 times of quality ethyl alcohol of addition into concentrate, after heating for dissolving, then 20 DEG C of crystallizations is cooled to 15 DEG C/h rate,
Centrifugal filtration is dried to obtain Co-Q10 finished product 30.76kg, and purity reaches 99.52%, total recovery 97.65%.
Embodiment 5:
1000L hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid (content 3150mg/L) is taken, plate is used
Bacterium powder is subjected to pneumatic conveying drying after frame filtering, the bacterium powder after drying is crushed to 30 μm of partial size or so with micronizer, is placed in extraction
It takes in tank, 10 times of quality petroleum ethers is added in 15 DEG C or so stirrings and extract 1h, extract liquor and bacteria residue are passed through cyclone separator progress
Separation, extract liquor are crossed silicagel column, are washed with 20% ethyl acetate-hexane solution, elute, and collect principal piece and are concentrated,
12 times of quality acetic acid ethyl esters are added into concentrate for recycled solvent, after being heated to 40 DEG C of dissolutions, then with 7 DEG C/h rate drop
Temperature to 0 DEG C of crystallization, centrifugal filtration is dried to obtain Co-Q10 finished product 3.02kg, and purity reaches 99.53%, total recovery 95.87%.
Embodiment 6:
1000L hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid (content 3150mg/L) is taken, plate is used
Bacterium powder is subjected to pneumatic conveying drying after frame filtering, the bacterium powder after drying is crushed to 2 μm of partial size or so with micronizer, is placed in extraction
In tank, 15 times of quality n-hexanes are added in 15 DEG C or so stirrings and extract 1.2h, extract liquor and bacteria residue are passed through cyclone separator progress
Separation, extract liquor are crossed silicagel column, are washed with 60% acetone-diethyl ether solution, elute, and collect principal piece and are concentrated, solvent
Recycle, 15 times of quality ethyl alcohol be added into concentrate, be heated to 45 DEG C dissolve after, then with 10 DEG C/h rate be cooled to 5 DEG C
Crystallization, centrifugal filtration are dried to obtain Co-Q10 finished product 3.01kg, and purity reaches 99.58%, total recovery 95.56%.
Embodiment 7:
Take 1000L capsula Rhodopseudomonas (Rhodopseudomonas capsulata) fermentation liquid (content 2260mg/
L), bacterium powder being subjected to pneumatic conveying drying with after plate-frame filtering, the bacterium powder after drying is crushed to 10 μm of partial size or so with micronizer,
It is placed in extractor, 20 times of quality chloroforms is added in 20 DEG C or so stirrings and extract 1.5h, extract liquor and bacteria residue are passed through cyclonic separation
Device is separated, and extract liquor crosses silicagel column, is washed with 10% normal propyl alcohol-pentamethylene solution, elution, is collected principal piece and is carried out
15 times of quality acetic acid methyl esters are added into concentrate for concentration, recycled solvent, after being heated to 50 DEG C of dissolutions, then with 15 DEG C/h
Rate is cooled to 15 DEG C of crystallizations, and centrifugal filtration is dried to obtain Co-Q10 finished product 2.19kg, and purity reaches 99.63%, total recovery
96.83%.
Embodiment 8:
Take 90m3Pseuomonas denitrifican (Pseudomonas denitrificans) fermentation liquid (content 2030mg/L),
Bacterium powder is subjected to pneumatic conveying drying with after plate-frame filtering, the bacterium powder after drying is crushed to 15 μm of partial size or so with micronizer, sets
In extractor, 5 times of quality acetone are added in 30 DEG C or so stirrings and extract 1h, extract liquor and bacteria residue are passed through cyclone and are divided
From, extract liquor crosses silicagel column, is washed with 5% ethyl acetate-light petrol solution, elutes, and it collects principal piece and is concentrated, solvent
It recycles, 10 times of quality methanol-ethyl acetate mixed solutions is added into concentrate, after being heated to 50 DEG C of dissolutions, then with 5
DEG C/h rate is cooled to 15 DEG C of crystallizations, centrifugal filtration is dried to obtain Co-Q10 finished product 177.2kg, and purity reaches 99.58%, always
Yield 97.01%.
Embodiment 9:
1000L candida tropicalis (Candida tropicalis) fermentation liquid (content 1780mg/L) is taken, sheet frame is used
Bacterium powder is subjected to pneumatic conveying drying after filtering, the bacterium powder after drying is crushed to 35 μm of partial size or so with micronizer, is placed in extraction
In tank, 10 times of quality petroleum ethers are added in 15 DEG C or so stirrings and extract 1h, extract liquor and bacteria residue are passed through cyclone separator and are divided
From extract liquor crosses silicagel column, is washed with 20% ethyl acetate-hexane solution, elutes, and collects principal piece and is concentrated, molten
Agent recycles, and 12 times of quality acetic acid ethyl esters are added into concentrate, after being heated to 40 DEG C of dissolutions, then with the cooling of 7 DEG C/h rate
It is crystallized to 0 DEG C, centrifugal filtration is dried to obtain Co-Q10 finished product 1.70kg, and purity reaches 99.50%, total recovery 95.24%.
Comparative example 1 (alkali alcohol saponification method):
Hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid 1000L (content 3150mg/L) is taken, plate is used
Bacterium powder is subjected to pneumatic conveying drying after frame filtering, the bacterium powder after drying is put into extraction column, and temperature is controlled at 30 DEG C or so, to bacterium powder
Middle Co-Q10 carries out refluxing extraction with petroleum ether, flows back after 2h, extracting solution is transferred in reaction kettle, and alkali methanol (PH7.8 is added
~8.0) saponification of alkali alcohol is carried out, is stirred 20 minutes, stands, layering, separates upper layer extracting solution, carries out purifies and separates into silicagel column,
Eluent is petroleum ether, collects the eluent of section containing Co-Q10, and vacuum distillation obtains purifying Co-Q10 concentrate, by concentrate
It is transferred in dissolving tank, 10 times of quality dehydrated alcohols is added, be heated to 48~50 DEG C, after stirring lower dissolution, filtered fluid is slowly dropped
Temperature is added Co-Q10 crystal seed, precipitates crystal to 30 DEG C, and temperature controls 23~25 DEG C, keeps the temperature 3h, filters, and wet crystal is dry through depressurizing
It is dry, obtain Co-Q10 sterling 2.58kg, purity 98.36%, total recovery 82.00%.
Comparative example 2 (alkalization saponification method):
Hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid 1000L (content 3150mg/L) is taken, plate is used
Bacterium powder is subjected to pneumatic conveying drying after frame filtering, the bacterium powder after drying is put into extraction column, and temperature is controlled at 30 DEG C or so, to bacterium powder
Middle Co-Q10 carries out refluxing extraction with petroleum ether, flows back after 2h, extracting solution is transferred in reaction kettle, and liquid alkaline (5% hydrogen-oxygen is added
Change sodium) it is saponified, it stirs 20 minutes, stands, layering, separate upper layer extracting solution, carry out purifies and separates, eluent into silicagel column
For petroleum ether, the eluent of section containing Co-Q10 is collected, vacuum distillation obtains purifying Co-Q10 concentrate, concentrate is transferred to molten
It solves in tank, 10 times of quality dehydrated alcohols is added, be heated to 48~50 DEG C, after stirring lower dissolution, by filtered fluid slow cooling to 30
DEG C, Co-Q10 crystal seed is added, precipitates crystal, temperature controls 23~25 DEG C, keeps the temperature 3h, filtering, and wet crystal is dried under reduced pressure, is obtained
To Co-Q10 sterling 2.75kg, purity 98.56%, total recovery 87.32%.
Comparative example 3 (microfiltration of ceramic membrane and spray drying extraction method):
Take hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid 1000L (content 3150mg/L), with into
Bacterium powder is spray-dried after microfiltration of ceramic membrane film (aperture 0.1um) filtering, the bacterium powder after drying is added in distiller, is added
0.6 ‰ (w/v) zeolites are added in 3.5 times of vol acetones, are to slowly warm up to 50 DEG C, flow back after 2.2h, filter while hot through ceramic element
Device filters (aperture 0.5um), and extracting solution carries out purifies and separates into silicagel column after filtering, and eluent is petroleum ether, collects and contains coenzyme
Q10 sections of eluents, vacuum distillation obtain purifying Co-Q10 concentrate, concentrate are transferred in dissolving tank, 10 times of quality are added
Dehydrated alcohol is heated to 48~50 DEG C, and after stirring lower dissolution, by filtered fluid slow cooling to 30 DEG C, Co-Q10 crystal seed is added,
It precipitating crystal, temperature controls 23~25 DEG C, keeps the temperature 3h, filtering, and wet crystal is dried under reduced pressure, Co-Q10 sterling 2.79kg is obtained,
Purity is 99.26%, total recovery 88.45%.
Comparative example 4 (extraction without ultramicro grinding):
1000L hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid (content 3150mg/L) is taken, plate is used
Bacterium powder is subjected to pneumatic conveying drying after frame filtering, the bacterium powder after drying is placed in extractor, and 10 times of quality petroleum ethers are added at 15 DEG C
Left and right stirring extraction 1h, extract liquor and bacteria residue are passed through cyclone separator and are separated, and extract liquor crosses silicagel column, with 20% acetic acid
Ethyl ester-hexane solution is washed, elution, is collected principal piece and is concentrated, and 12 times are added into concentrate for recycled solvent
Quality acetic acid ethyl ester after being heated to 40 DEG C of dissolutions, then with 7 DEG C/h rate is cooled to 0 DEG C of crystallization, and centrifugal filtration is dried to obtain auxiliary
Enzyme Q10 finished product 2.81kg, purity reach 99.18%, total recovery 89.32%.
Comparative example 5 (pulverizes extraction-partial size 0.1um):
1000L hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid (content 3150mg/L) is taken, plate is used
Bacterium powder is subjected to pneumatic conveying drying after frame filtering, the bacterium powder after drying is crushed to 0.1 μm of partial size or so with micronizer, is placed in extraction
It takes in tank, 10 times of quality petroleum ethers is added in 15 DEG C or so stirrings and extract 1h, extract liquor and bacteria residue are passed through cyclone separator progress
Separation, extract liquor are crossed silicagel column, are washed with 20% ethyl acetate-hexane solution, elute, and collect principal piece and are concentrated,
12 times of quality acetic acid ethyl esters are added into concentrate for recycled solvent, after being heated to 40 DEG C of dissolutions, then with 7 DEG C/h rate drop
Temperature to 0 DEG C of crystallization, centrifugal filtration is dried to obtain Co-Q10 finished product 2.89kg, and purity reaches 99.34%, total recovery 91.77%.
Comparative example 6 (pulverizes extraction-partial size 1um):
1000L hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid (content 3150mg/L) is taken, plate is used
Bacterium powder is subjected to pneumatic conveying drying after frame filtering, the bacterium powder after drying is crushed to 1 μm of partial size or so with micronizer, is placed in extraction
In tank, 10 times of quality petroleum ethers are added in 15 DEG C or so stirrings and extract 1h, extract liquor and bacteria residue are passed through cyclone separator and are divided
From extract liquor crosses silicagel column, is washed with 20% ethyl acetate-hexane solution, elutes, and collects principal piece and is concentrated, molten
Agent recycles, and 12 times of quality acetic acid ethyl esters are added into concentrate, after being heated to 40 DEG C of dissolutions, then with the cooling of 7 DEG C/h rate
It is crystallized to 0 DEG C, centrifugal filtration is dried to obtain Co-Q10 finished product 2.93kg, and purity reaches 99.39%, total recovery 92.97%.
Comparative example 7 (pulverizes extraction-partial size 40um):
1000L hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid (content 3150mg/L) is taken, plate is used
Bacterium powder is subjected to pneumatic conveying drying after frame filtering, the bacterium powder after drying is crushed to 40 μm of partial size or so with micronizer, is placed in extraction
It takes in tank, 10 times of quality petroleum ethers is added in 15 DEG C or so stirrings and extract 1h, extract liquor and bacteria residue are passed through cyclone separator progress
Separation, extract liquor are crossed silicagel column, are washed with 20% ethyl acetate-hexane solution, elute, and collect principal piece and are concentrated,
12 times of quality acetic acid ethyl esters are added into concentrate for recycled solvent, after being heated to 40 DEG C of dissolutions, then with 7 DEG C/h rate drop
Temperature to 0 DEG C of crystallization, centrifugal filtration is dried to obtain Co-Q10 finished product 2.90kg, and purity reaches 99.44%, total recovery 92.18%.
Comparative example 8 (pulverizes extraction-partial size 50um):
1000L hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid (content 3150mg/L) is taken, plate is used
Bacterium powder is subjected to pneumatic conveying drying after frame filtering, the bacterium powder after drying is crushed to 50 μm of partial size or so with micronizer, is placed in extraction
It takes in tank, 10 times of quality petroleum ethers is added in 15 DEG C or so stirrings and extract 1h, extract liquor and bacteria residue are passed through cyclone separator progress
Separation, extract liquor are crossed silicagel column, are washed with 20% ethyl acetate-hexane solution, elute, and collect principal piece and are concentrated,
12 times of quality acetic acid ethyl esters are added into concentrate for recycled solvent, after being heated to 40 DEG C of dissolutions, then with 7 DEG C/h rate drop
Temperature to 0 DEG C of crystallization, centrifugal filtration is dried to obtain Co-Q10 finished product 2.87kg, and purity reaches 99.52%, total recovery 91.01%.
Comparative example 9 (pulverizes extraction-partial size 70um):
1000L hydrogenlike silicon ion (Rhodobacter sphaeroides) fermentation liquid (content 3150mg/L) is taken, plate is used
Bacterium powder is subjected to pneumatic conveying drying after frame filtering, the bacterium powder after drying is crushed to 70 μm of partial size or so with micronizer, is placed in extraction
It takes in tank, 10 times of quality petroleum ethers is added in 15 DEG C or so stirrings and extract 1h, extract liquor and bacteria residue are passed through cyclone separator progress
Separation, extract liquor are crossed silicagel column, are washed with 20% ethyl acetate-hexane solution, elute, and collect principal piece and are concentrated,
12 times of quality acetic acid ethyl esters are added into concentrate for recycled solvent, after being heated to 40 DEG C of dissolutions, then with 7 DEG C/h rate drop
Temperature to 0 DEG C of crystallization, centrifugal filtration is dried to obtain Co-Q10 finished product 2.84kg, and purity reaches 99.58%, total recovery 90.13%.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art are not departing from the principle of the present invention and objective
In the case where can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.
Claims (7)
1. a kind of method of the separation and Extraction Co-Q10 from microbial fermentation solution, comprising the following steps:
The preparation of bacterium powder: microbial fermentation solution is filtered, bacterium powder is dried to obtain；
Bacterium powder crushes: bacterium powder being ground into the Co-Q10 micro mist that partial size is 2-35 μm using superfine communication technique；
It extracts: will be extracted after solvent 1 and Co-Q10 micro mist stirring；
It is separated by solid-liquid separation: the above-mentioned extract liquor 1 being obtained by extraction and bacteria residue being entered into cyclone separator, carried out using cyclone separation process
Isolated extract liquor 2；
Chromatography: extract liquor 2 carries out column chromatography, and chromatographic column therein is washed with eluant, eluent, elutes, Fractional Collections eluent,
Reduced pressure steams organic solvent, obtains Co-Q10 concentrate；
Crystallization: solvent 2 being added into the Co-Q10 concentrate, crystallizes, and filtering obtains Co-Q10 finished product.
2. as described in claim 1 from microbial fermentation solution separation and Extraction Co-Q10 method, which is characterized in that it is described micro-
Bio-fermented liquid is the microbial fermentation solution for producing Co-Q10；Preferably, for hydrogenlike silicon ion, aspergillus fumigatus, Fermentation with Rhizobium radiobacter,
Capsula Rhodopseudomonas, mucilage binding red pseudomonas, false calico are strangled and play spore yeast, candida tropicalis, Pseuomonas denitrifican, this paddy
At least one of pseudomonad, Paracoccus denitrificans, fermentation liquid of nucleoprotamine Bacillus.
3. as described in claim 1 from microbial fermentation solution separation and Extraction Co-Q10 method, which is characterized in that the bacterium
Powder pulverising step refers to be crushed with micronizer, and temperature when crushing is at 10-45 DEG C.
4. as described in claim 1 from microbial fermentation solution separation and Extraction Co-Q10 method, which is characterized in that it is described to mention
It takes in step, Co-Q10 micro mist is conveyed into extractor, be passed through solvent 1 and be stirred extraction, speed of agitator is controlled in 15-
20r/min stirs 0.5-3h；
Optional, the solvent 1 is acetone, chloroform, ether, n-hexane, ethyl alcohol or petroleum ether；
Optional, the feed liquid mass ratio in the Co-Q10 micro mist and solvent 1 is 1:4-1:20；
Optional, the Extracting temperature control is at 15-45 DEG C.
5. as described in claim 1 from microbial fermentation solution separation and Extraction Co-Q10 method, which is characterized in that it is described solid
In liquid separating step, extract liquor 1 extracted and bacteria residue are passed through cyclone separator and separated, control cyclone separator charging
Pressure is 0.1-0.5MPa, and the cyclone separator is made of primary cyclone or multistage cyclonic fluid separator is composed in series.
6. as described in claim 1 from microbial fermentation solution separation and Extraction Co-Q10 method, which is characterized in that the layer
It analyses in step, the filler of chromatographic column is aluminium oxide or silica gel；
Optional, eluant, eluent includes component A and component B, and wherein component A is petroleum ether, ether, isopropyl ether, diisopropyl ether, ethyl
The one or more of butyl ether, n-hexane, normal heptane, normal octane, pentamethylene, methyl cyclopentane, hexamethylene, hexahydrotoluene；
Component B is acetone, butanone, methanol, ethyl alcohol, normal propyl alcohol, isopropanol, methyl formate, Ethyl formate, propyl formate, acetic acid
The one or more of ethyl ester, methyl acetate；
Optional, the volumetric concentration of component A and component B are than control at (97-30): (3-70)；
Optional, the eluent refers to eluent of the purity of Co-Q10 at 90%-98% sections.
7. as described in claim 1 from microbial fermentation solution separation and Extraction Co-Q10 method, which is characterized in that the knot
In brilliant step, the dosage of the solvent 2 is 2-15 times of concentrate quality；
Optional, the solvent 2 is acetone, butanone, methanol, ethyl alcohol, normal propyl alcohol, isopropanol, methyl formate, Ethyl formate, first
Propyl propionate, ethyl acetate, methyl acetate, oily ether, ether, isopropyl ether, diisopropyl ether, ethyl-butyl ether, n-hexane, normal heptane,
One or more of normal octane；
Optional, the crystallization, filtering, drying are that heating is dissolved, and solution temperature control is stirred again after 40-65 DEG C, dissolution
Cooling is mixed, speed of agitator control is controlled in 5-15 DEG C/h, decrease temperature crystalline, final temperature in 0- in 15-20r/min, rate of temperature fall control
25 DEG C, centrifuge filtering is dry.
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| CN113636924A (en) * | 2021-09-14 | 2021-11-12 | 湖北诺克特药业股份有限公司 | Extraction and purification method for producing coenzyme Q10 by fermentation method |
| CN113754526A (en) * | 2021-09-27 | 2021-12-07 | 神舟生物科技有限责任公司 | High-purity coenzyme Q10 purification process |
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