CN109824496A - A method of the extraction purification farnoquinone from broken wall bafillus natto thallus - Google Patents
A method of the extraction purification farnoquinone from broken wall bafillus natto thallus Download PDFInfo
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Abstract
A method of the extraction purification farnoquinone from broken wall bafillus natto thallus, microorganism after fermentation is separated by solid-liquid separation with ultrafiltration and centrifugation, obtain wet thallus, after carrying out broken wall with Ion Beam Treatment wet thallus, it is extracted with dehydrated alcohol, can not only obtain a large amount of farnoquinone, also extract less grease, thick purifying has been carried out during the extraction process, provides good solution for the purifying of single step high efficiency below.By extract by column extracting, abstraction purification is carried out using the different organic reagent of polarity, the impurity extraction of opposed polarity is come out, so that the purity of solution be made to be greatly improved.Its freezing and crystallizing can be obtained into the farnoquinone crystal that purity is 95% later.This method eliminates the process of drying and thallus crushing in conventional method, improves the purity of extraction efficiency and extraction.And the higher vitamin K of purity is just obtained by single step purifying2。
Description
Technical field
The present invention relates to a kind of methods of extraction purification farnoquinone from broken wall bafillus natto thallus, belong to biology
Engineering and bio-separation engineering field.
Background technique
Vitamin K2It is a series of isoprene knots containing 2- methyl-1,4-naphthaquinone parent nucleus and C3 with number not etc.
The general designation of the terpenes side chain compound of structure unit, according to the number of carbon on terpenes side chain, can be divided into K2 (10), K2 (20),
K2 (35), K2 (40) etc..Vitamin K2It is a kind of liposoluble vitamin, the derivative of the naphthoquinones group with phylloquinone bioactivity
Object is one of indispensable important vitamin in human body.It with the research to its function, has been demonstrated that, vitamin K2Have
The effect of the diseases such as pre- preventing bone rarefaction, angiocarpy, Parkinson, liver cancer.Therefore, functional food, medical products, cosmetics etc. are led
Domain is to vitamin K2Demand increasingly increase, good market prospect.
Vitamin K2With chiral structure, trans-vitamin K2With bioactivity, cis vitamin K2It is living without biology
Property.Vitamin K made from chemical synthesis2Contain cis and trans simultaneously；In contrast, microbe fermentation method, it is available
Alltrans vitamin K2.Therefore, it has provided in the world, for the vitamin K in functional food2Microbial fermentation must be derived from.
Currently, bacillus natto to ferment prepares vitamin K2It is a kind of microbial fermentation production vitamin K generally used2Method.
Bacillus natto to ferment prepares vitamin K2In, the metabolite of bafillus natto is more, simultaneously because its
Aqtocytolysis can generate plurality of impurities, such as foreign protein, nucleic acid, grease, cause to obtain High Purity vitamin K2Difficulty compared with
Greatly.
Currently, organic solvent, which is widely used, extracts vitamin K from bacillus natto to ferment liquid2, this method need into
Pretreatment operations, the subsequent purification process such as the crushing of row thallus, dehydration and drying also need to carry out macroreticular resin and reverse phase silica gel etc.
Processing, integrated artistic is complicated, and expensive, recovery rate is lower.Extraction purification technology, it has also become bacillus natto to ferment preparation
Vitamin K2Bottleneck problem.
Summary of the invention
The purpose of the present invention is to provide the methods of the extraction purification farnoquinone from broken wall bafillus natto thallus.
In order to achieve the above objects and other related objects, present invention provide the technical scheme that from broken wall natto gemma bar
The method of extraction purification farnoquinone in bacterium thallus, including the following steps:
Step 1: bacillus natto to ferment liquid being filtered with the ceramic membrane that aperture is 5nm-10um, obtained concentration
Liquid is centrifuged 8-20min with the revolving speed of 5000-18000rpm, discards supernatant liquid, the deposit of collection is bafillus natto
New fresh thalli；
Step 2: by the new fresh thalli merging ion beam apparatus of bafillus natto, bombarding thallus, institute with nitrogen ion beam
It is 15keV-35keV with the energy of ion, the ionic weight of injection is 1 × 1015-5×1019N+/cm2, 10s is injected, injection is stopped
5s operates 30-60min repeatedly, the bafillus natto thallus after obtaining broken wall；
Step 3: being extracted including first time extraction and second；
It extracts for the first time: the bafillus natto thallus after broken wall is placed in the dehydrated alcohol of 5-25 times of its quality,
10min~40min is extracted at 15-25 DEG C of temperature；After the completion of extraction, 5-10min is centrifuged with the revolving speed of 3000-8000rpm, respectively
Collect supernatant and sediment；
Second of extraction: obtained sediment will be extracted for the first time and is placed in the dehydrated alcohol of 5-25 times of its quality, in temperature
10min~40min is extracted at 25-50 DEG C of degree；After the completion of extraction, 5-10min is centrifuged with the revolving speed of 3000-8000rpm, in collection
Clear liquid；
Step 4: the supernatant for merging the supernatant obtained after extracting for the first time and obtaining after extracting for second is depressurized
Substance after being concentrated after concentration；
Step 5: obtaining hexane solution after the substance after being concentrated under reduced pressure with n-hexane dissolution, the dimension of the hexane solution is raw
Plain K2Concentration range be 60-200mg/L then hexane solution is fitted into extraction column and carries out column extracting；
The extraction column includes a container, and the bottom of the container is equipped with filter membrane, and the liquid outlet of the container is equipped with switch；
The charge weight of hexane solution is 33.3-50.0% volume of a container in the extraction column, and hexane solution is packed into
After container, highly polar impurity is removed to extract firstly, ultrapure water is added into container, the dosage of ultrapure water is hexane solution
1-3 times, time 8-25min；Then, it is miscellaneous to extract removing low pole that 10%~95% ethanol solution is added into container column
Matter；
Step 6: the hexane solution after the column extracting obtained to step 5 carries out reduced pressure and handles to obtain concentrate, so
Ethanol solution is added in backward concentrate, until dissolving the homogenate substance of concentration all, is then placed into -30~-0 DEG C of environment
Middle standing 24-48h, collects the yellow crystals of generation.
Preferred technical solution are as follows: the thallus content of the bacillus natto to ferment liquid is 8-15g/L, residual sugar content is
0.01-2g/L, residual nitrogen content are 3-8g/L, surface tension 18-24Mn/m.
Preferred technical solution are as follows: in the equipment running process of the ceramic membrane, with pumping out following for hydraulic pressure 0.01-0.3MPa
Ring water-cooled, the running temperature of the ceramic membrane are 15-60 DEG C, and the flux of the ceramic membrane is 40-80L/m2·h。
Preferred technical solution are as follows: in step 4, carry out reduced pressure processing with Rotary Evaporators, control pressure is 0.01-
0.05MPa, rotation speed 40-90rpm/min.
Preferred technical solution are as follows: the ratio of height to diameter of the extraction column is 7.5-40.0.
Preferred technical solution are as follows: container is added in such a way that gradient concentration reduces in described 10%~95% ethanol solution
In.
Since above-mentioned technical proposal is used, the present invention has the advantage, that compared with prior art
The present invention prepares vitamin K for existing bacillus natto to ferment2In extraction purification technology deficiency, provide
One kind abstraction purification vitamin K from the broken wall bafillus natto thallus2Method, this method is by fresh natto gemma bar
Bacterium thallus carries out ion beam broken wall treatment, then directly obtains high-purity vitamin K using column extractive2.The method of the present invention, tool
There are high-efficient thallus broken wall, simple process, vitamin K2The advantages that recovery rate is high.
Detailed description of the invention
Fig. 1 is extraction ethyl alcohol volume optimization for the first time.
Fig. 2 is second of extraction ethyl alcohol volume optimization.
Fig. 3 is the temperature of wet extraction and the optimization of agitating mode.
Specific embodiment
Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this explanation
Content disclosed by book is understood other advantages and efficacy of the present invention easily.
Please refer to Fig. 1-Fig. 3.It should be clear that this specification structure depicted in this specification institute accompanying drawings, ratio, size etc., only to
Cooperate the revealed content of specification, so that those skilled in the art understands and reads, being not intended to limit the invention can be real
The qualifications applied, therefore do not have technical essential meaning, the tune of the modification of any structure, the change of proportionate relationship or size
It is whole, in the case where not influencing the effect of present invention can be generated and the purpose that can reach, it should all still fall in disclosed skill
Art content obtains in the range of capable of covering.Meanwhile in this specification it is cited as "upper", "lower", "left", "right", " centre " and
The term of " one " etc. is merely convenient to being illustrated for narration, rather than to limit the scope of the invention, relativeness
It is altered or modified, under the content of no substantial changes in technology, when being also considered as the enforceable scope of the present invention.
Embodiment 1: a method of the extraction purification farnoquinone from broken wall bafillus natto thallus
The purchase of high performance liquid chromatographs used in the embodiment of the present invention is equipped with ultraviolet from Japanese Shimadzu Corporation
Detector and reverse phase C18 column (4.6mm I.D × 250mm), mobile phase: ethanol/methylene=4:1, Detection wavelength: 248nm,
Column temperature: 35 DEG C, sample volume: 20ul, flow velocity: 1ml/min.
Reagent used in the embodiment of the present invention, in addition to high performance liquid chromatography is chromatographically pure, remaining is all analysis
It is pure.
Fermentation liquid handled by the present embodiment is bafillus natto by glycerol 69.6g/L, glucose 34.5g/L,
K2HPO44.0g/L, peptone 20g/L in culture medium composed by yeast leachate 25g/L, cultivate five at 250rpm, 37 DEG C
It is obtained.
Fermentation liquid is added in device for ultrafiltration membrane, each valve is opened, startup power supply starts to be concentrated, when the object being added
Material all completes concentration by circulating pump with concentrate outflow, that is, obtains the concentrate rich in bafillus natto thallus.
The filter membrane of device for ultrafiltration membrane is ceramic membrane.The aperture for the ceramic membrane that the present embodiment uses is 1 micron.
Above-mentioned concentrate is centrifuged at 4 DEG C, revolving speed 11000rpm, the time of centrifugation is 14min, after centrifugation
Liquid is outwelled, wet thallus is obtained.
The centrifuge tube of 3 50ml is taken, above-mentioned wet thallus 1g is packed into every centrifuge tube, lysozyme is configured to 1mg/mL's
Solution, the lysozyme soln of 800uL-1mL is added into wet thallus, and broken wall 30min is centrifuged later, 20ml is added into thallus
Ethyl alcohol, half an hour extraction time take supernatant to carry out high performance liquid chromatography detection after centrifugal filtration.Every group three groups of parallel test.
The centrifuge tube of 3 50ml is taken, above-mentioned wet thallus 1g is packed into every centrifuge tube, is freezed in -70 DEG C of refrigerator
After 10min, the 10min that thaws quickly is put into 70 DEG C of water-bath, repeatedly three times.20ml second is added in the backward thallus of freeze thawing
Alcohol, half an hour extraction time take supernatant to carry out high performance liquid chromatography detection after centrifugal filtration.Every group three groups of parallel test.
The centrifuge tube of 3 50ml is taken, above-mentioned wet thallus 1g is packed into every centrifuge tube, the water of 5-10mL is added, ultrasound
Ultrasound 15-30min under 350W (every ultrasound 5s suspends 3s).After the thallus of ultrasonication is centrifuged, the middle addition 20ml into precipitating
Ethyl alcohol, half an hour extraction time take supernatant to carry out high performance liquid chromatography detection after centrifugal filtration.Every group three groups of parallel test.
Broken wall is carried out with organic reagent, takes the centrifuge tube of 6 50ml, above-mentioned wet thallus 1g is packed into every centrifuge tube, to
It is wherein separately added into the methanol or ethyl alcohol of 5-10ml, after mixing evenly, 30min is reacted at 25 DEG C, carries out broken wall.It is centrifuged later,
Organic phase is poured out, 20ml ethyl alcohol is added into the thallus of broken wall, half an hour extraction time takes supernatant to carry out high after centrifugal filtration
Effect liquid phase chromatogram detection.Every group three groups of parallel test.
It takes the wet thallus 1g handled without above-mentioned steps to be laid in culture dish, is put into the injection of ion beam irradiation
In target chamber, the vacuum degree for injecting target chamber is 10-3Pa.Broken wall treatment is carried out to bafillus natto with ion beam.Wherein it is used from
The energy of son is 25keV, and injection rate is 1 × 1017N+/cm2.Into the above-mentioned bafillus natto by ion beam broken wall treatment
20ml ethyl alcohol is added, after mixing evenly, extracts 30min, supernatant is taken to carry out high performance liquid chromatography detection after centrifugal filtration.Every group flat
Three groups of row test.
Experimental result is as shown in Table 1, and ion beam can not only make clasmatosis, and the wall-breaking method common with other
It compares, best with ion beam shell-broken effect, shell-broken effect improves 30%, has reached 0.65mg/g.
Table 1: the effect of different broken wall modes
5ml, 10ml, 15ml, the anhydrous second of 20ml, 25ml are separately added into in by ion beam irradiation treated thallus
Alcohol stirs evenly, and after 25 DEG C of extraction 30min, 15000rpm is centrifuged 10min, takes supernatant, high performance liquid chromatography (HPLC) detection meter
Calculate extracted amount.
As shown in Figure 1, the effect of extraction is become better and better with the increase of dehydrated alcohol amount, when volume used be 20ml with
25ml phase difference is less, in order to reduce the use of organic reagent, therefore selects 20ml as the volume of our broken walls.
According to above-mentioned implementation method, extracted to the dehydrated alcohol that 20ml is added in wet thallus after ion beam broken wall treatment
Afterwards, it is centrifuged, collects supernatant, add 5-20m dehydrated alcohol in the wet thallus into precipitating to extract more VK2 intracellular
Afterwards, after 25 DEG C of static extraction 30min, 15000rpm is centrifuged 10min, collects supernatant, and high performance liquid chromatography (HPLC) detection calculates
Extracted amount.Repeat three groups of test.Optimization extraction organic reagent used.
As shown in Fig. 2, when ethyl alcohol volume used is 10ml, extracted amount can more reach 0.85mg/g, with addition nothing
The increase of water-ethanol, extracted amount is without significant change, therefore the volume for selecting the ethyl alcohol of 10ml to extract as our second steps.
The wet thallus that will be obtained, and three centrifuge tube wet thallus are taken, it is freeze-dried, obtains dry mycelium, divide into dry mycelium
Not Jia Ru 10ml methanol extraction 1h, 15000rpm is centrifuged 10min, collects organic phase, repeat the above steps three times, will extract three times
Liquid is collected, liquid phase detection, and three groups of parallel test.As a control group.
30min will be extracted by 20ml ethyl alcohol is added in the wet thallus after ion beam broken wall, is centrifuged, collects later
Supernatant.10ml dehydrated alcohol is added into precipitating；
The static 30min under the conditions of 25 DEG C, 30 DEG C, 40 DEG C, 50 DEG C respectively by the sample of above-mentioned addition dehydrated alcohol, from
The heart measures recovery rate；
At the highest temperature of recovery rate, sample is stirred 30min, collects supernatant after centrifugation；
The extracting solution of dehydrated alcohol under above-mentioned different condition is collected, high performance liquid chromatography detection.Three groups of parallel test.With
Above-mentioned dry mycelium extracting solution comparison.
As shown in figure 3, as the temperature rises, recovery rate also increases, when temperature reaches 40 DEG C, temperature is to recovery rate
Influence no longer changes.Therefore, by sample stir process at 40 DEG C, as seen from the figure, the recovery rate of stir process is obtained very
Big to improve, this is bigger related with solid-liquid contact area in the case of stirring.And compared with extracting dry mycelium with methanol, extract
Rate improves 26%.
Ethanol extract and dehydrated alcohol leaching liquor are collected and are concentrated under reduced pressure, n-hexane dissolution is added, obtains farnoquinone
Highly concentrated solution of the concentration in 60mg/L-200mg/L.It is 1cm-3.5cm to diameter, is highly the glass column of 25cm-40cm
Filter membrane is added in bottom, and the hexane solution of one third column volume is added thereto, is respectively added slowly to the super of different volumes
Pure water is extracted, and speed control is in 1ml/min.The purpose is to remove impurity highly polar in mother liquor, with ultrapure water used
The increase of volume, the impurity extracted is also with increasing, but when increasing to certain volume, effect of extracting increase it is unobvious, because
This, is extracted with 2.5 times of ultrapure water, removes highly polar impurity.
Speed into raffinate phase with 1ml/min sequentially adds 95%~10% ethyl alcohol, removes the impurity of opposed polarity.
It is successively are as follows: 95% ethyl alcohol is extracted with 2BV, and 70% ethyl alcohol is extracted with 2BV, and 30% ethyl alcohol is extracted with 2BV, and 10% ethyl alcohol is with 1BV
It extracts, the raffinate phase purity after column extracting reaches 85.6%.During the experiment we have found that extraction process does not emulsify now
As this is slowly related with the speed that we extract, and the rate of recovery of whole process is very high.
Raffinate phase is collected, is concentrated under reduced pressure and removes n-hexane, be homogenized, 95% a small amount of ethyl alcohol is added into homogenate,
Saturated solution is obtained, -20 DEG C of saturated solution freezings are obtained into crystal for 24 hours, supernatant is sucked out, molten, the purity of ethyl alcohol weight is added in crystal
Reach 95%.
Table two: the purity of each step
Embodiment 2: a method of the extraction purification farnoquinone from broken wall bafillus natto thallus
A method of the abstraction purification farnoquinone from broken wall bacillus natto to ferment product, including the following steps:
Step 1: bacillus natto to ferment liquid was carried out with the device for ultrafiltration membrane for the ceramic membrane for being 5nm with aperture
Filter, obtained concentrate are centrifuged 20min with the revolving speed of 5000rpm, discard supernatant liquid, the deposit of collection is natto gemma
The new fresh thalli of bacillus；
Step 2: by the new fresh thalli merging ion beam apparatus of bafillus natto, bombarding thallus, institute with nitrogen ion beam
It is 15keV with the energy of ion, the ionic weight of injection is 1 × 1015N+/cm2, 10s is injected, stops injection 5s, operates repeatedly
60min, the bafillus natto thallus after obtaining broken wall；
Step 3: being extracted including first time extraction and second；
It extracts: the bafillus natto thallus after broken wall being placed in the dehydrated alcohol of 5 times of its quality, in temperature for the first time
40min is extracted at 25 DEG C；After the completion of extraction, 10min is centrifuged with the revolving speed of 3000rpm, collects supernatant and sediment respectively；
Second of extraction: obtained sediment will be extracted for the first time and is placed in the dehydrated alcohol of 5 times of its quality, in temperature 50
40min is extracted at DEG C；After the completion of extraction, 10min is centrifuged with the revolving speed of 8000rpm, collects supernatant；
Step 4: the supernatant for merging the supernatant obtained after extracting for the first time and obtaining after extracting for second is depressurized
Substance after being concentrated after concentration；
Step 5: obtaining hexane solution after the substance after being concentrated under reduced pressure with n-hexane dissolution, the dimension of the hexane solution is raw
Plain K2Concentration range be 200mg/L then hexane solution is fitted into extraction column and carries out column extracting；
The extraction column includes a container, and the bottom of the container is equipped with filter membrane, and the liquid outlet of the container is equipped with switch；
The charge weight of hexane solution is 33.3% volume of a container in the extraction column, and hexane solution is packed into container
Afterwards, highly polar impurity being removed to extract firstly, ultrapure water is added into container, the dosage of ultrapure water is 1 times of hexane solution,
Time is 8min；Then, 10% ethanol solution is added into container column and removes low pole impurity to extract；
Step 6: the hexane solution after the column extracting obtained to step 5 carries out reduced pressure and handles to obtain concentrate, so
Ethanol solution is added in backward concentrate, until dissolving the homogenate substance of concentration all, is then placed into quiet in -30 DEG C of environment
Set the yellow crystals for collecting generation for 24 hours.
Preferred embodiment are as follows: the thallus content of the bacillus natto to ferment liquid is 8g/L, residual sugar content is
0.01g/L, residual nitrogen content are 3g/L, surface tension 18Mn/m.
Preferred embodiment are as follows: in the equipment running process of the ceramic membrane, with the recirculated water for pumping out hydraulic pressure 0.01MPa
Heat dissipation, the running temperature of the ceramic membrane are 15 DEG C, and the flux of the ceramic membrane is 40L/m2·h。
Preferred embodiment are as follows: in step 4, carry out reduced pressure processing with Rotary Evaporators, control pressure is
0.01MPa, rotation speed 40rpm/min.
Preferred embodiment are as follows: the ratio of height to diameter of the extraction column is 7.5.
Preferred embodiment are as follows: described 10% ethanol solution is added to the container in such a way that gradient concentration reduces.
Embodiment 3: a method of the extraction purification farnoquinone from broken wall bafillus natto thallus
A method of the abstraction purification farnoquinone from broken wall bacillus natto to ferment product, including the following steps:
Step 1: bacillus natto to ferment liquid is filtered with the ceramic membrane that aperture is 10um, obtained liquid with
The revolving speed of 18000rpm is centrifuged 20min, discards supernatant liquid, the deposit of collection is the new fresh thalli of bafillus natto；
Step 2: by the new fresh thalli merging ion beam apparatus of bafillus natto, bombarding thallus, institute with nitrogen ion beam
It is 35keV with the energy of ion, the ionic weight of injection is 5 × 1019N+/cm2, 10s is injected, stops injection 5s, operates repeatedly
30min, the bafillus natto thallus after obtaining broken wall；
Step 3: being extracted including first time extraction and second；
It extracts: the bafillus natto thallus after broken wall being placed in the dehydrated alcohol of 25 times of its quality, in temperature for the first time
10minmin is extracted at 15 DEG C of degree；After the completion of extraction, 5min is centrifuged with the revolving speed of 3000rpm, collects supernatant and precipitating respectively
Object；
Second of extraction: obtained sediment will be extracted for the first time and is placed in the dehydrated alcohol of 25 times of its quality, in temperature
10minmin is extracted at 25 DEG C；After the completion of extraction, 5min is centrifuged with the revolving speed of 3000rpm, collects supernatant；
Step 4: the supernatant for merging the supernatant obtained after extracting for the first time and obtaining after extracting for second is depressurized
Substance after being concentrated after concentration；
Step 5: obtaining hexane solution after the substance after being concentrated under reduced pressure with n-hexane dissolution, the dimension of the hexane solution is raw
Plain K2Concentration range be 200mg/L then hexane solution is fitted into extraction column and carries out column extracting；
The extraction column includes a container, and the bottom of the container is equipped with filter membrane, and the liquid outlet of the container is equipped with switch；
The charge weight of hexane solution is 50.0% volume of a container in the extraction column, and hexane solution is packed into container
Afterwards, highly polar impurity being removed to extract firstly, ultrapure water is added into container, the dosage of ultrapure water is 3 times of hexane solution,
Time is 25min；Then, 95% ethanol solution is added into container column and removes low pole impurity to extract；
Step 6: the hexane solution after the column extracting obtained to step 5 carries out reduced pressure and handles to obtain concentrate, so
Ethanol solution is added in backward concentrate, until dissolving the homogenate substance of concentration all, is then placed into quiet in -30 DEG C of environment
48h is set, the yellow crystals of generation are collected.
Preferred embodiment are as follows: the thallus content of the bacillus natto to ferment liquid is 15g/L, residual sugar content is
2g/L, residual nitrogen content are 8g/L, surface tension 24Mn/m.
Preferred embodiment are as follows: in the equipment running process of the ceramic membrane, with the recirculated water for pumping out hydraulic pressure 0.3MPa
Heat dissipation, the running temperature of the ceramic membrane are 60 DEG C, and the flux of the ceramic membrane is 80L/m2·h。
Preferred embodiment are as follows: in step 4, carry out reduced pressure processing with Rotary Evaporators, control pressure is
0.05MPa, rotation speed 90rpm/min.
Preferred embodiment are as follows: the ratio of height to diameter of the extraction column is 40.0.
Preferred embodiment are as follows: described 95% ethanol solution is added to the container in such a way that gradient concentration reduces.
As described above is only to be not intended to tool to explain the preferred embodiments of the invention to do any shape to the present invention
Limitation in formula should all wrap therefore all have any modification or change for making the related present invention under identical spirit
It includes in the scope that the invention is intended to protect.
Claims (6)
1. a kind of method of the extraction purification farnoquinone from broken wall bafillus natto thallus, it is characterised in that: including following
Step:
Step 1: bacillus natto to ferment liquid is filtered with the ceramic membrane that aperture is 5nm-10um, obtained concentrate with
The revolving speed of 5000-18000rpm is centrifuged 8-20min, discards supernatant liquid, the deposit of collection is the new of bafillus natto
Fresh thalli；
Step 2: by the new fresh thalli of bafillus natto merging ion beam apparatus, bombard thallus with nitrogen ion beam, it is used from
The energy of son is 15keV-35keV, and the ionic weight of injection is 1 × 1015-5×1019N+/cm2, 10s is injected, stops injection 5s, instead
30-60min is operated again, the bafillus natto thallus after obtaining broken wall；
Step 3: being extracted including first time extraction and second；
It extracts: the bafillus natto thallus after broken wall being placed in the dehydrated alcohol of 5-25 times of its quality, in temperature for the first time
10min~40min is extracted at 15-25 DEG C；After the completion of extraction, 5-10min is centrifuged with the revolving speed of 3000-8000rpm, is collected respectively
Supernatant and sediment；
Second of extraction: obtained sediment will be extracted for the first time and is placed in the dehydrated alcohol of 5-25 times of its quality, in temperature 25-
10min~40min is extracted at 50 DEG C；After the completion of extraction, 5-10min is centrifuged with the revolving speed of 3000-8000rpm, collects supernatant；
Step 4: the supernatant for merging the supernatant obtained after extracting for the first time and obtaining after extracting for second is concentrated under reduced pressure
Substance after being concentrated afterwards；
Step 5: obtaining hexane solution after the substance after being concentrated under reduced pressure with n-hexane dissolution, the vitamin K of the hexane solution2
Concentration range be 60-200mg/L then hexane solution is fitted into extraction column and carries out column extracting；
The extraction column includes a container, and the bottom of the container is equipped with filter membrane, and the liquid outlet of the container is equipped with switch；
The charge weight of hexane solution is 33.3-50.0% volume of a container in the extraction column, and hexane solution is packed into container
Afterwards, highly polar impurity is removed to extract firstly, ultrapure water is added into container, the dosage of ultrapure water is the 1-3 of hexane solution
Times, time 8-25min；Then, 10%~95% ethanol solution is added into container column and removes low pole impurity to extract；
Step 6: the hexane solution after the column extracting obtained to step 5 carries out reduced pressure and handles to obtain concentrate, then to
Ethanol solution is added in concentrate, until dissolving the homogenate substance of concentration all, is then placed into quiet in -30 ~ -0 DEG C of environment
24-48h is set, the yellow crystals of generation are collected.
2. the method for the extraction purification farnoquinone according to claim 1 from broken wall bafillus natto thallus, special
Sign is: the thallus content of the bacillus natto to ferment liquid is 8-15g/L, residual sugar content is 0.01-2g/L, residual nitrogen content
For 3-8g/L, surface tension 18-24Mn/m.
3. the method for the extraction purification farnoquinone according to claim 1 from broken wall bafillus natto thallus, special
Sign is: in the equipment running process of the ceramic membrane, with the circulation water-cooled for pumping out hydraulic pressure 0.01-0.3MPa, the ceramics
The running temperature of film is 15-60 DEG C, and the flux of the ceramic membrane is 40-80L/m2·h。
4. the method for the extraction purification farnoquinone according to claim 1 from broken wall bafillus natto thallus, special
Sign is: in step 4, carrying out reduced pressure processing with Rotary Evaporators, control pressure is 0.01-0.05MPa, and rotation speed is
40-90rpm/min。
5. the method for the extraction purification farnoquinone according to claim 1 from broken wall bafillus natto thallus, special
Sign is: the ratio of height to diameter of the extraction column is 7.5-40.0.
6. the method for the extraction purification farnoquinone according to claim 1 from broken wall bafillus natto thallus, special
Sign is: described 10%~95% ethanol solution is added to the container in such a way that gradient concentration reduces.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910183305.3A CN109824496B (en) | 2019-03-12 | 2019-03-12 | Method for extracting and purifying vitamin K2 from wall-broken bacillus natto thalli |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910183305.3A CN109824496B (en) | 2019-03-12 | 2019-03-12 | Method for extracting and purifying vitamin K2 from wall-broken bacillus natto thalli |
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| CN109824496A true CN109824496A (en) | 2019-05-31 |
| CN109824496B CN109824496B (en) | 2021-07-27 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113004134A (en) * | 2021-03-04 | 2021-06-22 | 中国科学院合肥物质科学研究院 | Method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract |
| CN114315549A (en) * | 2021-12-31 | 2022-04-12 | 南通励成生物工程有限公司 | Vitamin K2Is extracted by |
| CN116283531A (en) * | 2023-02-27 | 2023-06-23 | 广东新达瑞生物科技有限公司 | Extraction process of vitamin K2 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106631748A (en) * | 2016-12-16 | 2017-05-10 | 中国科学院合肥物质科学研究院 | Method for separating and purifying vitamin K2 in bacillus subtilis natto |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106631748A (en) * | 2016-12-16 | 2017-05-10 | 中国科学院合肥物质科学研究院 | Method for separating and purifying vitamin K2 in bacillus subtilis natto |
Non-Patent Citations (1)
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| LIU YAN等: "Improvement of Vitamin K2 Production by Escherichia sp. with Nitrogen Ion Beam Implantation Induction", 《PLASMA SCIENCE AND TECHNOLOGY》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113004134A (en) * | 2021-03-04 | 2021-06-22 | 中国科学院合肥物质科学研究院 | Method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract |
| CN113004134B (en) * | 2021-03-04 | 2023-04-07 | 中国科学院合肥物质科学研究院 | Method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract |
| CN114315549A (en) * | 2021-12-31 | 2022-04-12 | 南通励成生物工程有限公司 | Vitamin K2Is extracted by |
| CN116283531A (en) * | 2023-02-27 | 2023-06-23 | 广东新达瑞生物科技有限公司 | Extraction process of vitamin K2 |
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| CN109824496B (en) | 2021-07-27 |
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