CN109824496B - Method for extracting and purifying vitamin K2 from wall-broken bacillus natto thalli - Google Patents
Method for extracting and purifying vitamin K2 from wall-broken bacillus natto thalli Download PDFInfo
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Abstract
A method for extracting and purifying vitamin K2 from wall-broken bacillus natto thalli comprises the steps of carrying out solid-liquid separation on fermented microorganisms through ultrafiltration and centrifugation to obtain wet thalli, carrying out wall breaking on the wet thalli through ion beam treatment, and extracting with absolute ethyl alcohol, so that not only can a large amount of vitamin K2 be obtained, but also less oil can be extracted, coarse purification is carried out in the extraction process, and a good solution is provided for the following one-step high-efficiency purification. The extract is extracted by a column, organic reagents with different polarities are adopted for extraction and purification, and impurities with different polarities are extracted out, so that the purity of the solution is greatly improved. Then the vitamin K2 crystal with the purity of 95 percent can be obtained by freezing and crystallizing the vitamin K2 crystal. The method omits the drying and thallus pulverizing process in the traditional method, and improves the extraction efficiency and the extraction purity. And the vitamin K with higher purity can be obtained by one-step purification2。
Description
Technical Field
The invention relates to a method for extracting and purifying vitamin K2 from cell wall-broken bacillus natto thalli, and belongs to the field of bioengineering and bioseparation engineering.
Background
Vitamin K2The terpene side chain compounds are a general term for a series of terpene side chain compounds containing 2-methyl-1, 4-naphthoquinone parent nucleus and having an unequal number of isoprene structural units at the C3 position, and are classified into K2(10), K2(20), K2(35), K2(40), and the like, according to the number of carbon elements on the terpene side chain. Vitamin K2Is a fat-soluble vitamin, a derivative of naphthoquinone group with phylloquinone bioactivity, and is one of essential important vitamins in human body. With the study of its function, it has been demonstrated that vitamin K2Has effects in preventing osteoporosis, cardiovascular diseases, Parkinson's disease, and hepatocarcinoma. Therefore, vitamin K is used in the fields of functional foods, medical products, cosmetics and the like2The demand is increasing day by day, and the market prospect is good.
Vitamin K2Having a chiral structure, trans-vitamin K2Has biological activity, cis-vitamin K2Has no biological activity. Vitamin K prepared by chemical synthesis method2Contains cis and trans simultaneously; in contrast, the microorganism fermentation method can obtain all-trans vitamin K2. Thus, vitamin K has been internationally prescribed for use in functional foods2Must be derived from microbial fermentation. At present, the fermentation of Bacillus natto is used for preparing vitamin K2Is a commonly used method for producing vitamin K by microbial fermentation2The method of (1).
Preparation of vitamin K by fermentation of bacillus natto2In the method, the bacillus natto has more metabolites, and meanwhile, due to autolysis of cells of the bacillus natto, various impurities such as hybrid protein, nucleic acid, grease and the like can be generated, so that high-purity vitamin K can be obtained2The difficulty of (2) is great.
At present, organic solvent is widely used for extracting vitamin K from bacillus natto fermentation liquor2The method needs pretreatment operations such as thallus crushing, dehydration and drying, and the subsequent purification method also needs macroporous resin andthe treatment of reverse phase silica gel and the like has complex integral process, high cost and low extraction rate. An extraction and purification technology for preparing vitamin K by fermenting the bacillus natto2The bottleneck problem of (2).
Disclosure of Invention
The invention aims to provide a method for extracting and purifying vitamin K2 from cell wall-broken bacillus natto.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: the method for extracting and purifying vitamin K2 from the cell wall-broken bacillus natto comprises the following steps:
step 1: filtering the bacillus natto fermentation liquor by using a ceramic membrane with the aperture of 5nm-10um, centrifuging the obtained concentrated solution for 8-20min at the rotating speed of 5000-18000rpm, discarding the supernatant, and collecting the precipitate, namely the fresh bacillus natto;
step 2: placing fresh Bacillus natto thallus into ion beam equipment, bombarding thallus with nitrogen ion beam, wherein the energy of the ion is 15keV-35keV, and the amount of the implanted ion is 1 × 1015-5×1019N+/cm2Injecting for 10s, stopping injecting for 5s, and repeating for 30-60min to obtain cell wall-broken Bacillus natto thallus;
and step 3: comprises a first leaching and a second leaching;
leaching for the first time: placing the cell wall-broken bacillus natto in absolute ethyl alcohol with the mass 5-25 times of the cell wall-broken bacillus natto, and leaching for 10-40 min at the temperature of 15-25 ℃; after the leaching is finished, centrifuging for 5-10min at the rotating speed of 3000-;
and (3) leaching for the second time: putting the precipitate obtained by the first leaching into absolute ethyl alcohol with the mass 5-25 times of that of the precipitate, and leaching for 10-40 min at the temperature of 25-50 ℃; after the leaching is finished, centrifuging for 5-10min at the rotating speed of 3000-;
and 4, step 4: mixing the supernatant obtained after the first leaching and the supernatant obtained after the second leaching, and concentrating under reduced pressure to obtain a concentrated substance;
and 5: dissolving with n-hexanePressing the concentrated material to obtain n-hexane solution, which contains vitamin K2The concentration range of the n-hexane solution is 60-200mg/L, and then the n-hexane solution is filled into an extraction column for column extraction;
the extraction column comprises a container, a filter membrane is arranged at the bottom of the container, and a liquid outlet of the container is provided with a switch;
the loading amount of the normal hexane solution in the extraction column is 33.3-50.0% of the volume of the container, and after the normal hexane solution is loaded into the container, firstly, ultrapure water is added into the container to extract and remove strong-polarity impurities, wherein the using amount of the ultrapure water is 1-3 times of that of the normal hexane solution, and the time is 8-25 min; then, adding 10-95% ethanol solution into the container column to extract and remove the weak polar impurities;
step 6: and (3) carrying out reduced pressure concentration treatment on the n-hexane solution obtained after the column extraction in the step (5) to obtain a concentrated solution, then adding an ethanol solution into the concentrated solution until the concentrated homogenate substance is completely dissolved, then standing for 24-48h in an environment of-30 to-0 ℃, and collecting the generated yellow crystals.
The preferable technical scheme is as follows: the bacillus natto fermentation liquor has the thallus content of 8-15g/L, the residual sugar content of 0.01-2g/L, the residual nitrogen content of 3-8g/L and the surface tension of 18-24 Mn/m.
The preferable technical scheme is as follows: in the operation process of the ceramic membrane equipment, circulating water with the pump-out water pressure of 0.01-0.3MPa is used for heat dissipation, the operation temperature of the ceramic membrane is 15-60 ℃, and the flux of the ceramic membrane is 40-80L/m2·h。
The preferable technical scheme is as follows: and 4, carrying out reduced pressure concentration treatment by using a rotary evaporator, wherein the pressure is controlled to be 0.01-0.05MPa, and the rotating speed is 40-90 rpm/min.
The preferable technical scheme is as follows: the height-diameter ratio of the extraction column is 7.5-40.0.
The preferable technical scheme is as follows: the 10% -95% ethanol solution is added into the container in a gradient concentration reduction mode.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
the inventionAiming at the existing method for preparing vitamin K by fermenting bacillus natto2The deficiency of the extraction and purification technology in (1) provides a method for extracting and purifying vitamin K from the cell wall-broken bacillus natto2The method comprises the steps of carrying out ion beam wall breaking treatment on fresh bacillus natto thalli, and then directly utilizing column type extraction to obtain high-purity vitamin K2. The method has the advantages of high thallus wall breaking efficiency, simple process and vitamin K2High extraction rate and the like.
Drawings
Figure 1 is a first extraction ethanol volume optimization.
Figure 2 is a second extraction ethanol volume optimization.
Figure 3 is an optimization of the temperature and agitation pattern of the wet extraction.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Please refer to fig. 1-3. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.
Example 1: method for extracting and purifying vitamin K2 from wall-broken bacillus natto thalli
The HPLC chromatograph used in the examples of the present invention was purchased from Shimadzu corporation, Japan, equipped with an ultraviolet detector and a reversed-phase C18 column (4.6mm I.D. times 250mm), mobile phase: methanol/dichloromethane ═ 4:1, detection wavelength: 248nm, column temperature: 35 ℃, sample introduction: 20ul, flow rate: 1 ml/min.
The reagents used in the examples of the present invention were analytically pure except for the high performance liquid chromatography.
The fermentation liquid treated by the embodiment is prepared by mixing 69.6g/L of glycerol, 34.5g/L of glucose and K by using bacillus natto2HPO44.0g/L, peptone 20g/L, and yeast extract 25g/L, at 250rpm, 37 ℃ for five days.
And (3) adding the fermentation liquor into ultrafiltration membrane equipment, opening each valve, starting a power supply, starting concentration, and finishing concentration when the added materials completely flow out as concentrated solution through a circulating pump, so that the concentrated solution rich in bacillus natto thallus is obtained. The filter membrane of the ultrafiltration membrane equipment is a ceramic membrane. The pore size of the ceramic membrane used in this example was 1 μm.
Centrifuging the concentrated solution at 4 deg.C at 11000rpm for 14min, and pouring out the liquid to obtain wet thallus.
Taking 350 mL centrifuge tubes, filling 1g of the wet bacteria into each centrifuge tube, preparing the lysozyme into a solution of 1mg/mL, adding 800uL-1mL of lysozyme solution into the wet bacteria, breaking the walls for 30min, centrifuging, adding 20mL of ethanol into the bacteria, extracting for half an hour, centrifuging, filtering, and taking the supernatant to perform high performance liquid chromatography detection. Three groups were tested in parallel per group.
Taking 350 ml centrifuge tubes, loading 1g of the wet thallus into each centrifuge tube, freezing in a refrigerator at-70 deg.C for 10min, quickly placing in a water bath at 70 deg.C for thawing for 10min, and repeating the above steps three times. And (3) adding 20ml of ethanol into the thalli after freeze thawing, extracting for half an hour, centrifuging, filtering, and taking the supernatant for high performance liquid chromatography detection. Three groups were tested in parallel per group.
Taking 350 mL centrifuge tubes, loading 1g of the wet thallus into each centrifuge tube, adding 5-10mL of water, and performing ultrasonic treatment at 350W for 15-30min (5 s per ultrasonic treatment, 3s pause). Centrifuging the ultrasonic wall-broken thallus, adding 20ml ethanol into the precipitate, extracting for half an hour, centrifuging, filtering, and collecting the supernatant for high performance liquid chromatography detection. Three groups were tested in parallel per group.
And (3) breaking the wall by using an organic reagent, taking 6 centrifuge tubes of 50ml, filling 1g of the wet thalli into each centrifuge tube, respectively adding 5-10ml of methanol or ethanol into the centrifuge tubes, uniformly stirring, reacting at 25 ℃ for 30min, and breaking the wall. And centrifuging, pouring out the organic phase, adding 20ml of ethanol into the wall-broken thallus, extracting for half an hour, centrifuging, filtering, and taking the supernatant for high performance liquid chromatography detection. Three groups were tested in parallel per group.
Spreading 1g of wet thallus which has not been processed in the above steps in a culture dish, placing into an injection target chamber of an ion beam irradiation device, wherein the vacuum degree of the injection target chamber is 10-3Pa. And (3) carrying out wall breaking treatment on the bacillus natto by using ion beams. Wherein the energy of the ions is 25keV and the implantation amount is 1 × 1017N+/cm2. Adding 20ml ethanol into the Bacillus natto subjected to ion beam wall breaking treatment, stirring uniformly, extracting for 30min, centrifuging, filtering, and taking supernatant for high performance liquid chromatography detection. Three groups were tested in parallel per group.
The experimental result is shown in table one, the ion beam can not only break the cell, but also has the best wall breaking effect by the ion beam compared with other common wall breaking methods, the wall breaking effect is improved by 30 percent, and the wall breaking effect reaches 0.65 mg/g.
Table 1: effect of different wall breaking modes
Adding 5ml,10ml,15ml,20ml and 25ml of absolute ethyl alcohol into the thallus subjected to ion beam irradiation treatment, stirring uniformly, extracting at 25 ℃ for 30min, centrifuging at 15000rpm for 10min, taking supernatant, and detecting and calculating the extraction amount by High Performance Liquid Chromatography (HPLC).
As shown in FIG. 1, the extraction effect is better and better with the increase of the amount of absolute ethanol, the difference is less when the used volume is 20ml and 25ml, and in order to reduce the use of organic reagents, 20ml is selected as the wall breaking volume.
According to the implementation method, 20ml of absolute ethyl alcohol is added into wet thalli subjected to ion beam wall breaking treatment for extraction, centrifugation is carried out, supernatant is collected, 5-20m of absolute ethyl alcohol is added into wet thalli in precipitation to extract more intracellular VK2, the mixture is subjected to static extraction at 25 ℃ for 30min and then is centrifuged at 15000rpm for 10min, the supernatant is collected, and the extraction amount is calculated through High Performance Liquid Chromatography (HPLC) detection. Three sets of experiments were repeated. The organic reagent used for leaching is optimized.
As shown in FIG. 2, the extraction amount reached more 0.85mg/g when the volume of ethanol used was 10ml, and there was no significant change in the extraction amount with the addition of absolute ethanol, so 10ml of ethanol was selected as the volume of our second extraction.
And (3) taking the obtained wet thalli and three centrifuge tubes of wet thalli, freeze-drying to obtain dry thalli, adding 10ml of methanol into the dry thalli respectively, extracting for 1h, centrifuging at 15000rpm for 10min, collecting an organic phase, repeating the steps for three times, collecting three extracting solutions, detecting a liquid phase, and performing three groups of parallel tests. As a control group.
And adding 20ml of ethanol into the wet thalli subjected to ion beam wall breaking for extraction for 30min, centrifuging, and collecting supernatant. Adding 10ml of absolute ethyl alcohol into the precipitate;
standing the sample added with the absolute ethyl alcohol at 25 ℃, 30 ℃, 40 ℃ and 50 ℃ for 30min, centrifuging and measuring the extraction rate;
stirring the sample for 30min at the temperature with the highest extraction rate, centrifuging and collecting the supernatant;
collecting the above extractive solutions of anhydrous ethanol under different conditions, and detecting by high performance liquid chromatography. Three groups were tested in parallel. Comparing with the above dried thallus extract.
As shown in fig. 3, the extraction rate increased with the increase in temperature, and when the temperature reached 40 ℃, the effect of temperature on the extraction rate did not change. Therefore, the sample was stirred at 40 ℃ and, as can be seen from the figure, the extraction rate of the stirring treatment was greatly improved, which is related to the larger solid-liquid contact area with stirring. And compared with the extraction of dry thalli by methanol, the extraction rate is improved by 26 percent.
Collecting ethanol extractive solution and anhydrous ethanol extractive solution, concentrating under reduced pressure, adding n-hexane for dissolving to obtain high concentration solution of vitamin K2 with concentration of 60mg/L-200 mg/L. Adding a filter membrane at the bottom of a glass column with the diameter of 1cm-3.5cm and the height of 25cm-40cm, adding one third of column volume of n-hexane solution, and slowly adding different volumes of ultrapure water respectively for extraction at the speed of 1 ml/min. The purpose is to remove the impurities with strong polarity in the mother liquor, the extracted impurities are increased along with the increase of the volume of the used ultrapure water, but the extraction effect is not obviously increased when the volume is increased to a certain volume, so the impurities with strong polarity are removed by extracting with 2.5 times of ultrapure water.
And (3) sequentially adding 95-10% of ethanol into the raffinate phase at the speed of 1ml/min to remove impurities with different polarities. Which is sequentially as follows: the extraction with 95% ethanol at 2BV, the extraction with 70% ethanol at 2BV, the extraction with 30% ethanol at 2BV, the extraction with 10% ethanol at 1BV, the raffinate phase purity after column extraction reached 85.6%. In the experimental process, the extraction process is not emulsified, which is related to the slow extraction speed of the extraction process, and the recovery rate of the whole process is high.
Collecting the raffinate, concentrating under reduced pressure to remove n-hexane to obtain homogenate, adding a small amount of 95% ethanol into the homogenate to obtain saturated solution, freezing the saturated solution at-20 deg.C for 24 hr to obtain crystals, sucking out the supernatant, adding ethanol into the crystals, and redissolving to obtain the product with purity of 95%.
Table two: purity of each step
Example 2: method for extracting and purifying vitamin K2 from wall-broken bacillus natto thalli
A method for extracting and purifying vitamin K2 from a fermentation product of wall-broken bacillus natto comprises the following steps:
step 1: filtering the Bacillus natto fermentation liquor with an ultrafiltration membrane device with a ceramic membrane with the aperture of 5nm, centrifuging the obtained concentrated solution at the rotating speed of 5000rpm for 20min, discarding the supernatant, and collecting the precipitate which is the fresh thallus of the Bacillus natto;
step 2: placing fresh Bacillus natto thallus into ion beam equipment, bombarding thallus with nitrogen ion beam, wherein the energy of the ion is 15keV, and the amount of the implanted ion is 1 × 1015N+/cm2Injecting for 10s, stopping injecting for 5s, and repeating for 60min to obtain cell wall-broken Bacillus natto thallus;
and step 3: comprises a first leaching and a second leaching;
leaching for the first time: placing the cell wall-broken Bacillus natto thallus into 5 times of anhydrous ethanol, and leaching at 25 deg.C for 40 min; after leaching, centrifuging at 3000rpm for 10min, and respectively collecting supernatant and precipitate;
and (3) leaching for the second time: placing the precipitate obtained by the first leaching into anhydrous ethanol with 5 times of the mass of the precipitate, and leaching at 50 deg.C for 40 min; after the leaching is finished, centrifuging for 10min at the rotating speed of 8000rpm, and collecting supernatant;
and 4, step 4: mixing the supernatant obtained after the first leaching and the supernatant obtained after the second leaching, and concentrating under reduced pressure to obtain a concentrated substance;
and 5: dissolving the concentrated substance under reduced pressure with n-hexane to obtain n-hexane solution, and dissolving vitamin K in the n-hexane solution2The concentration range of the solution is 200mg/L, and then the normal hexane solution is filled into an extraction column for column extraction;
the extraction column comprises a container, a filter membrane is arranged at the bottom of the container, and a liquid outlet of the container is provided with a switch;
the loading amount of the normal hexane solution in the extraction column is 33.3% of the volume of the container, after the normal hexane solution is loaded into the container, firstly, ultrapure water is added into the container to extract and remove strong-polarity impurities, the using amount of the ultrapure water is 1 time of that of the normal hexane solution, and the time is 8 min; then, 10% ethanol solution is added into the container column to extract and remove the weak polar impurities;
step 6: and (3) carrying out reduced pressure concentration treatment on the n-hexane solution obtained after the column extraction in the step (5) to obtain a concentrated solution, then adding an ethanol solution into the concentrated solution until the concentrated homogenate substance is completely dissolved, then standing for 24h in an environment of-30 ℃, and collecting the generated yellow crystals.
The preferred embodiment is: the bacillus natto fermentation liquor has the thallus content of 8g/L, the residual sugar content of 0.01g/L, the residual nitrogen content of 3g/L and the surface tension of 18 Mn/m.
The preferred embodiment is: in the operation process of the ceramic membrane equipment, circulating water with the pump-out water pressure of 0.01MPa is used for heat dissipation, the operation temperature of the ceramic membrane is 15 ℃, and the flux of the ceramic membrane is 40L/m2·h。
The preferred embodiment is: and 4, carrying out reduced pressure concentration treatment by using a rotary evaporator, wherein the pressure is controlled to be 0.01MPa, and the rotating speed is 40 rpm/min.
The preferred embodiment is: the height-diameter ratio of the extraction column is 7.5.
The preferred embodiment is: the 10% ethanol solution was added to the vessel in a gradient of decreasing concentration.
Example 3: method for extracting and purifying vitamin K2 from wall-broken bacillus natto thalli
A method for extracting and purifying vitamin K2 from a fermentation product of wall-broken bacillus natto comprises the following steps:
step 1: filtering the bacillus natto fermentation liquor by using a ceramic membrane with the aperture of 10um, centrifuging the obtained liquor for 20min at the rotating speed of 18000rpm, removing supernatant, and collecting precipitate, namely the fresh thallus of the bacillus natto;
step 2: placing fresh Bacillus natto thallus into ion beam equipment, bombarding thallus with nitrogen ion beam, wherein the energy of the ion is 35keV, and the amount of the implanted ion is 5 × 1019N+/cm2Injecting for 10s, stopping injecting for 5s, and repeating for 30min to obtain cell wall-broken Bacillus natto thallus;
and step 3: comprises a first leaching and a second leaching;
leaching for the first time: placing the cell wall-broken Bacillus natto thallus into anhydrous ethanol with the mass of 25 times of the Bacillus natto thallus, and extracting for 10min at 15 ℃; after leaching, centrifuging at 3000rpm for 5min, and respectively collecting supernatant and precipitate;
and (3) leaching for the second time: placing the precipitate obtained by the first leaching into absolute ethyl alcohol with the mass of 25 times of that of the precipitate, and leaching for 10min at the temperature of 25 ℃; after leaching, centrifuging at 3000rpm for 5min, and collecting supernatant;
and 4, step 4: mixing the supernatant obtained after the first leaching and the supernatant obtained after the second leaching, and concentrating under reduced pressure to obtain a concentrated substance;
and 5: dissolving the concentrated substance under reduced pressure with n-hexane to obtain n-hexane solution, and dissolving vitamin K in the n-hexane solution2The concentration range of the solution is 200mg/L, and then the normal hexane solution is filled into an extraction column for column extraction;
the extraction column comprises a container, a filter membrane is arranged at the bottom of the container, and a liquid outlet of the container is provided with a switch;
the loading amount of the normal hexane solution in the extraction column is 50.0% of the volume of the container, after the normal hexane solution is loaded into the container, firstly, ultrapure water is added into the container to extract and remove strong-polarity impurities, the using amount of the ultrapure water is 3 times of that of the normal hexane solution, and the time is 25 min; then, 95% ethanol solution is added into the container column to extract and remove the weak polar impurities;
step 6: and (3) carrying out reduced pressure concentration treatment on the n-hexane solution obtained after the column extraction in the step (5) to obtain a concentrated solution, then adding an ethanol solution into the concentrated solution until the concentrated homogenate substance is completely dissolved, then standing for 48h in an environment of-30 ℃, and collecting the generated yellow crystals.
The preferred embodiment is: the bacillus natto fermentation liquid has the thallus content of 15g/L, the residual sugar content of 2g/L, the residual nitrogen content of 8g/L and the surface tension of 24 Mn/m.
The preferred embodiment is: in the operation process of the ceramic membrane equipment, circulating water with the water outlet pressure of 0.3MPa is used for heat dissipationThe operating temperature of the ceramic membrane is 60 ℃, and the flux of the ceramic membrane is 80L/m2·h。
The preferred embodiment is: and 4, carrying out reduced pressure concentration treatment by using a rotary evaporator, wherein the pressure is controlled to be 0.05MPa, and the rotating speed is controlled to be 90 rpm/min.
The preferred embodiment is: the height-diameter ratio of the extraction column is 40.0.
The preferred embodiment is: the 95% ethanol solution was added to the vessel in a gradient of decreasing concentration.
The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.
Claims (6)
1. A method for extracting and purifying vitamin K2 from cell wall-broken bacillus natto thalli is characterized by comprising the following steps: comprises the following steps:
step 1: filtering the bacillus natto fermentation liquor by using a ceramic membrane with the aperture of 5nm-10um, centrifuging the obtained concentrated solution for 8-20min at the rotating speed of 5000-18000rpm, discarding the supernatant, and collecting the precipitate, namely the fresh bacillus natto;
step 2: placing fresh Bacillus natto thallus into ion beam equipment, bombarding thallus with nitrogen ion beam, wherein the energy of the ion is 15keV-35keV, and the amount of the implanted ion is 1 × 1015-5×1019N+/cm2Injecting for 10s, stopping injecting for 5s, and repeating for 30-60min to obtain cell wall-broken Bacillus natto thallus;
and step 3: comprises a first leaching and a second leaching;
leaching for the first time: placing the cell wall-broken bacillus natto in absolute ethyl alcohol with the mass 5-25 times of the cell wall-broken bacillus natto, and leaching for 10-40 min at the temperature of 15-25 ℃; after the leaching is finished, centrifuging for 5-10min at the rotating speed of 3000-;
and (3) leaching for the second time: putting the precipitate obtained by the first leaching into absolute ethyl alcohol with the mass 5-25 times of that of the precipitate, and leaching for 10-40 min at the temperature of 25-50 ℃; after the leaching is finished, centrifuging for 5-10min at the rotating speed of 3000-;
and 4, step 4: mixing the supernatant obtained after the first leaching and the supernatant obtained after the second leaching, and concentrating under reduced pressure to obtain a concentrated substance;
and 5: dissolving the concentrated substance under reduced pressure with n-hexane to obtain n-hexane solution, and dissolving vitamin K in the n-hexane solution2The concentration range of the n-hexane solution is 60-200mg/L, and then the n-hexane solution is filled into an extraction column for column extraction;
the extraction column comprises a container, a filter membrane is arranged at the bottom of the container, and a liquid outlet of the container is provided with a switch;
the loading amount of the normal hexane solution in the extraction column is 33.3-50.0% of the volume of the container, and after the normal hexane solution is loaded into the container, firstly, ultrapure water is added into the container to extract and remove strong-polarity impurities, wherein the using amount of the ultrapure water is 1-3 times of that of the normal hexane solution, and the time is 8-25 min; then, adding 10-95% ethanol solution into the container column to extract and remove the weak polar impurities;
step 6: and (3) carrying out reduced pressure concentration treatment on the n-hexane solution obtained after the column extraction in the step (5) to obtain a concentrated solution, then adding an ethanol solution into the concentrated solution until all concentrated homogenate substances are dissolved, then standing for 24-48h in an environment of minus 30 to minus 0 ℃, and collecting the generated yellow crystals.
2. The method for extracting and purifying vitamin K2 from the cell wall-broken Bacillus natto strain according to claim 1, wherein the method comprises the following steps: the bacillus natto fermentation liquor has the thallus content of 8-15g/L, the residual sugar content of 0.01-2g/L, the residual nitrogen content of 3-8g/L and the surface tension of 18-24 Mn/m.
3. The method for extracting and purifying vitamin K2 from the cell wall-broken Bacillus natto strain according to claim 1, wherein the method comprises the following steps: in the operation process of the ceramic membrane equipment, the circulation with the water pumping pressure of 0.01-0.3MPa is usedCirculating water for heat dissipation, wherein the operating temperature of the ceramic membrane is 15-60 ℃, and the flux of the ceramic membrane is 40-80L/m2·h。
4. The method for extracting and purifying vitamin K2 from the cell wall-broken Bacillus natto strain according to claim 1, wherein the method comprises the following steps: and 4, carrying out reduced pressure concentration treatment by using a rotary evaporator, wherein the pressure is controlled to be 0.01-0.05MPa, and the rotating speed is 40-90 rpm/min.
5. The method for extracting and purifying vitamin K2 from the cell wall-broken Bacillus natto strain according to claim 1, wherein the method comprises the following steps: the height-diameter ratio of the extraction column is 7.5-40.0.
6. The method for extracting and purifying vitamin K2 from the cell wall-broken Bacillus natto strain according to claim 1, wherein the method comprises the following steps: the 10% -95% ethanol solution is added into the container in a gradient concentration reduction mode.
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| CN114315549A (en) * | 2021-12-31 | 2022-04-12 | 南通励成生物工程有限公司 | Vitamin K2Is extracted by |
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| 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 |
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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)
| Title |
|---|
| Improvement of Vitamin K2 Production by Escherichia sp. with Nitrogen Ion Beam Implantation Induction;LIU Yan等;《Plasma Science and Technology》;20150228;第17卷(第2期);159-166页 * |
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