CN113004134A - Method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract - Google Patents
Method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract Download PDFInfo
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Abstract
A method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract comprises the steps of separating the fermentation liquor to obtain wet thalli, freeze-drying to obtain dry thalli, mixing the dry thalli with the palm oil extract for 2-4 hours, and centrifuging to obtain supernatant; loading the pretreated macroporous resin into a column, putting the supernatant into a separation column filled with the macroporous resin, removing miscellaneous oil substances by using a first eluent, and eluting vitamin K2 from the macroporous resin by using a first resolving agent to obtain an eluent; concentrating the obtained eluent to obtain a concentrated solution; and placing the concentrated solution into a separation column containing reversed phase silica gel, removing impurities by using a second eluent, eluting by using a second resolving agent to obtain a high-purity vitamin K2 product, and performing freeze crystallization to obtain a vitamin K2 crystal. The palm oil extract used in the invention exists in nature, is easy to obtain, does not generate organic waste liquid, and greatly reduces the pollution to the environment.
Description
Technical Field
The invention belongs to the field of biochemical engineering, and relates to a method for separating and purifying vitamin K2 in fermentation liquor by using a palm oil extract, in particular to a method for green and efficient extraction of vitamin K2 in bacillus natto and bacillus subtilis by using reagents such as the palm oil extract (such as medium chain triglyceride) and butyl acetate as solvents.
Background
Vitamin K2 is a series of derivatives of 2-methyl-3-alkyl-1, 4-naphthoquinone, which can be classified into 14 types according to the number of isoprene at C-3 position, and is represented by MK-n (n is the number of isoprene units in the side chain). With the deep research on the function of vitamin K2, the vitamin K2 can not only promote the blood coagulation and prevent and treat osteoporosis, but also has better prevention and treatment effects on neurodegenerative diseases and various tumors. In Japan and Europe and America, it has been already sold as functional foods and medicines, and it is expected that the sales of vitamin K will reach more than 80 billion dollars in 2025, and the market prospect is wide.
Because the two methods of naturally obtaining vitamin K2 and chemically synthesizing vitamin K2 have the problems of low yield, more byproducts, complex reaction conditions and the like, the method for producing vitamin K2 with better effect at present is a microbial fermentation method, a large amount of vitamin K2 is produced after bacillus natto and bacillus subtilis are fermented, in order to obtain high-purity MK-7, the MK-7 needs to be separated from raw materials, and various purification technologies are applied to obtain a high-purity VK2 product. The separation and purification are the most costly part of the biological processing process, and products obtained by microbial fermentation are generally treated by methods such as concentration, centrifugation, drying and the like, and then samples with different purities are obtained by extraction and purification of a large amount of organic reagents. Most of the separation and purification solvents used in the past are organic reagents with high toxicity, such as methanol, tetrahydrofuran, dichloromethane and the like, which are used as solvents, so that vitamin K2 with high purity can be obtained only by multi-step operation, the pollution is serious in the process, a large amount of waste liquid is generated, the operation is complex, the cost is high, and the obtained product cannot be directly eaten by human bodies. Therefore, the separation and purification technology using natural oil and green organic reagents with high efficiency and low cost is very important in the industrial production of vitamin K2.
Disclosure of Invention
The invention aims to extract vitamin K2 in Bacillus natto or Bacillus subtilis dry bacteria by using palm oil extract (medium chain triglyceride) as a solvent, and then, the high-purity vitamin K2 is obtained in a green and high-efficiency two-step method by using ethanol and butyl acetate as solvents for separation and purification.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract comprises the following steps:
step 1: separating the fermentation liquor obtained by the microbial fermentation production method to obtain wet thalli, and freeze-drying the wet thalli to obtain dry thalli rich in vitamin K2;
step 2: mixing the dry thalli and the palm oil extract for 2-4 h, and then centrifuging the mixed solution to obtain a supernatant, namely the palm oil extract rich in vitamin K2;
and step 3: loading the pretreated macroporous resin into a column, putting the palm oil extract rich in vitamin K2 into a separation column filled with the macroporous resin, removing miscellaneous oil substances by using a first eluent, and eluting vitamin K2 from the macroporous resin by using a first resolving agent to obtain an eluent;
and 4, step 4: concentrating the eluent obtained in the step (3) to obtain a concentrated solution;
and 5: putting the concentrated solution into a separation column containing reversed phase silica gel, removing impurities by using a second eluent, and eluting by using a second resolving agent to obtain a high-purity vitamin K2 product;
step 6: and (3) freezing and crystallizing the high-concentration vitamin K2 solution to obtain the vitamin K2 crystal.
The preferable technical scheme is as follows: the mass ratio of the dry thallus to the palm oil extract is 1: 1-1: 20.
The preferable technical scheme is as follows: in the step 2, the dry thalli and the palm oil extract are mixed and then are placed on a shaking table to be processed for 2-4 hours at the rotating speed of 200-300 rpm/min.
The preferable technical scheme is as follows: and 2, placing the mixed solution in a floor type refrigerated centrifuge, and centrifuging at the rotating speed of 7500-9000 rpm for 10-20 min.
The preferable technical scheme is as follows: step 3, soaking macroporous resin in 95% ethanol by volume, then opening a cock at the lower end of a glass column, adding the macroporous resin into the glass column along a glass rod, after filling, lightly knocking the glass column to ensure that the filling is uniform, and enabling bubbles to escape; the macroporous resin is one of macroporous resin HPD417, macroporous resin HPD722, macroporous resin GDX-10 and macroporous resin GDX-502.
The preferable technical scheme is as follows: the first eluent is ethanol with the volume fraction of 80-100%, and the elution rate is 0.01-0.015 column volume/min; the first eluent is butyl acetate, and the elution rate is 0.05-0.06 column volume/min.
The preferable technical scheme is as follows: and 4, adopting a rotary evaporation concentration method, wherein the used instrument is a rotary evaporator, the evaporation flask is in a negative pressure state through a vacuum pump, the pressure is reduced to 400-600 mm Hg in a sealed manner, the water bath heating temperature is 75-95 ℃, the rotation speed is 60-120 rpm/min, and the rotary evaporation is finished until all ethyl acetate is evaporated.
The preferable technical scheme is as follows: in the step 5, the reversed phase silica gel needs to be dried for 0.5 h-2 h in advance in an environment of 130-140 ℃, and then is soaked for more than 2h by using butyl acetate.
The preferable technical scheme is as follows: the second eluent is a mixed solution composed of butyl acetate and ethanol according to a volume ratio of 5:2, and the flow rate is 0.001-0.0015 column volume/min.
The preferable technical scheme is as follows: the second resolving agent is a mixed solution composed of butyl acetate and ethanol according to the volume ratio of 3:2, and the flow rate is 0.001-0.0015 column volume/min.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
1. the existing vitamin K2 separation and purification method needs to use a large amount of toxic reagents (including methanol, tetrahydrofuran, n-hexane, dichloromethane and the like), can generate a large amount of organic waste liquid, and causes great pollution to the environment.
2. The palm oil extract (medium chain triglyceride) as an extraction agent of the invention is an edible oil, and the medium chain triglyceride of the extract is only composed of saturated fatty acid, has low freezing point, is liquid at room temperature and has low viscosity. Compared to soybean oil, medium chain triglycerides are completely odorless, colorless, transparent liquids. Compared with common oil and hydrogenated oil, the medium chain triglyceride has extremely low unsaturated fatty acid content, very good oxidation stability, and is particularly stable at high temperature and low temperature, and simultaneously, the medium chain triglyceride and vitamins have good intersolubility, and the product extracted by using the medium chain triglyceride as a solvent does not need to worry about the residue of organic chemical substances.
3. The existing vitamin K2 separation and purification method needs to carry out a plurality of steps, needs to use macroporous resin, molecular sieve, silica gel and other extraction intermediates, and uses more than four organic reagents.
Drawings
Figure 1 is a simplified representation of the medium chain triglyceride-caprylic acid structure.
Figure 2 is an HPLC liquid chromatography of palm oil extract enriched in vitamin K2.
FIG. 3 is a high performance liquid chromatogram of 1, in which vitamin K2 was separated from a macroporous resin using a butyl acetate solution as a resolving agent.
Fig. 4 is a high performance liquid chromatogram of 2 using butyl acetate: ethanol is 3:2 in reverse phase silica gel to purify vitamin K2.
Fig. 5 shows yellow crystals of vitamin K2 prepared according to the present invention.
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. 1-5. 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 separating and purifying vitamin K2 in fermentation liquor by using palm oil extract
The method mainly comprises the following steps of using palm oil extract (medium chain triglyceride) as a solvent to extract vitamin K2 in dry bacteria in one step, and separating and purifying high-purity vitamin K2 by a two-step method:
(1) and (5) strain seed culture. Before fermentation, the bacillus natto or the bacillus subtilis needs to be cultured by seed liquid to obtain a certain amount of pure strain liquid.
(2) And (4) preparing a fermentation medium. Continuously optimizing the components of the fermentation culture medium through the feedback result of the fermentation experiment, and selecting the optimal fermentation culture medium formula for fermentation.
(3) And (4) regulating and controlling the fermentation process. And (3) inoculating the seed liquid in the step (1) into the fermentation medium prepared in the step (2). The fermentation temperature is 35-40 ℃, the rotation speed is kept at 200-600 rpm, if air needs to be introduced, and the ventilation volume is controlled at 1-5 vvm. And (3) monitoring the fermentation temperature, the fermentation tank pressure and the growth rate of thalli in real time in the fermentation process, if more foams are found in the culture system, adding a sterilized defoaming agent at any time for defoaming, wherein the total adding volume is between 0.0001 and 0.001 percent of the culture system.
(4) Preparation of dried cells. Separating the bacillus natto or bacillus subtilis fermentation liquor obtained by a microbial fermentation production method to obtain wet thalli, and then putting the wet thalli into a vacuum freeze-drying machine for freeze-drying to obtain the bacillus natto dry thalli rich in vitamin K2.
(5) The palm oil extract is used for extracting vitamin K2 in the bacillus natto dry bacteria. Mixing the dry thalli and the palm oil extract according to a certain proportion, fully and uniformly mixing on a shaking table for 2-4 hours, placing the mixed solution in a floor type refrigerated centrifuge for centrifugation, and taking supernatant liquid, wherein the obtained supernatant liquid is the palm oil extract rich in vitamin K2.
(6) And (3) loading the pretreated macroporous resin into a column, putting the palm oil extract rich in vitamin K2 obtained in the step (5) into a separation column filled with the macroporous resin, removing miscellaneous oil substances by using an eluent I, and eluting the vitamin K2 from the macroporous resin by using a resolving agent I.
(7) Concentrating the vitamin K2-enriched butyl acetate solution obtained in step (6).
(8) And (4) putting the vitamin K2 solution obtained by concentration in the step (7) into a separation column filled with reverse phase silica gel, removing impurities by using an eluent II, and obtaining a high-purity vitamin K2 product by using a resolving agent II.
(9) And (4) collecting the high-concentration vitamin K2 solution obtained in the step (8), and then placing the solution in a refrigerator at the temperature of minus 20 ℃ for freezing and crystallizing to obtain the vitamin K2 crystal with high purity.
The seed liquid culture medium in the step (1) is 10g/L of glucose, 10g/L of peptone and 5g/L of NaCl, and the pH value is 7.0-7.5.
The culturing condition in the step (1) is that the temperature of the shaking table is 25-37 ℃, and the rotating speed of the shaking table is 100-250 rpm.
In the seed preparation process in the step (1), the number of passages is 2-4, and the inoculation amount in each passage is 1-5%.
The formula of the fermentation medium in the step (2) is 28g/L of glycerol, 55g/L of peptone and 6g/L of yeast extract, and K2HPO4 3g/L，MgSO4 1g/L，CaCl20.1g/L and a pH of 7.0 to 7.5.
The fermentation medium in the step (2) needs to be sterilized before use, and the main sterilization methods comprise high-temperature moist heat sterilization, sterilization membrane filtration and other methods.
The inoculation amount of fermentation in the step (3) is 1-5%.
When the shake flask is fermented in the step (3), the liquid loading of the shake flask is 15% -25%, and the rotation speed is 100-250 rpm.
When the fermentation tank is adopted for large-scale culture in the step (3), the liquid loading amount is 50-85%, the stirring speed is 200-500rpm, the ventilation amount is 1-10vvm when sterile air is introduced, and the fermentation temperature is 30-37 ℃.
The method for separating the wet thalli from the fermentation liquor in the step (4) mainly adopts a floor freezing centrifuge centrifugation method, 50-100 ml of the fermentation liquor is placed in a 400ml centrifuge cup, the rotor of a centrifuge is balanced, the centrifuge rotation speed is 8000-10000 rpm, the centrifugation time is 10-20 min, the supernatant is poured out, and the wet thalli precipitated at the bottom of the centrifuge cup are reserved.
The temperature of freeze drying in the step (4) is-20 ℃ to-80 ℃, the vacuum degree is 0-10 mtor, the freeze drying time is 24h to 72h, the wet thalli in the block shape are powdery dry thalli, and the water content after drying is 1-10%.
The ratio of the dry thalli to the palm oil extract in the step (5) is 1: 1-1: 20 (the dry thalli does not need pretreatment such as wall breaking, and most of thalli can be subjected to wall breaking by freezing vacuum drying at the temperature of-80 ℃ and ethanol solution in the next step).
The rotation speed of the shaking table culture in the step (5) is 200-300 rpm/min.
The rotating speed of the centrifuge in the step (5) is 7500-9000 rpm, and the centrifuging time is 10-20 min.
And (3) filling the column by using a wet method in the step (6), firstly soaking the macroporous resin by using an eluant, opening a cock at the lower end of the glass column, slowly adding the macroporous resin into the glass column along a glass rod, and after filling is finished, lightly knocking the column to ensure that the filling is uniform and bubbles escape. Because the wet-process column packing is not easy to generate bubbles and has better separation effect on substances, the wet-process column packing is selected by people.
In the step (6), the macroporous resin HPD722 needs to be soaked in 95% ethanol for more than 6 hours in advance.
The loading amount of the step (6) is 15-25ml (5-10% of the column volume).
And (3) the eluent I in the step (6) is 80-100% of ethanol, and the elution rate is 0.01-0.015 column volume/min.
And (3) the resolving agent I in the step (6) is butyl acetate, and the elution rate is 0.05-0.06 column volume/min.
The concentration method used in the step (7) is a rotary evaporation concentration method, and the used apparatus is a rotary evaporator. And (3) enabling the evaporation flask to be in a negative pressure state through a vacuum pump, sealing and reducing the pressure to 400-600 mm Hg, heating in a water bath at 75-95 ℃ at a rotating speed of 60-120 rpm/min, and performing rotary evaporation until ethyl acetate is completely evaporated.
The condition of rotary evaporation of butyl acetate in the step (7) is that the butyl acetate is heated to 85-95 ℃ in a water bath under a vacuum state.
The loading amount of the step (8) is 6-12 ml. (10-20% column volume)
In the step (8), the reversed phase silica gel needs to be dried for 0.5 h-2 h in advance in an environment of 130-140 ℃, and then is soaked for more than 2h by using butyl acetate.
And (3) eluting the second eluent in the step (8) with a mixed solution of butyl acetate and ethanol in a ratio of 5:2 at a flow rate of 0.001-0.0015 column volume/min.
And (3) the second resolving agent in the step (8) is a mixed solution of butyl acetate and ethanol in a ratio of 3:2, and the flow rate is 0.001-0.0015 column volume/min.
The crystallization method in the step (9) includes a low-temperature freezing method, a liquid phase diffusion method and the like. The freezing crystallization method used in the research is to concentrate a sample purified by reversed phase silica gel, add a small amount of methanol, and freeze the sample for 18 to 36 hours at a temperature of between 20 ℃ below zero and 25 ℃ below zero to obtain yellow crystals.
Physicochemical Properties and feedstock characteristics of the Medium chain Triglycerides used
| Acid value (mg KOH/g) | 0.02 | Relative density | 0.947 |
| Saponification number (mg KOH/g) | 333 | C8 (caprylic acid) content | 60.3％ |
| Hydrogen number (mg KOH/g) | 2 | C10 chain content (decanoic acid) | 39.6％ |
| Peroxide value (mg KOH/g) | 0 | Other impurities | 0.1％ |
| Moisture content | 0.02％ | Purity of | Food grade (99%) |
Palm oil extract (medium chain triglyceride) is used as an edible oil, and the medium chain triglyceride is composed of saturated fatty acids only, has low freezing point, is liquid at room temperature, and has low viscosity. Compared to soybean oil, medium chain triglycerides are completely odorless, colorless, transparent liquids. Compared with common oil and hydrogenated oil, the medium chain triglyceride has extremely low unsaturated fatty acid content, very good oxidation stability, and is particularly stable at high temperature and low temperature, and simultaneously, the medium chain triglyceride and vitamins have good intersolubility, and the product extracted by using the medium chain triglyceride as a solvent does not need to worry about the residue of organic chemical substances. Therefore, palm oil extract (medium chain triglyceride) is selected as the solvent for extracting the intracellular vitamin K2 of the bacillus natto or the bacillus subtilis. This experiment was applied to palm oil extract (medium chain triglycerides) produced by the dao-office biotechnology (shanghai) limited. The medium chain triglyceride-caprylic acid structure is shown schematically in figure 1.
Example 2: method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract
Firstly, fermenting bacillus natto, freezing and drying wet thalli obtained by fermentation to obtain bacillus natto dry thalli, extracting vitamin K2 in the bacillus natto dry thalli by using palm oil extract (medium chain triglyceride) as a solvent, and then using green nontoxic ethanol and butyl acetate as solvents for separation and purification to obtain high-purity vitamin K2 in a green and high-efficiency two-step method. The method comprises the following specific steps:
the bacillus natto is fermented and cultured in a 30L stirring reactor, and the liquid filling amount is 80 percent. Inoculating the seed culture solution with 10% inoculum size, fermenting at 37 deg.C and 250rpm, and culturing for 156 hr while monitoring thallus concentration (OD method). Centrifuging the bacillus natto or bacillus subtilis fermentation liquor obtained by a microbial fermentation production method by using a floor freezing centrifuge, wherein the centrifugal rotation speed is 10000rpm, and the centrifugation time is 15min to obtain wet thalli, and then placing the wet thalli in a vacuum freeze dryer for freeze drying for 48h to obtain the bacillus natto dry thalli rich in vitamin K2.
Mixing 10g of the fermented bacillus natto dry thallus with 100ml of palm oil extract, uniformly mixing the mixture on a shaking table for 2-4 hours generally, wherein the rotation speed of shaking table culture is 200-300 rpm/min, the culture temperature is 35-40 ℃, then placing the mixed solution in a floor freezing centrifuge for centrifugation, and taking supernatant, namely the palm oil extract rich in vitamin K2. The palm oil extract rich in vitamin K2 is detected by HPLC liquid chromatography, the concentration of vitamin K2 is determined by comparison with a standard product, the concentration of vitamin K2 in the palm oil solution is 323mg/L by calculation, and the effect of extracting vitamin K2 in the bacillus natto dry thalli by taking butyl acetate as a solvent is proved to be good. As shown in particular in fig. 2.
15ml of the obtained palm oil extract rich in vitamin K2 is put into a separation column filled with macroporous resin HPD722, about 150ml of 80% ethanol is used for removing the miscellaneous oil substances, the flow rate of the elution impurities is controlled at 0.5ml/min, then about 60ml of butyl acetate is used for eluting vitamin K2 from the macroporous resin, and the concentration and the purity of the vitamin K2 are determined by HPLC liquid chromatography. The vitamin K2 obtained by counting the experimental data and separating and purifying the macroporous resin by using ethanol and butyl acetate as eluent has the purity of 75 percent. As shown in fig. 3, the high performance liquid chromatogram showed that vitamin K2 was separated from the macroporous resin using a butyl acetate solution as the resolving agent.
In order to facilitate the next operation, the butyl acetate solution rich in vitamin K2 obtained in the previous step is subjected to vacuum rotary evaporation, the evaporation flask is in a negative pressure state through a vacuum pump, the pressure is sealed and reduced to 500 mm Hg, the heating temperature of a water bath is 85 ℃, the rotation speed is 90rpm/min, and the vitamin K2 solution is concentrated. Placing the concentrated solution in a separation column filled with reverse phase silica gel, wherein the weight ratio of butyl acetate: ethanol is 5: 1 as eluent, the impurities were removed at a flow rate of 0.001 column volume/min, followed by the use of butyl acetate: ethanol is 3: the vitamin K2 in the reversed phase silica gel is analyzed by the mixed solution of 2 as an analysis agent to obtain a vitamin K2 solution with the purity of more than 95%. The concentration of the vitamin K2 in the solution after the separation and purification operations is obtained by calculation to be 420 mg/L. High performance liquid chromatogram as shown in fig. 4, using butyl acetate: ethanol is 3:2 in reverse phase silica gel to purify vitamin K2.
Concentrating the sample purified by reverse phase silica gel, adding a small amount of methanol, and freezing at-25 deg.C for 18h to obtain yellow vitamin K2 crystal, as shown in FIG. 5.
Example 3: method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract
A method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract comprises the following steps:
step 1: separating the fermentation liquor obtained by the microbial fermentation production method to obtain wet thalli, and freeze-drying the wet thalli to obtain dry thalli rich in vitamin K2;
step 2: mixing the dry thallus with the palm oil extract for 2h, and then centrifuging the mixed solution to obtain a supernatant, wherein the obtained supernatant is the palm oil extract rich in vitamin K2;
and step 3: loading the pretreated macroporous resin into a column, putting the palm oil extract rich in vitamin K2 into a separation column filled with the macroporous resin, removing miscellaneous oil substances by using a first eluent, and eluting vitamin K2 from the macroporous resin by using a first resolving agent to obtain an eluent;
and 4, step 4: concentrating the eluent obtained in the step (3) to obtain a concentrated solution;
and 5: putting the concentrated solution into a separation column containing reversed phase silica gel, removing impurities by using a second eluent, and eluting by using a second resolving agent to obtain a high-purity vitamin K2 product;
step 6: and (3) freezing and crystallizing the high-concentration vitamin K2 solution to obtain the vitamin K2 crystal.
The preferred embodiment is: the mass ratio of the dry thallus to the palm oil extract is 1: 1.
The preferred embodiment is: in step 2, the dry thallus and the palm oil extract are mixed and then placed on a shaking table to be processed for 2 hours at the rotating speed of 200 rpm/min.
The preferred embodiment is: in step 2, the mixed solution is placed in a floor type refrigerated centrifuge and centrifuged for 10min at the rotating speed of 7500 rpm.
The preferred embodiment is: step 3, soaking macroporous resin in 95% ethanol by volume, then opening a cock at the lower end of a glass column, adding the macroporous resin into the glass column along a glass rod, after filling, lightly knocking the glass column to ensure that the filling is uniform, and enabling bubbles to escape; the macroporous resin is macroporous resin HPD 417.
The preferred embodiment is: the first eluent is ethanol with the volume fraction of 80%% and the elution rate is 0.01 column volume/min; the first eluent was butyl acetate and the elution rate was 0.05 column volume/min.
The preferred embodiment is: and 4, adopting a rotary evaporation concentration method, wherein the used instrument is a rotary evaporator, enabling the evaporation flask to be in a negative pressure state through a vacuum pump, sealing and reducing the pressure to 400 mm Hg, heating in water bath at 75 ℃, rotating at 60rpm/min, and finishing the rotary evaporation until all ethyl acetate is evaporated.
The preferred embodiment is: in the step 5, the reversed phase silica gel needs to be dried for 0.5h in advance in an environment of 130 ℃, and then is soaked for more than 2h by using butyl acetate.
The preferred embodiment is: the second eluent is a mixed solution of butyl acetate and ethanol according to the volume ratio of 5:2, and the flow rate is 0.001 column volume/min.
The preferred embodiment is: the second resolving agent is a mixed solution of butyl acetate and ethanol according to the volume ratio of 3:2, and the flow rate is 0.001 column volume/min.
Example 4: method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract
A method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract comprises the following steps:
step 1: separating the fermentation liquor obtained by the microbial fermentation production method to obtain wet thalli, and freeze-drying the wet thalli to obtain dry thalli rich in vitamin K2;
step 2: mixing the dry thallus with the palm oil extract for 4h, and then centrifuging the mixed solution to obtain a supernatant, wherein the obtained supernatant is the palm oil extract rich in vitamin K2;
and step 3: loading the pretreated macroporous resin into a column, putting the palm oil extract rich in vitamin K2 into a separation column filled with the macroporous resin, removing miscellaneous oil substances by using a first eluent, and eluting vitamin K2 from the macroporous resin by using a first resolving agent to obtain an eluent;
and 4, step 4: concentrating the eluent obtained in the step (3) to obtain a concentrated solution;
and 5: putting the concentrated solution into a separation column containing reversed phase silica gel, removing impurities by using a second eluent, and eluting by using a second resolving agent to obtain a high-purity vitamin K2 product;
step 6: and (3) freezing and crystallizing the high-concentration vitamin K2 solution to obtain the vitamin K2 crystal.
The preferred embodiment is: the mass ratio of the dry thallus to the palm oil extract is 1: 20.
The preferred embodiment is: in step 2, the dry thallus and the palm oil extract are mixed and then placed on a shaking table to be processed for 4 hours at the rotating speed of 300 rpm/min.
The preferred embodiment is: in step 2, the mixture was placed in a floor standing refrigerated centrifuge and centrifuged at 9000rpm for 20 min.
The preferred embodiment is: step 3, soaking macroporous resin in 95% ethanol by volume, then opening a cock at the lower end of a glass column, adding the macroporous resin into the glass column along a glass rod, after filling, lightly knocking the glass column to ensure that the filling is uniform, and enabling bubbles to escape; the macroporous resin is macroporous resin HPD 722.
The preferred embodiment is: the first eluent is ethanol with the volume fraction of 100 percent, and the elution rate is 0.015 column volume/min; the first eluent was butyl acetate and the elution rate was 0.06 column volumes/min.
The preferred embodiment is: and 4, adopting a rotary evaporation concentration method, wherein the adopted instrument is a rotary evaporator, the evaporation flask is in a negative pressure state through a vacuum pump, the pressure is reduced to 600 mm Hg in a sealed manner, the water bath heating temperature is 95 ℃, the rotation speed is 120rpm/min, and the rotary evaporation is finished until all ethyl acetate is evaporated.
The preferred embodiment is: in the step 5, the reversed phase silica gel needs to be dried for 2 hours in advance in an environment of 140 ℃, and then is soaked for more than 2 hours by using butyl acetate.
The preferred embodiment is: the second eluent is a mixed solution of butyl acetate and ethanol according to the volume ratio of 5:2, and the flow rate is 0.0015 column volume/min.
The preferred embodiment is: the second resolving agent is a mixed solution of butyl acetate and ethanol according to the volume ratio of 3:2, and the flow rate is 0.0015 column volume/min.
Example 5: method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract
A method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract comprises the following steps:
step 1: separating the fermentation liquor obtained by the microbial fermentation production method to obtain wet thalli, and freeze-drying the wet thalli to obtain dry thalli rich in vitamin K2;
step 2: mixing the dry thallus with the palm oil extract for 3h, and then centrifuging the mixed solution to obtain supernatant, wherein the obtained supernatant is the palm oil extract rich in vitamin K2;
and step 3: loading the pretreated macroporous resin into a column, putting the palm oil extract rich in vitamin K2 into a separation column filled with the macroporous resin, removing miscellaneous oil substances by using a first eluent, and eluting vitamin K2 from the macroporous resin by using a first resolving agent to obtain an eluent;
and 4, step 4: concentrating the eluent obtained in the step (3) to obtain a concentrated solution;
and 5: putting the concentrated solution into a separation column containing reversed phase silica gel, removing impurities by using a second eluent, and eluting by using a second resolving agent to obtain a high-purity vitamin K2 product;
step 6: and (3) freezing and crystallizing the high-concentration vitamin K2 solution to obtain the vitamin K2 crystal.
The preferred embodiment is: the mass ratio of the dry thallus to the palm oil extract is 1: 10.
The preferred embodiment is: in step 2, the dry thallus and the palm oil extract are mixed and then placed on a shaker at the rotating speed of 250rpm/min for processing for 3 hours.
The preferred embodiment is: in step 2, the mixed solution is placed in a floor type refrigerated centrifuge and centrifuged for 15min at the rotation speed of 8000 rpm.
The preferred embodiment is: step 3, soaking macroporous resin in 95% ethanol by volume, then opening a cock at the lower end of a glass column, adding the macroporous resin into the glass column along a glass rod, after filling, lightly knocking the glass column to ensure that the filling is uniform, and enabling bubbles to escape; the macroporous resin is macroporous resin GDX-10.
The preferred embodiment is: the first eluent is ethanol with the volume fraction of 90 percent, and the elution rate is 0.012 column volume/min; the first eluent was butyl acetate and the elution rate was 0.055 column volume/min.
The preferred embodiment is: and 4, adopting a rotary evaporation concentration method, wherein the adopted instrument is a rotary evaporator, the evaporation flask is in a negative pressure state through a vacuum pump, the pressure is reduced to 500 mm Hg in a sealed manner, the water bath heating temperature is 85 ℃, the rotation speed is 90rpm/min, and the rotary evaporation is finished until all ethyl acetate is evaporated.
The preferred embodiment is: in the step 5, the reversed phase silica gel needs to be dried for 1 hour in advance in an environment of 135 ℃, and then is soaked for more than 2 hours by using butyl acetate.
The preferred embodiment is: the second eluent is a mixed solution of butyl acetate and ethanol according to the volume ratio of 5:2, and the flow rate is 0.0012 column volume/min.
The preferred embodiment is: the second resolving agent is a mixed solution of butyl acetate and ethanol according to the volume ratio of 3:2, and the flow rate is 0.0012 column volume/min.
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 (10)
1. A method for separating and purifying vitamin K2 in fermentation liquor by using palm oil extract is characterized by comprising the following steps: comprises the following steps:
step 1: separating the fermentation liquor obtained by the microbial fermentation production method to obtain wet thalli, and freeze-drying the wet thalli to obtain dry thalli rich in vitamin K2;
step 2: mixing the dry thalli and the palm oil extract for 2-4 h, and then centrifuging the mixed solution to obtain a supernatant, namely the palm oil extract rich in vitamin K2;
and step 3: loading the pretreated macroporous resin into a column, putting the palm oil extract rich in vitamin K2 into a separation column filled with the macroporous resin, removing miscellaneous oil substances by using a first eluent, and eluting vitamin K2 from the macroporous resin by using a first resolving agent to obtain an eluent;
and 4, step 4: concentrating the eluent obtained in the step (3) to obtain a concentrated solution;
and 5: putting the concentrated solution into a separation column containing reversed phase silica gel, removing impurities by using a second eluent, and eluting by using a second resolving agent to obtain a high-purity vitamin K2 product;
step 6: and (3) freezing and crystallizing the high-concentration vitamin K2 solution to obtain the vitamin K2 crystal.
2. The method for separating and purifying vitamin K2 in the fermentation broth by using palm oil extract as claimed in claim 1, wherein: the mass ratio of the dry thallus to the palm oil extract is 1: 1-1: 20.
3. The method for separating and purifying vitamin K2 in the fermentation broth by using palm oil extract as claimed in claim 1, wherein: in the step 2, the dry thalli and the palm oil extract are mixed and then are placed on a shaking table to be processed for 2-4 hours at the rotating speed of 200-300 rpm/min.
4. The method for separating and purifying vitamin K2 in the fermentation broth by using palm oil extract as claimed in claim 1, wherein: and 2, placing the mixed solution in a floor type refrigerated centrifuge, and centrifuging at the rotating speed of 7500-9000 rpm for 10-20 min.
5. The method for separating and purifying vitamin K2 in the fermentation broth by using palm oil extract as claimed in claim 1, wherein: step 3, soaking macroporous resin in 95% ethanol by volume, then opening a cock at the lower end of a glass column, adding the macroporous resin into the glass column along a glass rod, after filling, lightly knocking the glass column to ensure that the filling is uniform, and enabling bubbles to escape; the macroporous resin is one of macroporous resin HPD417, macroporous resin HPD722, macroporous resin GDX-10 and macroporous resin GDX-502.
6. The method for separating and purifying vitamin K2 in the fermentation broth by using palm oil extract as claimed in claim 1, wherein: the first eluent is ethanol with the volume fraction of 80-100%, and the elution rate is 0.01-0.015 column volume/min; the first eluent is butyl acetate, and the elution rate is 0.05-0.06 column volume/min.
7. The method for separating and purifying vitamin K2 in the fermentation broth by using palm oil extract as claimed in claim 1, wherein: and 4, adopting a rotary evaporation concentration method, wherein the used instrument is a rotary evaporator, the evaporation flask is in a negative pressure state through a vacuum pump, the pressure is reduced to 400-600 mm Hg in a sealed manner, the water bath heating temperature is 75-95 ℃, the rotation speed is 60-120 rpm/min, and the rotary evaporation is finished until all ethyl acetate is evaporated.
8. The method for separating and purifying vitamin K2 in the fermentation broth by using palm oil extract as claimed in claim 1, wherein: in the step 5, the reversed phase silica gel needs to be dried for 0.5 to 2 hours in advance in an environment of 130 to 140 ℃, and then is soaked for more than 2 hours by using butyl acetate.
9. The method for separating and purifying vitamin K2 in the fermentation broth by using palm oil extract as claimed in claim 1, wherein: the second eluent is a mixed solution composed of butyl acetate and ethanol according to a volume ratio of 5:2, and the flow rate is 0.001-0.0015 column volume/min.
10. The method for separating and purifying vitamin K2 in the fermentation broth by using palm oil extract as claimed in claim 1, wherein: the second resolving agent is a mixed solution composed of butyl acetate and ethanol according to the volume ratio of 3:2, and the flow rate is 0.001-0.0015 column volume/min.
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