CN209885294U - Vitamin K2 organic solution extraction column - Google Patents

Abstract

Vitamin K₂An organic solution extraction column, characterized in that: the extraction column comprises a container, a filter membrane is arranged at the bottom of the container, and a liquid layer is arranged above the filter membrane; a switch is arranged at the liquid outlet of the container; the side part of the container is provided with a filling opening which is communicated with a micro-droplet injection device through a pipeline, and the inner cavity of the container is provided with a piston in a sliding way, and the piston is in driving connection with a reciprocating device. The utility model has the advantage of high extraction efficiency.

Description

Vitamin K2Organic solution extraction column

Technical Field

The utility model relates to a vitamin K₂An organic solution extraction column belongs to the technical field of vitamin K2 extraction equipment.

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 treatment of macroporous resin, reversed phase silica gel and the like, and has complex overall 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). Therefore, it is necessary to design a vitamin K₂Organic solution extraction column.

Disclosure of Invention

In order to overcome the defects in the prior art, the utility model aims to provide a vitamin K₂Organic solution extraction column.

In order to achieve the above objects and other related objects, the present invention provides a technical solution: vitamin K₂The organic solution extraction column comprises a container, wherein a filter membrane is arranged at the bottom of the container, and a liquid layer is arranged above the filter membrane; a switch is arranged at the liquid outlet of the container; the side part of the container is provided with a filling opening which is communicated with a micro-droplet injection device through a pipeline, and the inner cavity of the container is provided with a piston in a sliding way, and the piston is in driving connection with a reciprocating device.

The preferable technical scheme is as follows: the pore size of the filter membrane is 0.45 micron.

The preferable technical scheme is as follows: the height-diameter ratio of the extraction column is 7.5-40.0: 1.

the preferable technical scheme is as follows: the micro-droplet injection device is a syringe.

The preferable technical scheme is as follows: the liquid layer is made of n-hexane.

Because of the application of the technical scheme, compared with the prior art, the utility model the advantage that has is:

the utility model has the advantage of high extraction efficiency.

Drawings

Fig. 1 is a schematic view of the present invention.

In the above figures, 1, container; 2. an extraction column; 3. a piston; 4. filtering the membrane; 5. a switch; 6. a syringe.

Detailed Description

The following description is provided for illustrative purposes, and other advantages and features of the present invention will become apparent to those skilled in the art from the following detailed description.

Please refer to fig. 1. It should be understood that the structure, ratio, size and the like shown in the drawings attached to the present specification are only used for matching with the content disclosed in the specification, so as to be known and read by those skilled in the art, and are not used for limiting the limit conditions that the present invention can be implemented, so that the present invention has no technical essential meaning, and any structure modification, ratio relationship change or size adjustment should still fall within the scope that the technical content disclosed in the present invention can cover without affecting the function that the present invention can produce and the purpose that the present invention can achieve. Meanwhile, the terms such as "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for convenience of description, and are not intended to limit the scope of the present invention, and changes or adjustments of the relative relationship thereof may be made without substantial technical changes, and the present invention is also regarded as the scope of the present invention.

As shown in figure 1, a vitamin K₂The organic solution extraction column comprises a container 1, wherein a filter membrane 4 is arranged at the bottom of the container, and a liquid layer is arranged above the filter membrane; a liquid outlet of the container is provided with a switch 5; the side part of the container is provided with a filling opening which is communicated with a micro-droplet injection device through a pipeline, the inner cavity of the container is provided with a piston 3 in a sliding way, and the piston is in driving connection with a reciprocating device.

The preferred embodiment is: the pore size of the filter membrane is 0.45 micron.

The preferred embodiment is: the height-diameter ratio of the extraction column is 7.5-40.0: 1.

the preferred embodiment is: the micro-droplet injection device is a syringe 6.

The preferred embodiment is: the liquid layer is made of n-hexane.

The diameter of the container 1 is 3.5cm, the height is 25cm, the material is glass, a filter membrane (a support structure can be arranged for fixing the filter membrane) is added at the bottom of the glass column, and one third of the column volume of n-hexane is added. When in work: the loading of the n-hexane solution of vitamin K2 in the extraction column was 33.3% of the column volume. After the normal hexane solution is filled into an extraction column, firstly, slowly adding ultrapure water into the extraction column to extract and remove strong-polarity impurities, and adding positive pressure into a sample inlet, wherein the using amount of the ultrapure water is 2 times of that of the normal hexane solution, and the time is 8-25 min; then, slowly adding 10-95% ethanol into the extraction column to extract and remove the weak polar impurities, wherein the dosage of the ethanol with different concentrations is as follows: extracting with 95% ethanol at 2BV, extracting with 70% ethanol at 2BV, extracting with 30% ethanol at 2BV, extracting with 10% ethanol at 1BV for 25-45 min.

Adding ultrapure water and ethanol with different concentrations into an injector, adjusting the size of a dropping nozzle of the injector to control the size of a formed liquid drop, wherein the size of the liquid drop is 140 drops/ml-15 drops/ml, and 7ul-60ul of micro liquid drops can be formed.

Vitamin K₂The source of the n-hexane solution of (a):

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.

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 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, and the wall breaking effect is improved by 30 percent and reaches 0.65 mg/g. 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).

The extraction efficiency is better and better with the increase of the absolute ethanol amount, the difference is less when the volume is 20ml and 25ml, and 20ml is selected as the wall breaking volume for reducing the use of organic reagents.

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.

The extraction amount can reach 0.85mg/g more when the volume of ethanol used is 10ml, and the extraction amount has no obvious change along with the addition of absolute ethanol, so 10ml of ethanol is selected as the volume of the 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.

The extraction yield increases with increasing temperature, and when the temperature reaches 40 ℃, the effect of the temperature on the extraction yield does not change any more. 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 vitamin K₂The high-concentration solution with the concentration of 60mg/L to 200mg/L is the vitamin K₂N-hexane solution of (1).

The above embodiments are merely illustrative of the principles and effects of the present invention, and are not to be construed as limiting the invention. Modifications and variations can be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims (5)

1. Vitamin K₂An organic solution extraction column, characterized in that: the extraction column comprises a container, a filter membrane is arranged at the bottom of the container, and a liquid layer is arranged above the filter membrane; a switch is arranged at the liquid outlet of the container; the side part of the container is provided with a filling opening which is communicated with a micro-droplet injection device through a pipeline, and the inner cavity of the container is provided with a piston in a sliding way, and the piston is in driving connection with a reciprocating device.

2. Vitamin K according to claim 1₂An organic solution extraction column, characterized in that: the pore size of the filter membrane is 0.45 micron.

3. Vitamin K according to claim 1₂An organic solution extraction column, characterized in that: the height-diameter ratio of the extraction column is 7.5-40.0: 1.

4. vitamin K according to claim 1₂An organic solution extraction column, characterized in that: the micro-droplet injection device is a syringe.

5. Vitamin K according to claim 1₂An organic solution extraction column, characterized in that: the liquid layer is made of n-hexane.

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