Method for separating and purifying vitamin K2 in bacillus subtilis natto

A technology for separation and purification of Bacillus natto is applied in the field of bioengineering to achieve the effects of high biological activity, simple process operation and convenient industrial application.

Active Publication Date: 2017-05-10

HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI

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Abstract

The invention discloses a method for separating and purifying vitamin K2 in bacillus subtilis natto. The method comprises the following steps: acquiring vitamin K2 extract, acquiring a vitamin K2 column stock solution, acquiring a low-purity vitamin K2 concentrated solution, acquiring a high-purity vitamin K2 concentrated solution, acquiring high-purity vitamin K2, and acquiring vitamin K2 crystals. The method has the advantages that (1) an obtained vitamin K2 crystal product is high in purity, bioactivity and recovery rate; (2) a separation and purification process is easy to operate, mild in condition and high in treatment capability; (3) each organic solvent can be recycled for industrial application.

Application Domain

Quinone separation/purification

Technology Topic

Bacillus subtilis (natto)Chemistry +7

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Examples

Experimental program(3)

Example Embodiment

[0040] Example 1

[0041] A kind of separation and purification of vitamin K from Bacillus natto 2 Method such as figure 1 As shown, including the following steps:

[0042] (1) Vitamin K 2 Extraction

[0043] Using Bacillus natto fermentation broth as vitamin K 2 Extract the stock solution, centrifuge to obtain the bacteria, after drying, extract with ethanol to obtain vitamin K 2 Extraction solution: The extraction method is static extraction, the extraction time is 20 minutes, and the number of extractions is once.

[0044] (2) Vitamin K 2 Obtaining the upper column stock solution

[0045] Vitamin K 2 The extract is passed through a membrane separation device (membrane pore size≤0.45μm) to remove insoluble impurities, and the obtained vitamin K 2 The filtrate is evaporated to dryness under reduced pressure, and then re-dissolved in methanol to obtain vitamin K 2 Upper column stock solution;

[0046] (3) Low purity vitamin K 2 Obtaining concentrate

[0047] Vitamin K 2 The stock solution of the upper column is adsorbed by a macroporous adsorption resin column (aromatic adsorbent), and the column is loaded by a wet method (the column conditions are: the height-to-diameter ratio is 3:1, the flow rate is 0.025 times the column volume/min), and then the polarity Wash with organic solvent (the amount is 1 time of the column volume of the macroporous resin column), and finally elution with dichloromethane (the amount is 1 time of the column volume of the macroporous resin column), collect the corresponding eluate and carry out Concentrated to obtain low-purity vitamin K 2 Concentrate (purity less than 50%);

[0048] (4) Higher purity vitamin K 2 Obtaining concentrate

[0049] The low purity vitamin K 2 The concentrate is purified by a molecular weight exclusion chromatography column, and then eluted with benzene (the amount is 1 times the volume of the molecular weight exclusion chromatography column), and the corresponding eluate is collected and concentrated to obtain higher purity Vitamin K 2 Concentrated solution (50-80% purity);

[0050] Wherein, the molecular weight of the substance excluded by the molecular weight exclusion chromatography filler is 400Da-1000Da; the filler can be used as a column or packed in several sections in the same molecular weight exclusion chromatography column. The exclusion limit passes through the column packing in order from small to large or from large to small; the height-to-diameter ratio of the molecular weight exclusion chromatography column is 10:1, the flow rate is 0.004 times the column volume/min, and the column height is related to the vitamin K 2 The liquid height ratio of the concentrated liquid is 35:1;

[0051] (5) High purity vitamin K 2 Get

[0052] The higher purity vitamin K 2 After the concentrated solution is dissolved in methanol, it passes through a reversed-phase silica gel column (the particle size of the reversed-phase silica gel column is 10μm, the height-to-diameter ratio is 5:1, the flow rate is 0.015 times the column volume/min, the column height and the higher purity vitamin K 2 The concentrated liquid has a height ratio of 7:1) separation, then eluted with methanol and collected in sections to obtain high-purity vitamin K 2 (Purity is greater than 80%);

[0053] (6) Vitamin K 2 Crystal acquisition

[0054] Use analytically pure methanol to convert vitamin K 2 Dissolve at a high temperature of 40℃, crystallize at a low temperature of -40℃, and recrystallize to obtain vitamin K 2 Crystal (purity greater than or equal to 98.2%).

[0055] Preferably, the concentration method is reduced pressure concentration, the temperature of the reduced pressure concentration is 40° C., and the vacuum degree is less than -0.08 MPa.

Example Embodiment

[0056] Example 2

[0057] A kind of separation and purification of vitamin K from Bacillus natto 2 Method such as figure 1 As shown, including the following steps:

[0058] (1) Vitamin K 2 Extraction

[0059] Using Bacillus natto fermentation broth as vitamin K 2 Extract the stock solution, centrifuge to obtain the bacteria, after drying, extract with toluene to obtain vitamin K 2 Extraction solution: The extraction method is static extraction, the extraction time is 60 minutes, and the extraction times are 3 times.

[0060] (2) Vitamin K 2 Obtaining the upper column stock solution

[0061] Vitamin K 2 The extract is passed through a membrane separation device (membrane pore size≤0.45μm) to remove insoluble impurities, and the obtained vitamin K 2 The filtrate is evaporated to dryness under reduced pressure, and then re-dissolved in acetone to obtain vitamin K 2 Upper column stock solution;

[0062] (3) Low purity vitamin K 2 Obtaining concentrate

[0063] Vitamin K 2 The stock solution of the upper column is adsorbed by the non-polar macroporous adsorption resin column, and the column is loaded by dry method (the column conditions are: height-to-diameter ratio is 10:1, flow rate is 0.110 column volume/min), and then washed with polar organic solvent (Dosage is 3 times the column volume of the macroporous resin column), and finally eluted with isopentane (the amount is 4 times the column volume of the macroporous resin column), collect the corresponding eluate and concentrate it, thereby Get low purity vitamin K 2 Concentrate (purity less than 50%);

[0064] (4) Higher purity vitamin K 2 Obtaining concentrate

[0065] The low purity vitamin K 2 The concentrate is purified by a molecular weight exclusion chromatography column, and then eluted with trichlorobenzene (the amount is 4 times the column volume of the molecular weight exclusion chromatography column), and the corresponding eluate is collected and concentrated to obtain Higher purity vitamin K 2 Concentrated solution (50-80% purity);

[0066] Wherein, the molecular weight of the substance excluded by the molecular weight exclusion chromatography filler is 400Da-1000Da; the filler can be used as a column or packed in several sections in the same molecular weight exclusion chromatography column. The exclusion limit passes through the column packing in order from small to large or large to small; the height to diameter ratio of the molecular weight exclusion chromatography column is 25:1, the flow rate is 0.010 times the column volume/min, and the column height is Vitamin K 2 The liquid height ratio of the concentrated liquid is 50:1;

[0067] (5) High purity vitamin K 2 Get

[0068] The higher purity vitamin K 2 After the concentrated solution is dissolved in acetone, it passes through a reversed-phase silica gel column (the particle size of the reversed-phase silica gel column is 80μm, the height-to-diameter ratio is 25:1, the flow rate is 0.065 times the column volume/min, the column height and higher purity vitamin K 2 The height ratio of the concentrated liquid to the upper liquid is 30:1) separation, then eluted with acetone and collected in sections to obtain high-purity vitamin K 2 (Purity is greater than 80%);

[0069] (6) Vitamin K 2 Crystal acquisition

[0070] Vitamin K is added to methanol containing 30% ultrapure water 2 Vitamin K can be obtained by dissolving at a high temperature of 85℃, crystallizing at a low temperature of 0℃ and recrystallizing. 2 Crystal (purity greater than 98.2%).

[0071] Preferably, the concentration method is concentration under reduced pressure, the temperature of concentration under reduced pressure is 99° C., and the vacuum degree is less than -0.08 MPa.

Example Embodiment

[0072] Example 3

[0073] A kind of separation and purification of vitamin K from Bacillus natto 2 Method such as figure 1 As shown, including the following steps:

[0074] (1) Vitamin K 2 Extraction

[0075] Take Bacillus natto fermentation broth (composition: soybean meal 65g/L, corn meal 60g/L, peptone 50g/L, fermentation period: 7-9 days) as vitamin K 2 Extract the stock solution, centrifuge to obtain the bacteria, and after drying, extract according to the following steps:

[0076] ①Weigh several aliquots of 1.0g dry cells in a 50ml centrifuge tube, add 5ml different organic solvents to stand for 50min, extract 2 times, and make 3 parallel samples; the different organic solvents are methanol, ethanol, 1,3 propylene glycol, n-butanol, acetic acid, dichloromethane, isoamyl alcohol, n-hexane, ethyl acetate, chloroform, carbon tetrachloride, acetone, ether, toluene;

[0077] ②Fix the volume of the obtained extract to 5ml to obtain the test solution;

[0078] ③Use HPLC method (mobile phase: methanol:dichloromethane=4:1, flow rate 1mL/min, detection wavelength 248nm) to detect vitamin K in each extract 2 The concentration content of which image 3 Is the vitamin K in fermentation samples when n-butanol is used as the extractant 2 HPLC liquid spectrum of figure 2 Different organic solvents for extraction of vitamin K 2 Efficiency chart, where the extraction efficiency of methanol and 1,3 propylene glycol is extremely low, and it will not be the choice of the corresponding extractant in the later test;

[0079] (2) Vitamin K 2 Obtaining the upper column stock solution

[0080] Vitamin K 2 The extract is passed through a membrane separation device (membrane pore size≤0.45μm) to remove insoluble impurities, and the obtained vitamin K 2 The filtrate is evaporated to dryness under reduced pressure, and then re-dissolved in methanol to obtain vitamin K 2 Upper column stock solution;

[0081] (3) Low purity vitamin K 2 Obtaining concentrate

[0082] ①Weigh a certain amount of macroporous adsorption resin, soak it in 95% ethanol overnight, wet it on the column, and rinse with 95% ethanol until the mixture of effluent and distilled water (1:5, V/V) has no turbidity Then wash the column with 2 times the column volume of methanol to make the column height to diameter ratio 6:1;

[0083] ②Measure a certain amount of vitamin K 2 The methanol solution was applied to the column and passed through the column at a flow rate of 0.0283 times the column volume/min. Then the column was washed with 2 times the column volume of methanol at the same flow rate, and then vitamin K was eluted with 2 times the column volume of n-hexane at the same flow rate. 2;

[0084] ③ Repeat step 2 twice as a parallel experiment to obtain the second and third vitamin K2 eluates;

[0085] Measure each vitamin K 2 The volume of the eluent was measured by HPLC (mobile phase: methanol: dichloromethane=4:1, flow rate 1mL/min, detection wavelength 248nm) to detect vitamin K in each eluent 2 Content and grease impurities, calculate vitamin K 2 Then, combine the corresponding eluates and concentrate them to obtain low purity (purity less than 50%) vitamin K 2 Concentrate;

[0086] Among them, the obtained low-purity vitamin K 2 The concentrate does not contain or contains a very small amount of grease-like polar impurities (mass fraction <5%), and vitamin K 2 The recovery rate is calculated to be 80%-99%;

[0087] (4) Higher purity vitamin K 2 Obtaining concentrate

[0088] ①Weigh a certain amount of molecular weight exclusion chromatography packing material (the exclusion limit is 1000Da), and apply it to the column by wet method. Wash the column with 2 times the column volume of tetrahydrofuran. The height to diameter ratio of the prepared column is 17:1. ;

[0089] ②The low-purity vitamin K 2 The concentrate is re-dissolved in tetrahydrofuran solution, and a certain volume of vitamin K is measured. 2 The tetrahydrofuran solution is applied to the column, and the column is flushed with tetrahydrofuran. The flow rate is 0.0056 times the column volume/min. The height of the chromatography column is consistent with that of vitamin K. 2 The ratio of the upper liquid height of the concentrated liquid is 46:1;

[0090] ③ Collect each interception section by sections and concentrate them to obtain higher purity (50-80% purity) vitamin K 2 Concentrate, in which the vitamin K in each interception section is detected by HPLC 2 concentration;

[0091] ④Weigh a certain amount of molecular weight exclusion chromatography packing (the exclusion limit is 400Da) after pretreatment, and apply the wet method to the column, repeat ①-③, and obtain vitamin K after HPLC detection 2 Interception section.

[0092] Preferably, the obtained higher purity vitamin K 2 Macromolecular impurities in solution (molecular weight> 1000Da) and small molecule impurities (molecular weight <400Da) The sum of content ≤1%, vitamin K 2 The recovery rate is ≥98%. ;

[0093] (5) High purity vitamin K 2 Get

[0094] ① After pre-treatment of reversed-phase silica gel (50μm in particle size), the column is wet-processed, and then the column is washed with 2 times the column volume of methanol to make the column height to diameter ratio 12:1;

[0095] ②Higher purity vitamin K 2 The concentrated solution is re-dissolved with an appropriate amount of methanol, and an appropriate amount is applied to the column. The height of the chromatography column is the same as that of vitamin K. 2 The ratio of the upper liquid height of the concentrate is 10:1, and then elution is carried out with methanol at a flow rate of 0.0163 times the column volume/min;

[0096] ③Collect each interception section by section to obtain high purity (purity greater than 80%) vitamin K 2 , In which HPLC method is used to detect vitamin K in each cut-off section 2 Concentration and calculate the recovery rate;

[0097] (6) Vitamin K 2 Crystal acquisition

[0098] ①For the vitamin K obtained 2 The eluate is concentrated, and then re-dissolved with an appropriate amount of methanol containing 10% ultrapure water at 80°C;

[0099] ② will get vitamin K 2 Keep the methanol solution in a refrigerator at -20°C overnight;

[0100] ③ Filter the above solid-liquid mixture at 4°C to obtain vitamin K 2 Crystal, and recrystallize once;

[0101] ④The vitamin K will be obtained 2 The crystals were re-dissolved in HPLC loading solution (methanol:dichloromethane=4:1), and then HPLC method (mobile phase: methanol:dichloromethane=4:1, flow rate 1mL/min, detection wavelength 248nm) was used to detect vitamin K 2 The concentration and content of crystals, the detection diagram is as Figure 4 As shown, the test found that its purity reached 98.2%.

[0102] Preferably, the above-mentioned concentration methods are all concentrated under reduced pressure, the temperature is 40-99°C, and the vacuum degree is less than -0.08MPa.

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PUM

Property	Measurement	Unit
Molecular weight	400.0 ~ 1000.0
tensile		MPa
Particle size		Pa
strength	10

Description & Claims & Application Information

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Classification and recommendation of technical efficacy words

High purity
Improve biological activity

Method for separating and purifying vitamin K2 in bacillus subtilis natto

A technology for separation and purification of Bacillus natto is applied in the field of bioengineering to achieve the effects of high biological activity, simple process operation and convenient industrial application.

AI-Extracted Technical Summary

Problems solved by technology

Abstract

Image

Examples

Example Embodiment

Example Embodiment

Example Embodiment

PUM

Description & Claims & Application Information

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