CN103898175A - Bacillus subtilis natto and method for purifying vitamin menadione-7 by using bacterial strain - Google Patents
Bacillus subtilis natto and method for purifying vitamin menadione-7 by using bacterial strain Download PDFInfo
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Abstract
The invention provides bacillus subtilis natto ST188 with CGMCC No. 8400 and a high-yield method for purifying vitamin menadione-7 by using the bacterial strain. The high-yield method comprises the following steps: (1) carrying out spray drying on natto fermentation liquor, and extracting or leaching bacillus natto fermentation liquor spray drying powder by using a solvent so as to obtain a leaching solution; (2) concentrating the leaching solution obtained in the step (1) so as to obtain extract; (3) carrying out column chromatography on the extract obtained in the step (2), carrying out gradient or isocratic elution, and concentrating collected liquid, thus obtaining a menadione-7 crude product; and (4) crystallizing and purifying the menadione-7 crude product obtained in the step (3), thus obtaining the pure vitamin menadione-7 product.
Description
Technical field
The present invention relates to a kind of Bacillus subtilis natto (Bacillus subtilis natto) ST188, CGMCC No.8400, and by the method for this bacterial strain purification VITAMIN menaquinone-7.
Background technology
Because vitamin K has the effect that promotes blood coagulation, vitamin K is otherwise known as.Since nearly half a century, people only limit to its impact on blood coagulation system to the understanding of vitamin K, can promote the union of fracture of rabbit until nineteen sixty Bouckaert finds vitamin K.1975, after Pettifor etc. propose the hypothesis of vitamin K participation human body bone metabolism first, people just had deep understanding to vitamin K in the metabolic effect of bone.
Vitamin K family comprises several fat-soluble 2-MNQ derivatives.Natural vitamin K comprises vitamin K1 (being phylloquinone) and multiprenylmenaquinone (being aphthoquinone), and it is take No. 3 locational lipotropy side chains in 2-MNQ ring texture as feature.The side chain of phylloquinone is the aliphatic unsaturated hydrocarbon of determining length, and the side chain of vitamin k4 is the aliphatic unsaturated hydrocarbon of uncertain length.(vitamin K1 is that phylloquinone is a kind of compound, and multiprenylmenaquinone is a series of compounds)
Multiprenylmenaquinone does not singly have the blood coagulation of promotion, improves arteriosclerotic effect, has author studying it to liver cancer and leukemic effect abroad yet.China has entered aging society now, and sufferers of osteoporosis face sickness rate is higher.Great mass of data research shows, multiprenylmenaquinone lacks can cause the elderly's hip fracture and Decrease of Bone Mineral Density.Multiprenylmenaquinone lacks, and serum not carboxylation osteocalcin level reduces, and serum carboxylation osteocalcin level also may reduce, thereby may cause the danger of the elderly's Decrease of Bone Mineral Density generation hip fracture.
Multiprenylmenaquinone is as a kind of nutritive substance, and can be used as bone metabolism and adjust material, covers the effect of whole body; Side effect is less, is fat-soluble, can one after each meal, no matter all suitable uses of what age.(Zou Zhiqiang, the progress of multiprenylmenaquinone, Chinese osteoporosis magazine, 2005,11(3): 389-392)
Nineteen ninety-five, Japan started multiprenylmenaquinone preparation to sell as the medicine of the osteoporosis of improving the minimizing of bone amount, pain.(Wang Liming, multiprenylmenaquinone and osteoporosis, 2007,26(4): 293-295)
Multiprenylmenaquinone (2-methyl-3-alltrans-polypenthylene-1,4-naphthoquinone) or aphthoquinone are gang's isopentene group derivatives.The quantity of the isoprene residue being made up of 5 carbon atoms can be used for distinguishing vitamin k4 based compound.Its naming rule is the quantity based on prenyl, and for example " vitamin k4 " or " MK " add the quantity of group afterwards.Although be found to reach at most 15 prenyls on vitamin k4 side chain, the vitamin k4 that only contains 2 to 13 prenyls occurring in human and animal's tissue.The vitamin k4 of demethylation, on No. 2 carbon atom sites, unconjugated vitamin k4 is also found.
The general title of multiprenylmenaquinone or conventional vitamin k4 by name, its basal component is MK-4 (MK-4) and menaquinone-7 (MK-7).Known MK-7 is the one in the strongest active multiprenylmenaquinone.
Phylloquinone and 2 kinds of common vitamin k4 structure iron are as follows.
Vitamin K1
Molecular weight: 450
MK-4
Menaquinone-7
Molecular weight: 649
But at present, also there is no a kind of method of the multiprenylmenaquinone of can purifying simply, efficiently, the method for the MK-7 of the multiprenylmenaquinone of especially purifying.
Summary of the invention
The object of this invention is to provide a kind of Bacillus subtilis natto (Bacillus subtilis natto) ST188, CGMCC No.8400 and a kind of for this bacterial strain purification Vitamin A naphthoquinones-7(MK-7) method.The method is simply efficient, and can obtain purity and yield that vitamin(e) M K-7 is higher.
Bacillus subtilis natto provided by the present invention (Bacillus subtilis natto) ST188, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: on October 31st, 2013, preserving number is: CGMCC No.8400.
The method of purification VITAMIN menaquinone-7 provided by the invention comprises the following steps:
1) fermenting bacillus natto liquid spraying is dry, with solvent extraction or soak molten fermenting bacillus natto liquid spray powder, obtain and soak solution, described Bacillus natto is Bacillus subtilis natto (Bacillus subtilis natto) ST188, preserving number is: CGMCC No.8400;
2) what step 1) is obtained soaks the concentrated medicinal extract that obtains of solution;
3) by step 2) medicinal extract that obtains is by column chromatography, and carry out gradient or isocratic elution, and will collect liquid and concentrate, obtain menaquinone-7 crude product;
4) menaquinone-7 crude product crystallization and purification step 3) being obtained, obtains described VITAMIN menaquinone-7 sterling.
U.S. soybean outlet board of management (USSEC) definition natto is fermented soybean goods.The daily food of Asia Countries is fermented soybean goods, comprises soy sauce, miso, fermented bean curd, natto, day shellfish, fermented soya bean, mould bean dregs, sauce solid food etc.The difference of fermented soybean goods is, soybean is combined with cereal sometimes as enzymolysis matrix, and the microorganism that participates in fermenting process is also different.And natto is to ferment through genus bacillus or Bacillus subtilis natto from Traditional Japanese Food with soybean completely.Fermenting bacillus natto liquid described in the application is the fermented liquid obtaining through Bacillus subtilis natto (Bacillus subtilis natto) fermentation with analysis for soybean powder.Described Bacillus subtilis natto can separate and obtain from commercial japanese traditional food natto.
According to an embodiment of the invention, described fermenting bacillus natto liquid can be for to be seeded in described Bacillus subtilis natto ST188 in the substratum that contains 10-50g/1000mL analysis for soybean powder and 5-45g/1000mL sucrose, at 30-40 ℃, obtain fermenting bacillus natto liquid through fermentation in 20-48 hour, the inoculum density of wherein said Bacillus subtilis natto ST188 is 5-20%.
According to an embodiment of the invention; before the described fermenting bacillus natto liquid of spraying dry (being called for short in the present invention: spray is dry); by add soybean oil in described fermenting bacillus natto liquid; can effectively protect vitamin(e) M K-7 in described fermenting bacillus natto liquid not destroyed by the high temperature of the dry process of spray; therefore, contribute to improve the yield of MK-7.
According to an embodiment of the invention, as long as can be conducive to improve the yield of vitamin(e) M K-7 to the soybean oil of adding in described fermenting bacillus natto liquid.Under preferable case, to the soybean oil of adding 0.1-1 % by weight in fermenting bacillus natto liquid, be more conducive to improve the yield of vitamin(e) M K-7.
According to an embodiment of the invention, in step 1), spray solid carbon dioxide is divided and is controlled at below 3%, thereby be conducive to the extraction of MK-7.Meanwhile, adopt spray powder to extract, directly with organic solvent extraction, can reduce the consumption of 80% organic solvent than fermented liquid, simultaneously because less moisture and be conducive to concentrating under reduced pressure.
According to an implementation method of the present invention, in step 1), comprise by solvent extraction or the condition of soaking molten fermenting bacillus natto liquid spray powder: by fermenting bacillus natto liquid spray powder with solvent by 1: the mass ratio of 3-8 mixes, more preferably with 1: the mass ratio of 3-6 mixes, at the temperature of 15-40 ℃, stir 20-40min with 20-60rpm stir speed (S.S.), solid-liquid separation, gained clear liquid is and soaks solution.Wherein, solid-liquid separation can adopt any one solid-liquid separation method well known by persons skilled in the art, for example, staticly settle, the mode of any one or multiple combination in the method such as filtration, centrifugation.In addition, can repeatedly extract or soak molten (repeatedly extract or soak molten can improve as far as possible yield) for gained solid.
According to an embodiment of the invention, in step 1), described solvent is selected from one or more in methyl alcohol, sherwood oil, acetone, methylene dichloride, trichloromethane, Virahol, ethyl acetate, butylacetate and 75-100 volume % ethanol.More preferably in situation, described solvent is selected from one or more in sherwood oil, acetone, methylene dichloride, Virahol, ethyl acetate and 75-100 volume % ethanol.
According to an embodiment of the invention, in step 2) and step 3) in, described concentrated can be concentrating under reduced pressure or evaporation concentration.The preferred concentrating under reduced pressure of the present invention.The condition of described concentrating under reduced pressure comprises: temperature is 40-95 ℃, and vacuum tightness is less than 3000Pa.
According to one embodiment of the invention, by step 2) medicinal extract that obtains comprises with wet method upper prop decolouring: by silica gel and sherwood oil by 1: 1.5-2 mass ratio mixes rear dress post, then by step 2) medicinal extract of acquisition and sherwood oil be by 1: 0.5-1 mass ratio mixes rear upper prop.
According to one embodiment of the invention, in step 3), the method for described column chromatography comprises: first by step 2) medicinal extract that obtains is with wet method upper prop decolouring, and with the first eluent wash-out; Then the product after decolouring is carried out to purifying in Wet-dry method mixing upper prop mode, first with wet method dress post, again the product after decolouring is filled in to the upper strata of wet method dress post in the mode of dry column-packing, and with the second eluent wash-out, wherein, in the post of Wet-dry method mixing upper prop, the volume ratio of wet method dress post and dry column-packing is 1: 0.2-2.
According to an embodiment of the invention, in step 3), described the first eluent is non-polar solvent, is preferably one or more in sherwood oil, normal hexane and hexanaphthene.The consumption of described the first eluent is 2-3 times of chromatography column volume.
According to an embodiment of the invention, the product after decolouring is carried out to purifying in Wet-dry method mixing upper prop mode and comprises: first by silica gel and sherwood oil with 1: the mass ratio of 1.5-2 mixes rear dress post; Again by product and silica gel after decolouring and sherwood oil with 1: after the mass ratio of 3-4: 4-5 mixes, dries in the shade, load in the post formerly loading with wet method.
According to an embodiment of the invention, described in the post of Wet-dry method mixing upper prop, the volume ratio of described wet method dress post and described dry column-packing is 1: 0.2-2(is 1-10: 2) time, can be conducive to improve the yield of vitamin(e) M K-7, under preferable case, the volume ratio of described wet method dress post and described dry column-packing is 1: 0.5-1(is 1-2: 1), be more conducive to improve the yield of vitamin(e) M K-7.The present invention is by adopting the column chromatography of Wet-dry method mixings upper prop, can more effective absorption impurity, and separating effect is better, and the volume ratio that suitable raising wet method fills post and dry column-packing can improve product purity; In addition, when MK-7 purity and yield are higher, amount of filler is also less.
According to an embodiment of the invention, described column chromatography can be any one or its combination in silica gel column chromatography, macroporous resin column chromatography, Sephadex LH20 gel filtration chromatography and polyamide column chromatography.
According to an embodiment of the invention, the silica gel using in described wet method dress post and described dry column-packing can be the silica gel that can be used in chromatography column well known by persons skilled in the art.Under preferable case, described silica gel is between 100-400 order, aperture (for example, specific surface area>=550m
2/ g, pore volume≤0.7mL/g), (for example, specific surface area is for being greater than 300m for mesopore
2/ g is to being less than 550m
2/ g, pore volume is for being greater than 0.7mL/g to being less than 0.9mL/g) or gross porosity (for example, specific surface area is 200m
2/ g is to being less than 300m
2/ g, pore volume is>=0.85mL/g) silica gel.
According to an embodiment of the invention, in step 3), the method for column chromatography for separation can adopt method known to those skilled in the art, and the present invention does not have special requirement.For example column chromatography for separation can separate under low pressure (being less than 0.5MPa), middle pressure (0.5-5MPa) or high pressure (being greater than 5MPa to 40MPa).
According to an embodiment of the invention, in step 3), described the second eluent is selected from one or more in sherwood oil, normal hexane, hexanaphthene, methylene dichloride, trichloromethane, ethyl acetate, Virahol, propyl carbinol, acetone, methyl alcohol, ethanol, acetonitrile and water.More preferably in situation, described the second eluent is selected from the mixture of sherwood oil, methylene dichloride that volume ratio is 1: 9 and sherwood oil or trichloromethane that volume ratio is 1: 9 and the mixture of sherwood oil.The consumption of described the second eluent is: the consumption of described sherwood oil is 10-15 times of chromatography column volume, described volume ratio be the consumption of the mixture of the methylene dichloride of 1: 9 and sherwood oil be chromatography column volume 4-5 doubly, the consumption of the trichloromethane that described volume ratio is 1: 9 and the mixture of sherwood oil be chromatography column volume 4-5 doubly.
According to an embodiment of the invention, in step 4), described menaquinone-7 crude product crystallization and purification is comprised: described menaquinone-7 crude product solvent is carried out to crystallization and recrystallization.Under preferable case, described solvent can be selected from one or more in sherwood oil, normal hexane, hexanaphthene, methylene dichloride, trichloromethane, ethyl acetate, Virahol, propyl carbinol, acetone, methyl alcohol, ethanol, acetonitrile and water.
According to an embodiment of the invention, in step 4), can adopt evaporative crystallization or crystallisation by cooling, the temperature of crystallization can be-10 ℃ to 95 ℃.Preferably, after crystallization filters, wash crystallization, and carry out recrystallization, to improve the purity of menaquinone-7 of final acquisition.
The method according to this invention, can obtain the vitamin(e) M K-7 that purity and yield are higher.Under preferable case, by add the soybean oil of 0.1-1 % by weight in fermenting bacillus natto liquid, effectively protect vitamin(e) M K-7 in described fermenting bacillus natto liquid not destroyed by the high temperature of the dry process of spray, thereby improved the amount of vitamin(e) M K-7 in fermenting bacillus natto liquid, improved the yield of vitamin(e) M K-7; Meanwhile, by the mode that wet method upper prop decolours and Wet-dry method mixes upper column purification, simplify purification step, improved efficiency, and improved the purity of vitamin(e) M K-7.
Accompanying drawing explanation
Accompanying drawing is to be used to provide a further understanding of the present invention, and forms a part for specification sheets, is used from explanation the present invention, but is not construed as limiting the invention with embodiment one below.In the accompanying drawings:
Fig. 1 is according to the liquid chromatogram of the product of the method acquisition of the embodiment of the present invention 1.
Embodiment
Below will by embodiment, the invention will be further described, these descriptions are not that content of the present invention is further limited.One skilled in the art will understand that the replacement that is equal to that the technology of the present invention feature is done, or improve accordingly, within still belonging to protection scope of the present invention.
In following examples, agents useful for same is all from being purchased.
The purity of vitamin(e) M K-7 is measured by waters company of U.S. high performance liquid chromatograph.
The yield of vitamin(e) M K-7 calculates by following formula:
Yield=MK-7 sterling purity × MK-7 sterling weight × 100%/(MK-7 concentration × fermentating liquid volume in fermented liquid) of MK-7
Embodiment 1
Prepare fermenting bacillus natto liquid:
Natto (purchased from Japanese biotechnology company) is dissolved with sterilized water, (making method is referring to Wei Hua after dilution, to spread upon agarose-fibrin plate, Zhao Xiangying, Liu Jianjun, the determination of activity [J] of Nattokinase. Shandong Light Ind College journal, 2007, 21 (1) 60-63.) on, after 37 ℃ of cultivation 18h, produce transparent circle, select the bacterium colony that transparent circle is larger to utilize ultraviolet ray (15s) many for mutagenesis, obtain Bacillus subtilis natto (Bacillus subtilis natto) ST188(culture presevation at China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: on October 31st, 2013, preserving number is: CGMCC No.8400).
500mL gained Bacillus subtilis natto ST188 is seeded in the 10000mL substratum that contains 185g analysis for soybean powder and 185g sucrose, at 37 ℃, obtains fermenting bacillus natto liquid through fermentation in 24 hours.
(1) then dry 90 ℃ of sprays to the soybean oil of adding 5g in 1000g fermenting bacillus natto liquid, soak molten 40g fermenting bacillus natto liquid spray powder with the methyl alcohol of 240g, described soaking is dissolved in 20 ℃ and stirs under the conditions of 30min and carry out with 40rpm stir speed (S.S.), after solid-liquid separation, obtains and soaks solution; Adopt the identical molten condition of soaking to soak for the second time molten the solid of acquisition;
(2) what step (1) is obtained soaks after solution filter at 55 ℃, and concentrating under reduced pressure under 2600Pa, obtains medicinal extract;
(3) first 8g silica gel and sherwood oil are mixed to rear dress post by 1: 2 mass ratio, then 8g medicinal extract and sherwood oil that step (2) is obtained fill post with the quality of 1: 1 than wet method, and carry out wash-out (at 0.1MPa) 1 time as the first eluent with 40g sherwood oil;
(4) first silica gel and sherwood oil are mixed to rear wet method dress post with the mass ratio of 1: 2, in the post that after again step (3) being mixed, dried in the shade with the mass ratio of 1: 3: 4 with silica gel and sherwood oil with the decolouring product that the first eluent wash-out obtains, filling is formerly loaded with wet method, the volume ratio of wet method dress post and dry column-packing is 1: 1, and carry out wash-out (under 1MPa) 1 time as the second eluent with the mixture that 80g volume ratio is 1: 9 trichloromethane and sherwood oil, can single step purification just obtaining purity by this operation is 40% vitamin(e) M K-7;
(5) mixture step (4) being obtained dissolves with the propyl carbinol of its 30 times of quality, then evaporative crystallization, the crystal obtaining is carried out to recrystallization, the color atlas that the final product obtaining obtains through high performance liquid chromatograph as shown in Figure 1, contains a large amount of vitamin(e) M K-7 in the final product obtaining as seen from Figure 1.Determine that through stratographic analysis the purity of vitamin(e) M K-7 in product is 95%, the yield of gained vitamin(e) M K-7 is 93%.
Embodiment 2
Adopt the method for the embodiment 1 vitamin(e) M K-7 in fermenting bacillus natto liquid that purifies, different, in step (4), the volume ratio of wet method dress post and dry column-packing is 2: 1.
The final product obtaining determines that through stratographic analysis the purity of vitamin(e) M K-7 in product is 99%, and the yield of gained vitamin(e) M K-7 is 93%.
Embodiment 3
Adopt the method for the embodiment 1 vitamin(e) M K-7 in fermenting bacillus natto liquid that purifies, different, in step (4), the volume ratio of wet method dress post and dry column-packing is 1: 2.
The final product obtaining determines that through stratographic analysis the purity of vitamin(e) M K-7 in product is 93%, and the yield of gained vitamin(e) M K-7 is 92%.
Embodiment 4
Adopt the method for the embodiment 1 vitamin(e) M K-7 in fermenting bacillus natto liquid that purifies, different is, in step (4), first silica gel and sherwood oil are mixed to the part of rear wet method filling post with the mass ratio of 1: 1.5, then load in the post formerly loading with wet method after mixing, dry in the shade with the mass ratio of 1: 4: 5 with silica gel and sherwood oil after the mixture decolouring that step (3) is obtained.
The final product obtaining determines that through stratographic analysis the purity of vitamin(e) M K-7 in product is 94%, and the yield of gained vitamin(e) M K-7 is 93%.
Embodiment 5
Adopt the method for the embodiment 1 vitamin(e) M K-7 in fermenting bacillus natto liquid that purifies, different, in step (1), to the soybean oil of adding 10g in 1000g fermenting bacillus natto liquid.
The final product obtaining determines that through stratographic analysis the purity of vitamin(e) M K-7 in product is 96%, and the yield of gained vitamin(e) M K-7 is 95%.
Embodiment 6
Adopt the method for the embodiment 1 vitamin(e) M K-7 in fermenting bacillus natto liquid that purifies, different, in step (1), to the soybean oil of adding 1g in 1000g fermenting bacillus natto liquid.
The final product obtaining determines that through stratographic analysis the purity of vitamin(e) M K-7 in product is 95%, and the yield of gained vitamin(e) M K-7 is 90%.
Embodiment 7
Adopt the method for the embodiment 1 vitamin(e) M K-7 in fermenting bacillus natto liquid that purifies, different, in step (1), in fermenting bacillus natto liquid, do not add soybean oil.
The final product obtaining determines that through stratographic analysis the purity of vitamin(e) M K-7 in product is 93%, and the yield of gained vitamin(e) M K-7 is 80%.
Embodiment 8
Adopt the method for the embodiment 1 vitamin(e) M K-7 in fermenting bacillus natto liquid that purifies, different is, in step (4), chromatography column directly adopt dry column-packing be step (3) the product that obtains and silica gel and sherwood oil dry in the shade after mixing by 1: 4: 4 mass ratio, refill post, with the sherwood oil drip washing of 10 times of column volumes, the MK-7 purity after single step purification is 30%.In order to obtain higher product purity, carry out 3 times and repeat.
The final product obtaining determines that through stratographic analysis the purity of vitamin(e) M K-7 in product is 90%, and the yield of gained vitamin(e) M K-7 is 90%.
Can find out by above embodiment, adopt the method for purification vitamin(e) M K-7 provided by the invention, can obtain purity higher, the vitamin(e) M K-7 that yield is higher.
The application includes but not limited to above embodiment, and every any being equal to of carrying out under the principle of the application's spirit substitutes or local improvement, all will be considered as within the application's protection domain.
Claims (12)
1. a method for the VITAMIN of purifying menaquinone-7, the method comprises the following steps:
1) fermenting bacillus natto liquid spraying is dry, with solvent extraction or soak molten fermenting bacillus natto liquid spray powder, obtain and soak solution, described Bacillus natto is Bacillus subtilis natto (Bacillus subtilis natto) ST188, preserving number is: CGMCC No.8400;
2) what step 1) is obtained soaks the concentrated medicinal extract that obtains of solution;
3) by step 2) medicinal extract that obtains is by column chromatography, and carry out gradient or isocratic elution, and will collect liquid and concentrate, obtain menaquinone-7 crude product;
4) menaquinone-7 crude product crystallization and purification step 3) being obtained, obtains described VITAMIN menaquinone-7 sterling.
2. method according to claim 1, wherein, in step 1), fermenting bacillus natto liquid spraying is dry before, to the soybean oil of adding 0.1-1 % by weight in described fermenting bacillus natto liquid.
3. method according to claim 1, wherein, in step 3), the method for described column chromatography comprises: first by step 2) the described medicinal extract that obtains is with the decolouring of wet method upper prop, and with the first eluent wash-out; Then the product after decolouring is carried out to purifying in Wet-dry method mixing upper prop mode, first with wet method dress post, again the product after decolouring is filled in to the upper strata of wet method dress post in the mode of dry column-packing, and with the second eluent wash-out, wherein, in the post of Wet-dry method mixing upper prop, the volume ratio of described wet method dress post and described dry column-packing is 1: 0.2-2.
4. method according to claim 1, wherein, in step 1), described extraction or soak molten condition and comprise: by fermenting bacillus natto liquid spray powder with solvent by 1: the mass ratio of 3-8 mixes, at the temperature of 15-40 ℃, stir 20-40min with 20-60rpm stir speed (S.S.), solid-liquid separation, gained clear liquid is and soaks solution.
5. according to the method described in any one in claim 1-4, wherein, in step 1), described solvent is selected from one or more in methyl alcohol, sherwood oil, acetone, methylene dichloride, trichloromethane, Virahol, ethyl acetate, butylacetate and 75-100 volume % ethanol.
6. according to the method described in any one in claim 1-4, wherein, in step 2) and step 3) in, described simmer down to concentrating under reduced pressure, the condition of described concentrating under reduced pressure comprises: temperature is 40-95 ℃, and vacuum tightness is less than 3000Pa.
7. method according to claim 3, wherein, by step 2) the described medicinal extract that obtains comprises with wet method upper prop decolouring: by silica gel and sherwood oil by 1: 1.5-2 mass ratio mixes rear dress post, then by step 2) medicinal extract of acquisition and sherwood oil be by 1: 0.5-1 mass ratio mixes rear upper prop.
8. according to the method described in claim 3 or 7, wherein, described the first eluent is non-polar solvent, is preferably one or more in sherwood oil, normal hexane and hexanaphthene; The consumption of described the first eluent is 2-3 times of chromatography column volume.
9. method according to claim 3, wherein, the product after decolouring is carried out to purifying in Wet-dry method mixing upper prop mode and comprises: first by silica gel and sherwood oil with 1: the mass ratio of 1.5-2 mixes rear dress post; Again by the product after described decolouring and silica gel and sherwood oil with 1: after the mass ratio of 3-4:4-5 mixes, dries in the shade, filling is formerly in the post with wet method filling.
10. according to the method described in claim 3 or 9, wherein, described the second eluent is selected from the mixture of sherwood oil, methylene dichloride that volume ratio is 1: 9 and sherwood oil or trichloromethane that volume ratio is 1: 9 and the mixture of sherwood oil, wherein, the consumption of described sherwood oil is 10-15 times of chromatography column volume, described volume ratio be the consumption of the mixture of the methylene dichloride of 1: 9 and sherwood oil be chromatography column volume 4-5 doubly, the consumption of the trichloromethane that described volume ratio is 1: 9 and the mixture of sherwood oil be chromatography column volume 4-5 doubly.
11. according to the method described in any one in claim 1-4, wherein said fermenting bacillus natto liquid is that described Bacillus subtilis natto ST188 is seeded in the substratum that contains 10-50g/1000mL analysis for soybean powder and 5-45g/1000mL sucrose, at 30-40 ℃, obtain fermenting bacillus natto liquid through fermentation in 20-48 hour, the inoculum density of wherein said Bacillus subtilis natto ST188 is 5-20%.
12. 1 kinds of Bacillus subtilis nattos (Bacillus subtilis natto) ST188, preserving number is: CGMCC No.8400.
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| CN104694588A (en) * | 2015-02-12 | 2015-06-10 | 中国科学院合肥物质科学研究院 | Method for preparing vitamin MK-7 based on fermentation of bacillus natto |
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