CN103012514B - Membrane separation and extraction method for micronomicin sulfate - Google Patents
Membrane separation and extraction method for micronomicin sulfate Download PDFInfo
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- CN103012514B CN103012514B CN201210565158.4A CN201210565158A CN103012514B CN 103012514 B CN103012514 B CN 103012514B CN 201210565158 A CN201210565158 A CN 201210565158A CN 103012514 B CN103012514 B CN 103012514B
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Abstract
The invention relates to a membrane separation and extraction method for micronomicin sulfate. The membrane separation and extraction method adopts the technical scheme that the method comprises the steps that a micronomicin sulfate analytic solution is decolorized by an ultrafiltration membrane system, and concentrated by a nanofiltration membrane system, wherein the ultrafiltration membrane system comprises six ultrafiltration membranes, every three ultrafiltration membranes are in series connection to form a group; the two groups are in parallel connection; and the nanofiltration membrane system is formed by connecting three nanofiltration membranes in series. The membrane separation and extraction method has the advantages that a technique is simple and easy to operate; the loss of effective ingredients is less; the product quality is stable; and the energy and material consumption is reduced.
Description
Technical field
The present invention relates to a kind of membrane sepn extracting method, be specifically related to the membrane sepn extracting method of Micronomicin Sulfate.
Background technology
Micronomicin Sulfate is white or the porous solid of off-white color or powder; Odorless, have draw moist.It is easily molten in water, almost insoluble in methyl alcohol, ethanol, acetone, ethyl acetate or trichloromethane.For aminoglycoside antibiotics, its impede protein matter is synthesized, and shows anti-microbial activity to the sensitive organism of gram-negative bacteria and gram positive organism.For the various infection caused by gram negative bacilli, as microbemia, respiratory system, bile duct, urinary tract infections etc.Local eye drop treatment responsive microbial blepharitis, dacryocystitis, conjunctivitis, keratitis etc.Existing technique Micronomicin Sulfate is by deep red micromonospora 201-9-8-53-84-37
#(Micromonospora purpura 201-9-8-53-84-37
#) generate.Deep red micromonospora 201-9-8-53-84-37
#(Micromonospora purpura 201-9-8-53-84-37
#), be actinomyces, Micromonosporaceae, in China typical culture collection center preservation, preserving number has been: CCTCC NO:M 2012471; Depositary institution address: Wuhan, China Wuhan University, preservation date: on November 21st, 2012.
Existing Micronomicin Sulfate extractive technique, by Micronomicin Sulfate desorbed solution, through 711 type strongly basic anion exchange resin beds, again through film under vacuum upgrading tower after dynamic adsorption pigment, use steam heating evaporating solvent, obtain the concentrated solution of original volume 1/40, this concentrated solution finally with the residual pigment of powdered active carbon absorption, thus reaches processing requirement.This technical process is complicated, the cycle is long, energy consumption is high, and effective constituent runs off comparatively serious, and product yield is low, and the organic solvent used, not only increase extraction cost, and easily serious pollution is caused to environment.As selected traditional culture medium prescription and traditional technology in " producing 15 tons of Micronomicin Sulfate plant design per year ", Micronomicin Sulfate desorbed solution through pre-treatment, ion exchange resin absorption, steam concentrates, turn the technique such as salt, decolouring obtains product, annual about loss 20 tons of resins, cause a large amount of wastes.For optimization technological process, improve the quality of products and stability, cost-saving, seek new producing and manufacturing technique extremely urgent.
Background technology of the present invention is as follows:
Certain traditional technology first is as follows:
The preparation of A, micronomicin seed:
A1, female bottle slant culture: female bottle classical media formulation; 6-7 days is cultivated in 35 DEG C;
A2, sub-bottle slant culture: sub-bottle conventional medium; Cultivate 10-15 days in 37 DEG C, make spore, vacuum-drying saves backup;
A3, spore suspension: the spore of sub-bottle slant culture is made suspension;
The preparation of B, Micronomicin Sulfate fermented liquid:
B1, first class seed pot are cultivated: first order seed conventional medium; In 35-37 DEG C, ventilation 1:2V/V.m, stirring velocity 320-350rpm, PH nature, incubation time 40-45h;
B2, secondary seed tank are cultivated: secondary seed conventional medium; In 33-35 DEG C, ventilation 1:1-1.5V/V.m, stirring velocity 250-300rpm, PH nature, incubation time 13-15h;
B3, by cultivate seed with 10% inoculum size access fermentation flask, within 5-7 days, obtain fermented liquid, wherein power of agitator 2-4KW/m3, rotating speed 150-180r/min, air flow quantity 50-70m3/m3.h, tank pressure 0.03-0.05Mpa in 32-34 DEG C of cultivation;
The preparation of C, Micronomicin Sulfate desorbed solution:
C1, acidifying removal of impurities: 98% sulfuric acid acidation with fermented liquid quality 1 times amount filters to obtain acidizing fluid, removing miscellaneous bacteria and other impurity;
C2, neutralization: it is 7 that acidizing fluid is neutralized to PH by the NaOH adding 3mol/L;
C3,732 resin absorption;
Through 711 type strongly basic anion exchange resin beds, again through film under vacuum upgrading tower after dynamic adsorption pigment, use steam heating evaporating solvent, obtain the concentrated solution of original volume 1/40, this concentrated solution finally with the residual pigment of powdered active carbon absorption, thus reaches processing requirement.
Certain traditional technology second, as " producing 15 tons of Micronomicin Sulfate plant design per year ", has many similarities with certain traditional technology first.
For making up the defect of above-mentioned prior art, the problem needing most solution is at present: optimization technological process, reduces effective constituent loss, carries high product yield, stabilized product quality, reduces the energy and supplies consumption, and decreasing pollution thing discharges, and reduces production cost.
Summary of the invention
The object of this invention is to provide a kind of membrane sepn extracting method of Micronomicin Sulfate.Main step comprises: the acquisition of Micronomicin Sulfate desorbed solution, the ultra-filtration membrane decoloration process of Micronomicin Sulfate desorbed solution, the nanofiltration membrane concentration technology of Micronomicin Sulfate desorbed solution.
Object one of the present invention is the 711 type strongly basic anion exchange resin bed decoloration process substituting traditional technology with ultra-filtration membrane decoloration process, the steam concentration technology of traditional technology is substituted with nanofiltration membrane concentration technology, reduce the waste of resin, and a large amount of losses of the energy.
Object two of the present invention is by improving culture medium prescription, and be combined with appropriate ultra-filtration membrane decoloration process, nanofiltration membrane concentration technology, the molecular weight cut-off of such as ultra-filtration membrane, the aperture of nanofiltration membrane, working pressure, optimization through flow etc., not only can reduce the waste of resin, and a large amount of losses of energy consumption, the Micronomicin Sulfate that active constituent content is higher can also be prepared, tiring and purity of product can be improved.Under the prerequisite obtaining more efficient valency product, suitable culture medium prescription, effectively can reduce the content of impurity, and then reduces the link of ultra-filtration membrane decoloration process, and such as ultra-filtration membrane unit piece can be kept to six unit by ten unit.
Lay special stress on of the present invention: technical characteristics of the present invention is ultra-filtration membrane decoloration process, nanofiltration membrane concentration technology.The acquisition not technical characteristics of the present invention of Micronomicin Sulfate desorbed solution, Micronomicin Sulfate desorbed solution can obtain according to the technical clarification of " producing 15 tons of Micronomicin Sulfate plant design per year ", also can cultivate according to other culture medium prescriptions and be separated acquisition.
Micronomicin Sulfate belongs to amino acid glycoside Broad spectrum antibiotics, and it can by a strain deep red micromonospora 201-9-8-53-84-37
#(Micromonospora purpura 201-9-8-53-84-37
#) produce.Deep red micromonospora 201-9-8-53-84-37
#(Micromonospora purpura 201-9-8-53-84-37
#), be actinomyces, Micromonosporaceae, in China typical culture collection center preservation, preserving number has been: CCTCC NO:M 2012471; Depositary institution address: Wuhan, China Wuhan University, preservation date: on November 21st, 2012.
The principle of work of membrane separation technique is, with selectivity through film for separating medium, certain impellent in addition in film both sides.With making feed side compositional selecting through film, thus reach the object being separated or purifying.According to the selective penetrated property of film and varying in size of membrane pore size, selectively by the material concentrating and separating of different-grain diameter, useful matter can be reclaimed.The aperture of film is generally micron order, according to the difference (or being called molecular weight cut-off) in its aperture, film can be divided into microfiltration membrane (MF), ultra-filtration membrane (UF), nanofiltration membrane (NF) and reverse osmosis membrane (RO) etc.Wherein, ultra-filtration membrane is that a kind of aperture specification is consistent, and nominal pore scope is the micropore filtering film of 0.001-0.02 micron.And nanofiltration membrane is the semi-permeable membranes allowing solvent molecule or some low molecular weight solutes or low price ion permeable.
Do not undergo phase transition in membrane separating process, separation factor is comparatively large, energy-conservation, efficient, non-secondary pollution, can operate continuously at normal temperatures, is specially adapted to heat-sensitive substance.And this process is without the need to adding other material from the external world, like this while avoiding effective constituent destroyed, also cost-saved, decreasing pollution thing discharges.
Technical scheme of the present invention is as follows:
The preparation of A, micronomicin seed:
Get deep red micromonospora 201-9-8-53-84-37
#(Micromonospora purpura 201-9-8-53-84-37
#), implant carrier bottle is cultivated;
A1, female bottle slant culture: substratum is made up of the supplementary material of following weight proportioning, Zulkovsky starch 8-12 part, sodium-chlor 0.3-0.8 part, dipotassium hydrogen phosphate 0.2-0.5 part, saltpetre 0.8-1.2 part, asparagine 0.01-0.03 part, magnesium sulfate 0.3-0.7 part, wheat bran 15-20 part, glycerine 0.3-0.8 part, agar 16-20 part; 6-7 days is cultivated in 35 DEG C;
A2, sub-bottle slant culture: substratum is made up of the supplementary material of following weight proportioning, Zulkovsky starch 10-15 part, sodium-chlor 0.3-0.8 part, dipotassium hydrogen phosphate 0.3-0.6 part, saltpetre 1.0-1.5 part, asparagine 0.02-0.04 part, magnesium sulfate 0.3-0.7 part, wheat bran 16-22 part, glycerine 0.4-1.0 part, agar 18-22 part; Cultivate 10-15 days in 37 DEG C, make spore, vacuum-drying saves backup;
A3, spore suspension: the spore of sub-bottle slant culture is made suspension;
The preparation of B, Micronomicin Sulfate fermented liquid:
B1, first class seed pot are cultivated: substratum is made up of the supplementary material by following weight proportioning, soybean cake powder 12-18 part, glucose 0.8-1.5 part, Semen Maydis powder 18-24 part, fish meal 1-3 part, urea 0.8-1.2 part, calcium carbonate 0.8-1.2 part, starch 36-45 part; In 35-37 DEG C, ventilation 1:2V/V.m, stirring velocity 320-350rpm, PH nature, incubation time 40-45h;
B2, secondary seed tank are cultivated: substratum is made up of the supplementary material by following weight proportioning, groundnut meal 15-20 part, glucose 1.0-1.8 part, Semen Maydis powder 20-26 part, urea 0.8-1.2 part, saltpetre 0.8-1.2 part, calcium carbonate 0.8-1.2 part, starch 36-45 part; In 33-35 DEG C, ventilation 1:1-1.5V/V.m, stirring velocity 250-300rpm, PH nature, incubation time 13-15h;
B3, by cultivate seed with 10% inoculum size access fermentation flask, within 5-7 days, obtain fermented liquid, wherein power of agitator 2-4KW/m3, rotating speed 150-180r/min, air flow quantity 50-70m3/m3.h, tank pressure 0.03-0.05Mpa in 32-34 DEG C of cultivation;
The preparation of C, Micronomicin Sulfate desorbed solution:
C1, acidifying removal of impurities: 98% sulfuric acid acidation with fermented liquid quality 1 times amount filters to obtain acidizing fluid, removing miscellaneous bacteria and other impurity;
C2, neutralization: it is 7 that acidizing fluid is neutralized to PH by the NaOH adding 3mol/L;
C3,732 resin absorption;
D, bleaching process: Micronomicin Sulfate desorbed solution decolours through ultrafiltration membrane system;
Ultrafiltration membrane system wherein, comprises first group of ultra filtration unit, second group of ultra filtration unit; First group of ultra filtration unit is made up of first group of first step ultra filtration unit, first group of second stage ultra filtration unit, first group of third stage ultra filtration unit; Second group of ultra filtration unit is made up of second group of first step ultra filtration unit, second group of second stage ultra filtration unit, second group of third stage ultra filtration unit; Wherein: first group of first step ultra filtration unit, first group of second stage ultra filtration unit are connected successively with first group of third stage ultra filtration unit, second group of first step ultra filtration unit, second group of second stage ultra filtration unit are connected successively with second group of third stage ultra filtration unit; First group of ultra filtration unit and second group of ultra filtration unit are connected in parallel;
Decolorization process: Micronomicin Sulfate desorbed solution is parallel through first group of ultra filtration unit and second group of ultra filtration unit decolouring, namely Micronomicin Sulfate desorbed solution is abreast successively through first group of first step ultra filtration unit, first group of second stage ultra filtration unit, first group of third stage ultra filtration unit, with second group of first step ultra filtration unit, second group of second stage ultra filtration unit, second group of third stage ultra filtration unit, the Micronomicin Sulfate desorbed solution after decolouring enters enrichment process;
Ultrafiltration membrane system working parameter is as follows: ultra filtration unit selects aperture to be the ultra-filtration membrane retaining 1000-5000 molecular weight, and it is 2-5T/h that working pressure controls at 0.3-0.8Mpa, through flow;
E, enrichment process: the Micronomicin Sulfate desorbed solution after decolouring concentrates through nanofiltration membrane system;
Nanofiltration membrane system wherein, comprises first step nano-filtration unit, second stage nano-filtration unit, third stage nano-filtration unit; Wherein first step nano-filtration unit, second stage nano-filtration unit, third stage nano-filtration unit connect successively;
Enrichment step: the Micronomicin Sulfate desorbed solution after decolouring concentrates through first step nano-filtration unit, second stage nano-filtration unit, third stage nano-filtration unit successively; Obtain Micronomicin Sulfate.
Nanofiltration membrane system working parameter is as follows: nano-filtration unit selects aperture to be the nanofiltration membrane of 1nm-50nm; It is 2-5T/h that working pressure controls at 1.0-3.0Mpa, through flow.
Tool of the present invention has the following advantages:
1, simplify the technical process of traditional Micronomicin Sulfate separation and Extraction, shorten the production cycle;
2, avoid the loss of product effective ingredient in the course of processing or rotten, improve product yield;
3, stabilized product quality;
4, membrane sepn does not undergo phase transition, and carries out at normal temperatures, does not add other materials, reduces the energy and supplies consumption, and decreasing pollution thing discharges, and reduces production cost.
Accompanying drawing explanation
Fig. 1 is the structural representation of ultrafiltration membrane system of the present invention and nanofiltration membrane system.
Reference numeral: first group of first step ultra filtration unit, 1, first group of second stage ultra filtration unit, 2, first group of third stage ultra filtration unit, 3, second group of first step ultra filtration unit, 4, second group of second stage ultra filtration unit 5, second group of third stage ultra filtration unit 6, first step nano-filtration unit 7, second stage nano-filtration unit 8, third stage nano-filtration unit 9.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1,
The membrane sepn extracting method of Micronomicin Sulfate (Micronomicin Sulfate), obtains especially by following steps:
The preparation of A, micronomicin seed:
Get deep red micromonospora 201-9-8-53-84-37
#(Micromonospora purpura 201-9-8-53-84-37
#), implant carrier bottle is cultivated;
A1, female bottle slant culture: substratum is made up of the supplementary material of following weight proportioning, Zulkovsky starch 10 parts, 0.5 part, sodium-chlor, dipotassium hydrogen phosphate 0.3 part, 1.0 parts, saltpetre, asparagine 0.02 part, 0.5 part, magnesium sulfate, 18 parts, wheat bran, glycerine 0.6 part, 18 parts, agar; Cultivate 7 days in 35 DEG C;
A2, sub-bottle slant culture: substratum is made up of the supplementary material of following weight proportioning, Zulkovsky starch 13 parts, 0.5 part, sodium-chlor, dipotassium hydrogen phosphate 0.4 part, 1.3 parts, saltpetre, asparagine 0.03 part, 0.5 part, magnesium sulfate, 18 parts, wheat bran, glycerine 0.8 part, 20 parts, agar; Cultivate 12 days in 37 DEG C, make spore, vacuum-drying saves backup;
A3, spore suspension: the spore of sub-bottle slant culture is made suspension;
The preparation of B, Micronomicin Sulfate fermented liquid:
B1, first class seed pot are cultivated: substratum is made up of the supplementary material by following weight proportioning, soybean cake powder 15 parts, glucose 1.2 parts, Semen Maydis powder 22 parts, fish meal 2 parts, 1.0 parts, urea, 1.0 parts, calcium carbonate, starch 40 parts; In 37 DEG C, ventilation 1:2V/V.m, stirring velocity 350rpm, PH nature, incubation time 42h;
B2, secondary seed tank are cultivated: substratum is made up of the supplementary material by following weight proportioning, groundnut meal 18 parts, glucose 1.5 parts, Semen Maydis powder 24 parts, 1.0 parts, urea, 1.0 parts, saltpetre, 0.8 part, calcium carbonate, starch 42 parts; In 35 DEG C, ventilation 1:1.2V/V.m, stirring velocity 280rpm, PH nature, incubation time 15h;
B3, by cultivate seed with 10% inoculum size access fermentation flask, in 34 DEG C cultivate 6 days fermented liquid, wherein power of agitator 3KW/m3, rotating speed 160r/min, air flow quantity 60m3/m3.h, tank pressure 0.04Mpa;
The preparation of C, Micronomicin Sulfate desorbed solution:
C1, acidifying removal of impurities: 98% sulfuric acid acidation with fermented liquid quality 1 times amount filters to obtain acidizing fluid, removing miscellaneous bacteria and other impurity;
C2, neutralization: it is 7 that acidizing fluid is neutralized to PH by the NaOH adding 3mol/L;
C3, resin absorption removal of impurities;
D, bleaching process: Micronomicin Sulfate desorbed solution decolours through ultrafiltration membrane system;
Ultrafiltration membrane system wherein, comprises first group of ultra filtration unit, second group of ultra filtration unit; First group of ultra filtration unit is made up of first group of first step ultra filtration unit, 1, first group of second stage ultra filtration unit, 2, first group of third stage ultra filtration unit 3; Second group of ultra filtration unit is made up of second group of first step ultra filtration unit, 4, second group of second stage ultra filtration unit, 5, second group of third stage ultra filtration unit 6; Wherein: first group of first step ultra filtration unit, 1, first group of second stage ultra filtration unit 2 is connected successively with first group of third stage ultra filtration unit 3, second group of first step ultra filtration unit, 4, second group of second stage ultra filtration unit 5 is connected successively with second group of third stage ultra filtration unit 6; First group of ultra filtration unit and second group of ultra filtration unit are connected in parallel;
Decolorization process: Micronomicin Sulfate desorbed solution is parallel through first group of ultra filtration unit and second group of ultra filtration unit decolouring, namely Micronomicin Sulfate desorbed solution is abreast successively through first group of first step ultra filtration unit, 1, first group of second stage ultra filtration unit, 2, first group of third stage ultra filtration unit 3, with second group of first step ultra filtration unit, 4, second group of second stage ultra filtration unit, 5, second group of third stage ultra filtration unit 6, the Micronomicin Sulfate desorbed solution after decolouring enters enrichment process;
Ultrafiltration membrane system working parameter is as follows: first group of first step ultra filtration unit, 1, second group of first step ultra filtration unit 4 selects aperture to be the ultra-filtration membrane retaining 5000 molecular weight, first group of second stage ultra filtration unit, 2, second group of second stage ultra filtration unit 5 selects aperture to be the ultra-filtration membrane retaining 4000 molecular weight, first group of third stage ultra filtration unit composition, 3, second group of third stage ultra filtration unit 6 selects aperture to be the ultra-filtration membrane retaining 3000 molecular weight, and it is 3T/h that working pressure all controls at 0.5Mpa, through flow;
E, enrichment process: the Micronomicin Sulfate desorbed solution after decolouring concentrates through nanofiltration membrane system;
Nanofiltration membrane system wherein, comprises first step nano-filtration unit 7, second stage nano-filtration unit 8, third stage nano-filtration unit 9; Wherein first step nano-filtration unit 7, second stage nano-filtration unit 8, third stage nano-filtration unit 9 connect successively;
Enrichment step: the Micronomicin Sulfate desorbed solution after decolouring concentrates through first step nano-filtration unit 7, second stage nano-filtration unit 8, third stage nano-filtration unit 9 successively; Obtain Micronomicin Sulfate.
Nanofiltration membrane system working parameter is as follows: first step nano-filtration unit 7 selects aperture to be the nanofiltration membrane of 35nm, and second stage nano-filtration unit 8 selects aperture to be the nanofiltration membrane of 20nm, and third stage nano-filtration unit 9 selects aperture to be the nanofiltration membrane of 10nm; It is 3T/h that working pressure all controls at 1.5Mpa, through flow.
Embodiment 2,
Ultrafiltration membrane system working parameter is as follows: first group of first step ultra filtration unit, 1, second group of first step ultra filtration unit 4 selects aperture to be the ultra-filtration membrane retaining 3000 molecular weight, first group of second stage ultra filtration unit, 2, second group of second stage ultra filtration unit 5 selects aperture to be the ultra-filtration membrane retaining 2000 molecular weight, first group of third stage ultra filtration unit composition, 3, second group of third stage ultra filtration unit 6 selects aperture to be the ultra-filtration membrane retaining 1000 molecular weight, and it is 2T/h that working pressure all controls at 0.3Mpa, through flow; All the other are with embodiment 1.
Embodiment 3,
Ultrafiltration membrane system working parameter is as follows: first group of first step ultra filtration unit, 1, second group of first step ultra filtration unit 4 selects aperture to be the ultra-filtration membrane retaining 4000 molecular weight, first group of second stage ultra filtration unit, 2, second group of second stage ultra filtration unit 5 selects aperture to be the ultra-filtration membrane retaining 3000 molecular weight, first group of third stage ultra filtration unit composition, 3, second group of third stage ultra filtration unit 6 selects aperture to be the ultra-filtration membrane retaining 2000 molecular weight, and it is 5T/h that working pressure all controls at 0.8Mpa, through flow; All the other are with embodiment 1.
Embodiment 4,
Nanofiltration membrane system working parameter is as follows: first step nano-filtration unit 7 selects aperture to be the nanofiltration membrane of 20nm, and second stage nano-filtration unit 8 selects aperture to be the nanofiltration membrane of 10nm, and third stage nano-filtration unit 9 selects aperture to be the nanofiltration membrane of 5nm; It is 2T/h that working pressure all controls at 1.0Mpa, through flow; All the other are with embodiment 1.
Embodiment 5,
Nanofiltration membrane system working parameter is as follows: first step nano-filtration unit 7 selects aperture to be the nanofiltration membrane of 50nm, and second stage nano-filtration unit 8 selects aperture to be the nanofiltration membrane of 30nm, and third stage nano-filtration unit 9 selects aperture to be the nanofiltration membrane of 20nm; It is 5T/h that working pressure all controls at 3.0Mpa, through flow; All the other are with embodiment 1.
Comparative example 1,
The membrane sepn extracting method of Micronomicin Sulfate, obtains especially by following steps:
The preparation of A, micronomicin seed:
Micronomicin Sulfate belongs to amino acid glycoside Broad spectrum antibiotics, and it is by a strain deep red micromonospora 201-9-8-53-84-37
#(Micromonospora purpura 201-9-8-53-84-37
#) produce.
A1, female bottle slant culture: substratum is made up of the supplementary material of following weight proportioning, Zulkovsky starch 8 parts, 1 part, calcium carbonate, dipotassium hydrogen phosphate 0.5 part, potassium sulfate 1.0 parts, asparagine 0.02 part, 0.5 part, magnesium sulfate, 20 parts, wheat bran, 18 parts, agar; Cultivate 7 days in 35 DEG C;
A2, sub-bottle slant culture: substratum is made up of the supplementary material of following weight proportioning, Zulkovsky starch 10 parts, 1 part, calcium carbonate, dipotassium hydrogen phosphate 0.6 part, potassium sulfate 1.2 parts, asparagine 0.02 part, 0.5 part, magnesium sulfate, 22 parts, wheat bran, 16 parts, agar; Cultivate 12 days in 37 DEG C, make spore, vacuum-drying saves backup;
A3, spore suspension: the spore of sub-bottle slant culture is made suspension;
The preparation of B, Micronomicin Sulfate fermented liquid:
B1, first class seed pot are cultivated: substratum is made up of the supplementary material by following weight proportioning, soybean cake powder 0.5 part, glucose 4 parts, 0.5 part, calcium carbonate, starch 55 parts, 0.05 part, ammonium sulfate, 0.05 part, saltpetre, peptone 0.3 part; In 37 DEG C, ventilation 1:2V/V.m, stirring velocity 350rpm, PH nature, incubation time 42h;
B2, secondary seed tank are cultivated: substratum is made up of the supplementary material by following weight proportioning, 15 parts, Zulkovsky starch powder, glucose 3 parts, Semen Maydis powder 15 parts, analysis for soybean powder 20 parts, peptone 2 parts, 5 parts, calcium carbonate, yeast powder 2 parts; In 35 DEG C, ventilation 1:1.2V/V.m, stirring velocity 280rpm, PH nature, incubation time 15h;
B3, by cultivate seed with 10% inoculum size access fermentation flask, in 34 DEG C cultivate 6 days fermented liquid, wherein power of agitator 3KW/m3, rotating speed 160r/min, air flow quantity 60m3/m3.h, tank pressure 0.04Mpa;
The preparation of C, Micronomicin Sulfate desorbed solution:
C1, acidifying removal of impurities: 98% sulfuric acid acidation with fermented liquid quality 1 times amount filters to obtain acidizing fluid, removing miscellaneous bacteria and other impurity;
C2, neutralization: it is 7 that acidizing fluid is neutralized to PH by the NaOH adding 3mol/L;
C3, resin absorption removal of impurities;
D, bleaching process: Micronomicin Sulfate desorbed solution decolours through ultrafiltration membrane system;
Ultrafiltration membrane system wherein, comprises first group of ultra filtration unit, second group of ultra filtration unit; First group of ultra filtration unit is made up of first group of first step ultra filtration unit, 1, first group of second stage ultra filtration unit, 2, first group of third stage ultra filtration unit 3; Second group of ultra filtration unit is made up of second group of first step ultra filtration unit, 4, second group of second stage ultra filtration unit, 5, second group of third stage ultra filtration unit 6; Wherein: first group of first step ultra filtration unit, 1, first group of second stage ultra filtration unit 2 is connected successively with first group of third stage ultra filtration unit 3, second group of first step ultra filtration unit, 4, second group of second stage ultra filtration unit 5 is connected successively with second group of third stage ultra filtration unit 6; First group of ultra filtration unit and second group of ultra filtration unit are connected in parallel;
Decolorization process: Micronomicin Sulfate desorbed solution is parallel through first group of ultra filtration unit and second group of ultra filtration unit decolouring, namely Micronomicin Sulfate desorbed solution is abreast successively through first group of first step ultra filtration unit, 1, first group of second stage ultra filtration unit, 2, first group of third stage ultra filtration unit 3, with second group of first step ultra filtration unit, 4, second group of second stage ultra filtration unit, 5, second group of third stage ultra filtration unit 6, the Micronomicin Sulfate desorbed solution after decolouring enters enrichment process;
Ultrafiltration membrane system working parameter is as follows: first group of first step ultra filtration unit, 1, second group of first step ultra filtration unit 4 selects aperture to be the ultra-filtration membrane retaining 5000 molecular weight, first group of second stage ultra filtration unit, 2, second group of second stage ultra filtration unit 5 selects aperture to be the ultra-filtration membrane retaining 4000 molecular weight, first group of third stage ultra filtration unit composition, 3, second group of third stage ultra filtration unit 6 selects aperture to be the ultra-filtration membrane retaining 3000 molecular weight, and it is 3T/h that working pressure all controls at 0.5Mpa, through flow;
E, enrichment process: the Micronomicin Sulfate desorbed solution after decolouring concentrates through nanofiltration membrane system;
Nanofiltration membrane system wherein, comprises first step nano-filtration unit 7, second stage nano-filtration unit 8, third stage nano-filtration unit 9; Wherein first step nano-filtration unit 7, second stage nano-filtration unit 8, third stage nano-filtration unit 9 connect successively;
Enrichment step: the Micronomicin Sulfate desorbed solution after decolouring concentrates through first step nano-filtration unit 7, second stage nano-filtration unit 8, third stage nano-filtration unit 9 successively; Obtain Micronomicin Sulfate.
Nanofiltration membrane system working parameter is as follows: first step nano-filtration unit 7 selects aperture to be the nanofiltration membrane of 35nm, and second stage nano-filtration unit 8 selects aperture to be the nanofiltration membrane of 20nm, and third stage nano-filtration unit 9 selects aperture to be the nanofiltration membrane of 10nm; It is 3T/h that working pressure all controls at 1.5Mpa, through flow.
Comparative example 2,
The membrane sepn extracting method of Micronomicin Sulfate, obtains especially by following steps:
The preparation of A, micronomicin seed:
Micronomicin Sulfate belongs to amino acid glycoside Broad spectrum antibiotics, and it is by a strain deep red micromonospora 201-9-8-53-84-37
#(Micromonospora purpura 201-9-8-53-84-37
#) produce.
A1, female bottle slant culture: substratum is made up of the supplementary material of following weight proportioning, Zulkovsky starch 8 parts, 1 part, calcium carbonate, dipotassium hydrogen phosphate 0.5 part, potassium sulfate 1.0 parts, asparagine 0.02 part, 0.5 part, magnesium sulfate, 20 parts, wheat bran, 18 parts, agar; Cultivate 7 days in 35 DEG C;
A2, sub-bottle slant culture: substratum is made up of the supplementary material of following weight proportioning, Zulkovsky starch 10 parts, 1 part, calcium carbonate, dipotassium hydrogen phosphate 0.6 part, potassium sulfate 1.2 parts, asparagine 0.02 part, 0.5 part, magnesium sulfate, 22 parts, wheat bran, 16 parts, agar; Cultivate 12 days in 37 DEG C, make spore, vacuum-drying saves backup;
A3, spore suspension: the spore of sub-bottle slant culture is made suspension;
The preparation of B, Micronomicin Sulfate fermented liquid:
B1, first class seed pot are cultivated: substratum is made up of the supplementary material by following weight proportioning, soybean cake powder 0.5 part, glucose 4 parts, 0.5 part, calcium carbonate, starch 55 parts, 0.05 part, ammonium sulfate, 0.05 part, saltpetre, peptone 0.3 part; In 37 DEG C, ventilation 1:2V/V.m, stirring velocity 350rpm, PH nature, incubation time 42h;
B2, secondary seed tank are cultivated: substratum is made up of the supplementary material by following weight proportioning, 15 parts, Zulkovsky starch powder, glucose 3 parts, Semen Maydis powder 15 parts, analysis for soybean powder 20 parts, peptone 2 parts, 5 parts, calcium carbonate, yeast powder 2 parts; In 35 DEG C, ventilation 1:1.2V/V.m, stirring velocity 280rpm, PH nature, incubation time 15h;
B3, by cultivate seed with 10% inoculum size access fermentation flask, in 34 DEG C cultivate 6 days fermented liquid, wherein power of agitator 3KW/m3, rotating speed 160r/min, air flow quantity 60m3/m3.h, tank pressure 0.04Mpa;
The preparation of C, Micronomicin Sulfate desorbed solution:
C1, acidifying removal of impurities: 98% sulfuric acid acidation with fermented liquid quality 1 times amount filters to obtain acidizing fluid, removing miscellaneous bacteria and other impurity;
C2, neutralization: it is 7 that acidizing fluid is neutralized to PH by the NaOH adding 3mol/L;
C3, resin absorption removal of impurities;
D, bleaching process: Micronomicin Sulfate desorbed solution decolours through ultrafiltration membrane system;
Ultrafiltration membrane system wherein, comprises first group of ultra filtration unit, second group of ultra filtration unit; First group of ultra filtration unit is made up of first group of first step ultra filtration unit, first group of second stage ultra filtration unit, first group of third stage ultra filtration unit, first group of fourth stage ultra filtration unit, first group of level V ultra filtration unit; Second group of ultra filtration unit is made up of second group of first step ultra filtration unit, second group of second stage ultra filtration unit, second group of third stage ultra filtration unit, second group of fourth stage ultra filtration unit, second group of level V ultra filtration unit; Wherein: first group of first step ultra filtration unit, first group of second stage ultra filtration unit, first group of third stage ultra filtration unit, first group of fourth stage ultra filtration unit, first group of level V ultra filtration unit connect successively, second group of first step ultra filtration unit, second group of second stage ultra filtration unit, second group of third stage ultra filtration unit, second group of fourth stage ultra filtration unit, second group of level V ultra filtration unit connect successively; First group of ultra filtration unit and second group of ultra filtration unit are connected in parallel;
Decolorization process: Micronomicin Sulfate desorbed solution is parallel through first group of ultra filtration unit and second group of ultra filtration unit decolouring, namely Micronomicin Sulfate desorbed solution is abreast successively through first group of first step ultra filtration unit, first group of second stage ultra filtration unit, first group of third stage ultra filtration unit, first group of fourth stage ultra filtration unit, first group of level V ultra filtration unit, with second group of first step ultra filtration unit, second group of second stage ultra filtration unit, second group of third stage ultra filtration unit, second group of fourth stage ultra filtration unit, second group of level V ultra filtration unit, Micronomicin Sulfate desorbed solution after decolouring enters enrichment process,
Ultrafiltration membrane system working parameter is as follows: first group of first step ultra filtration unit, second group of first step ultra filtration unit selects aperture to be the ultra-filtration membrane retaining 5000 molecular weight, first group of second stage ultra filtration unit, second group of second stage ultra filtration unit selects aperture to be the ultra-filtration membrane retaining 4000 molecular weight, first group of third stage ultra filtration unit composition, first group of fourth stage ultra filtration unit, first group of level V ultra filtration unit, second group of third stage ultra filtration unit, second group of fourth stage ultra filtration unit, second group of level V ultra filtration unit selects aperture to be the ultra-filtration membrane retaining 3000 molecular weight, working pressure all controls at 0.5Mpa, be 3T/h through flow,
E, enrichment process: the Micronomicin Sulfate desorbed solution after decolouring concentrates through nanofiltration membrane system;
Nanofiltration membrane system wherein, comprises first step nano-filtration unit, second stage nano-filtration unit, third stage nano-filtration unit; Wherein first step nano-filtration unit, second stage nano-filtration unit, third stage nano-filtration unit connect successively;
Enrichment step: the Micronomicin Sulfate desorbed solution after decolouring concentrates through first step nano-filtration unit, second stage nano-filtration unit, third stage nano-filtration unit successively; Obtain Micronomicin Sulfate.
Nanofiltration membrane system working parameter is as follows: first step nano-filtration unit selects aperture to be the nanofiltration membrane of 35nm, and second stage nano-filtration unit selects aperture to be the nanofiltration membrane of 20nm, and third stage nano-filtration unit selects aperture to be the nanofiltration membrane of 10nm; It is 3T/h that working pressure all controls at 1.5Mpa, through flow.
Test example
Tiring of test example 1, Nephelometric Determination Micronomicin Sulfate
The Micronomicin Sulfate of Example 1-5 and comparative example 1 and comparative example 2 preparation respectively, precision weighing, the solution of 1000U/mg is made with aqua sterilisa, accurate this liquid of absorption is appropriate respectively, with sterile phosphate damping fluid (PH=7.0) dilution make 10, the height dosing solution of 20U/mg concentration, than being 2:1 between height dosage facility, precision measures each 1.0ml of above-mentioned solution respectively, put in sterilizing colorimetric cylinder, each 6 pipes, add 3% escherichia coli liquid culture medium 9.0ml respectively, shake up immediately, in turbidimetry, cultivate 3.5h.Use microorganism than turbid instrument, constant temperature culture, timing vibration, calculation result.Negative control: PH=7.0 phosphate buffer 1 .0ml, adds the substratum of the non-inoculation experiments bacterium of 9.0ml.The results are shown in Table 1.
The results contrast of tiring of table 1 Nephelometric Determination Micronomicin Sulfate
| Embodiment | Tire (μ g/mg) | Micronomicin Sulfate C2b percentage composition (%) |
| Embodiment 1 | 630 | 96.1 |
| Embodiment 2 | 582 | 85.9 |
| Embodiment 3 | 618 | 92.7 |
| Embodiment 4 | 594 | 86.2 |
| Embodiment 5 | 604 | 93.5 |
| Comparative example 1 | 533 | 75.4 |
| Comparative example 2 | 605 | 89.3 |
Conclusion: from above data, changes the membrane pore size in film separating system, working pressure, through flow, affects larger on product concentration.In embodiment, Micronomicin Sulfate content is all greater than 85%, and Micronomicin Sulfate prepared by embodiment 1 is tired and Micronomicin Sulfate C2b percentage composition is all optimum, and parameter selected by visible embodiment 1 is optimum.The each parameter of comparative example 1 is all not as embodiment 1-5, and the desorbed solution preparation method after visible improvement is better than traditional technology, can effectively improve the quality of products.Though the Micronomicin Sulfate C2b percentage composition of comparative example 2 is greater than 85%, uses ten ultra-filtration membrane unit in its decoloration process, added production cost and production time.
Claims (1)
1. the membrane sepn extracting method of Micronomicin Sulfate, is characterized in that: Micronomicin Sulfate desorbed solution decolours through ultrafiltration membrane system, and the Micronomicin Sulfate desorbed solution after decolouring concentrates through nanofiltration membrane system again;
Ultrafiltration membrane system wherein, comprises first group of ultra filtration unit, second group of ultra filtration unit; First group of ultra filtration unit is made up of first group of first step ultra filtration unit (1), first group of second stage ultra filtration unit (2), first group of third stage ultra filtration unit (3); Second group of ultra filtration unit is made up of second group of first step ultra filtration unit (4), second group of second stage ultra filtration unit (5), second group of third stage ultra filtration unit (6); Wherein: first group of first step ultra filtration unit (1), first group of second stage ultra filtration unit (2) are connected successively with first group of third stage ultra filtration unit (3), second group of first step ultra filtration unit (4), second group of second stage ultra filtration unit (5) are connected successively with second group of third stage ultra filtration unit (6); First group of ultra filtration unit and second group of ultra filtration unit are connected in parallel;
Decolorization process: Micronomicin Sulfate desorbed solution is parallel through first group of ultra filtration unit and second group of ultra filtration unit decolouring, namely Micronomicin Sulfate desorbed solution is abreast successively through first group of first step ultra filtration unit (1), first group of second stage ultra filtration unit (2), first group of third stage ultra filtration unit (3), with second group of first step ultra filtration unit (4), second group of second stage ultra filtration unit (5), second group of third stage ultra filtration unit (6), the Micronomicin Sulfate desorbed solution after decolouring enters enrichment process;
Ultrafiltration membrane system working parameter is as follows: first group of first step ultra filtration unit (1), second group of first step ultra filtration unit (4) select aperture to be the ultra-filtration membrane retaining 5000 molecular weight, first group of second stage ultra filtration unit (2), second group of second stage ultra filtration unit (5) select aperture to be the ultra-filtration membrane retaining 4000 molecular weight, and first group of third stage ultra filtration unit composition, 3, second group of third stage ultra filtration unit (6) selects aperture to be the ultra-filtration membrane retaining 3000 molecular weight;
Nanofiltration membrane system, comprises first step nano-filtration unit (7), second stage nano-filtration unit (8), third stage nano-filtration unit (9); Wherein first step nano-filtration unit (7), second stage nano-filtration unit (8), third stage nano-filtration unit (9) connect successively;
Enrichment step: the Micronomicin Sulfate desorbed solution after decolouring concentrates through first step nano-filtration unit (7), second stage nano-filtration unit (8), third stage nano-filtration unit (9) successively; Obtain Micronomicin Sulfate;
Nanofiltration membrane system working parameter is as follows: first step nano-filtration unit (7) selects aperture to be the nanofiltration membrane of 35nm, second stage nano-filtration unit (8) selects aperture to be the nanofiltration membrane of 20nm, and third stage nano-filtration unit (9) selects aperture to be the nanofiltration membrane of 10nm;
It is 3T/h that working pressure in ultrafiltration membrane system working parameter all controls at 0.5MPa, through flow; It is 3T/h that working pressure in nanofiltration membrane system working parameter all controls at 1.5MPa, through flow;
Micronomicin Sulfate desorbed solution obtains as follows,
The preparation of A, micronomicin seed:
Get deep red micromonospora, implant carrier bottle is cultivated;
A1, female bottle slant culture: substratum is made up of the supplementary material of following weight proportioning, Zulkovsky starch 10 parts, 0.5 part, sodium-chlor, dipotassium hydrogen phosphate 0.3 part, 1.0 parts, saltpetre, asparagine 0.02 part, 0.5 part, magnesium sulfate, 18 parts, wheat bran, glycerine 0.6 part, 18 parts, agar; Cultivate 7 days in 35 DEG C;
A2, sub-bottle slant culture: substratum is made up of the supplementary material of following weight proportioning, Zulkovsky starch 13 parts, 0.5 part, sodium-chlor, dipotassium hydrogen phosphate 0.4 part, 1.3 parts, saltpetre, asparagine 0.03 part, 0.5 part, magnesium sulfate, 18 parts, wheat bran, glycerine 0.8 part, 20 parts, agar; Cultivate 12 days in 37 DEG C, make spore, vacuum-drying saves backup;
A3, spore suspension: the spore of sub-bottle slant culture is made suspension;
The preparation of B, Micronomicin Sulfate fermented liquid:
B1, first class seed pot are cultivated: substratum is made up of the supplementary material by following weight proportioning, soybean cake powder 15 parts, glucose 1.2 parts, Semen Maydis powder 22 parts, fish meal 2 parts, 1.0 parts, urea, 1.0 parts, calcium carbonate, starch 40 parts; In 37 DEG C, ventilation 1:2V/V.m, stirring velocity 350rpm, pH nature, incubation time 42h;
B2, secondary seed tank are cultivated: substratum is made up of the supplementary material by following weight proportioning, groundnut meal 18 parts, glucose 1.5 parts, Semen Maydis powder 24 parts, 1.0 parts, urea, 1.0 parts, saltpetre, 0.8 part, calcium carbonate, starch 42 parts; In 35 DEG C, ventilation 1:1.2V/V.m, stirring velocity 280rpm, pH nature, incubation time 15h;
B3, by cultivate seed with 10% inoculum size access fermentation flask, in 34 DEG C cultivate 6 days fermented liquid, wherein power of agitator 3KW/m3, rotating speed 160r/min, air flow quantity 60m3/m3.h, tank pressure 0.04MPa;
The preparation of C, Micronomicin Sulfate desorbed solution:
C1, acidifying removal of impurities: 98% sulfuric acid acidation with fermented liquid quality 1 times amount filters to obtain acidizing fluid, removing miscellaneous bacteria and other impurity;
C2, neutralization: it is 7 that acidizing fluid is neutralized to pH by the NaOH adding 3mol/L;
C3, resin absorption removal of impurities.
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