CN105616447A - Mycobacterium phlei injection and preparation method thereof - Google Patents
Mycobacterium phlei injection and preparation method thereof Download PDFInfo
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- CN105616447A CN105616447A CN201610070768.5A CN201610070768A CN105616447A CN 105616447 A CN105616447 A CN 105616447A CN 201610070768 A CN201610070768 A CN 201610070768A CN 105616447 A CN105616447 A CN 105616447A
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- mycobacterium phlei
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention provides a preparation method of a Mycobacterium phlei injection. The preparation method includes: adopting a three-stage culture mode of original strain, master seed lot and working seed lot; subjecting the working seed lot to fermentation culture, separation, purification and sterilization to obtain Mycobacterium phlei stoste; using a diluent prepared from polysorbate and sodium chloride to dilute the stoste to directly obtain the Mycobacterium phlei injection. The preparation method has the advantages of good strain retentivity, high safety level, low cost and high production efficiency.
Description
Technical field
The present invention relates to Mycobacterium phlei, particularly to the preparation method of a kind of Mycobacterium phlei injection.
Background technology
Mycobacterium phlei is the one in antiacid mycobacteria, based on its biological affinity with mycobacterium tuberculosis, can get involved the immunologic process of human body, regulate the immunocompetence of body immune system. The Mycobacterium phlei of inactivation can stimulate T lymphocyte to discharge multiple lymphokine such as MAF, MIF, MCF after entering human body, MMF etc., these factors act on mononuclear phagocyte system, so as to assemble to lesions position, activate, pathogen is swallowed, kill and removes; Meanwhile, NKT (NK) cell, bone-marrow-derived lymphocyte also activate, increase; IgM, IgG become normal, regulate cell immune system and produce immunologic function, enhancing human body immunity ability. Clinical results proves, in immunologic function test, t lymphocyte subset group, T3, T4, T8, T4/T8 substantially increase, significant difference notable (P < 0.001-0.002), natural killer cell, the equal significant change of immunoglobulin, statistics has significant difference (P < 0.01), has notable immunological enhancement.
Immunostimulant or even anti-cancer function for Mycobacterium phlei, substantial amounts of research has all been carried out at present in pharmacy, clinic, in document DE3728367C1, disclose method HIV person being carried out immunity inoculation with Mycobacterium phlei, improve the survival state of HIV person.
In " experiment of new immunostimulant Mycobacterium phlei and clinical research " (practical Journal of Cancer, 2003.3, Vol.18, No.2) literary composition, the play-by-play Mycobacterium phlei clinically inhibitory action to Lovo human colon cancer cell ".
In document CN103396958A, the preparation method disclosing a kind of animal Mycobacterium phlei and immunostimulant thereof, after being inoculated on seed culture medium by strain to carry out seed culture, it is directly entered fermentation culture. Although this training method ensure that the purity of strain to a certain extent and reduces variation probability, but, also limit its yield.
In order to expand the use scope of Mycobacterium phlei, it is necessary to a kind of can the preparation method of Mycobacterium phlei injection of industrialized production.
Summary of the invention
The preparation method that the present invention proposes a kind of Mycobacterium phlei injection, comprises the following steps:
A) just the original freeze-drying lactobacillus of Mycobacterium phlei dissolves, leads to culture medium dilution, subpackage with the Soviet Union of dilution, completes the foundation that primordial seed is criticized, and preserves under-70 degrees Celsius of environment;
B) primordial seed is criticized strain to check, and qualified primordial seed is criticized strain thaw, filter, dilute, be inoculated on modified Russell medium under the environment of 30-40 degree Celsius cultivate 120-150 hour;
C) it is inoculated on modified Russell medium after the thalline obtained in step b) being ground, dilutes, cultivates 168-196 hour under the environment of 30-40 degree Celsius, through eluting, grinding, subpackage, form main seed lot strain, and test;
D) by checking qualified main seed lot strain to be inoculated on modified Russell medium, cultivate 196-240 hour under the environment of 30-40 degree Celsius, through eluting, grinding, subpackage, form working seed lots strain, and check;
E) working seed lots strain is formed work seed liquor by recovering, expanding, work seed liquor is inoculated in the fermentation medium in fermentation tank by work seed liquor according to the inoculum concentration of volume ratio 1-5%, condition of culture is 35-42 degree Celsius, ventilation is 0.5-1.5L/ minute, with 50-300rpm rotating speed when cultivate 360-450 hour, it is rinsed with the NaCl flushing liquor of 0.8-1.0%, filters after collection, after purification, sterilizing, namely obtain Mycobacterium phlei stock solution;
F) mix with polyoxyethylene sorbitan monoleate and sodium chloride and water for injection, filtration is configured to diluent, in described diluent, the percent by volume of polyoxyethylene sorbitan monoleate is 0.05-0.4%, weight sodium chloride percentage ratio is 0.7-1.1%, diluent after wherein filtering mixes with Mycobacterium phlei stock solution, it is thus achieved that Mycobacterium phlei medicinal liquid;
G) Mycobacterium phlei medicine liquid irrigation is enclosed in the ampoule bottle after cleaning sterilizing, after terminal sterilization leak detection, is Mycobacterium phlei injection.
Preparation method as above, it is characterised in that:
The formula of the logical culture medium of described Soviet Union is (unit: g/L): asparagine 4g, citrate 2g, dipotassium hydrogen phosphate 0.5g, MgSO4.7H2O0.5g, ferric ammonium citrate 0.5g, glycerol-6ml;
Regulating pH value to alkalescence, then adopt moist hear heat test to carry out secondary inactivation, step is:
The logical culture medium of the Soviet Union prepared is carried out inactivation treatment 30 minutes under 121 degree celsius temperature;
Culture medium is led in Soviet Union after once having inactivated, stands 4 hours at normal temperatures, then again carries out inactivation treatment 35 minutes with the temperature of 121 degrees Celsius, complete secondary inactivation treatment.
As above arbitrary described method, it is characterised in that:
The formula of described modified Russell medium is (unit: g/L):
Potassium dihydrogen phosphate 2.4, magnesium sulfate 0.24, magnesium citrate 0.6, L-sodium 7.2, peacock green 0.4, potato starch 30, and regulate pH value to alkalescence, then adopt moist hear heat test to carry out secondary inactivation, step is:
The modified Russell medium prepared is carried out inactivation treatment 30 minutes under 121 degree celsius temperature;
Modified Russell medium after once having inactivated, stands 4 hours at normal temperatures, then again carries out inactivation treatment 30 minutes with the temperature of 121 degrees Celsius, complete secondary inactivation treatment.
As above arbitrary described method, it is characterised in that:
The formula of described fermentation medium is (unit: g/L): potassium dihydrogen phosphate 1.5, disodium hydrogen phosphate 1.5, ammonium sulfate 0.5, magnesium sulfate 0.055, copper sulfate 0.001, monosodium glutamate 0.5, sodium citrate 0.45, agar powder 15, glycerol 6, yeast extract 5, potato starch 15, glucose 5, then carrying out inactivation treatment by moist hear heat test again, concrete grammar is:
The fermentation medium prepared is carried out inactivation treatment 30 minutes under 121 degree celsius temperature.
The invention allows for a kind of Mycobacterium phlei injection, described Mycobacterium phlei injection is to be prepared from by as above arbitrary described method.
The preparation method of Mycobacterium phlei injection proposed by the invention, is suitable for the commercial production of scale, and reduces cost significantly, it is possible to fully meet patient demand.
Accompanying drawing explanation
The preparation flow figure of Fig. 1 Mycobacterium phlei stock solution
Fig. 2 preparation flow figure from stock solution to injection
Specific implementation method
Embodiment 1
One, the preparation of Mycobacterium phlei stock solution
As it is shown in figure 1, the pattern that Mycobacterium phlei stock solution adopts third stage culture is prepared, specifically comprise the following steps that
The foundation that primordial seed is criticized
The original freeze-drying lactobacillus of Mycobacterium phlei is dissolved, leads to culture medium dilution, subpackage with the Soviet Union of dilution, complete the foundation that primordial seed is criticized, and preserve under-70 degrees Celsius of environment.
The formula of the logical culture medium of described Soviet Union is (unit: g/L): asparagine 4g, citrate 2g, dipotassium hydrogen phosphate 0.5g, MgSO4.7H2O0.5g, ferric ammonium citrate 0.5g, glycerol-6ml;
Regulating pH value to alkalescence, then adopt moist hear heat test to carry out secondary inactivation, step is:
The logical culture medium of the Soviet Union prepared is carried out inactivation treatment 30 minutes under 121 degree celsius temperature;
Culture medium is led in Soviet Union after once having inactivated, stands 4 hours at normal temperatures, then again carries out inactivation treatment 35 minutes with the temperature of 121 degrees Celsius, complete secondary inactivation treatment.
The foundation of main seed lot strain
Primordial seed is criticized strain check, and qualified primordial seed is criticized strain thaw, filter, dilute, be inoculated on modified Russell medium under the environment of 30-40 degree Celsius cultivate 120-150 hour;
Then it is inoculated on modified Russell medium after the thalline of acquisition being ground, dilutes, cultivates 168-196 hour under the environment of 30-40 degree Celsius, through eluting, grinding, subpackage, form main seed lot strain, and test.
The formula of described modified Russell medium is (unit: g/L):
Potassium dihydrogen phosphate 2.4, magnesium sulfate 0.24, magnesium citrate 0.6, L-sodium 7.2, peacock green 0.4, potato starch 30, and regulate pH value to alkalescence, then adopt moist hear heat test to carry out secondary inactivation, step is:
The modified Russell medium prepared is carried out inactivation treatment 30 minutes under 121 degree celsius temperature;
Modified Russell medium after once having inactivated, stands 4 hours at normal temperatures, then again carries out inactivation treatment 30 minutes with the temperature of 121 degrees Celsius, complete secondary inactivation treatment.
The foundation of working seed lots strain
Check qualified main seed lot strain to be inoculated on modified Russell medium, cultivate 196-240 hour under the environment of 30-40 degree Celsius, through eluting, grinding, subpackage, form working seed lots strain, and check.
The formation of Mycobacterium phlei stock solution
Working seed lots strain forms work seed liquor by recovering, expanding, work seed liquor is inoculated in the fermentation medium in fermentation tank by work seed liquor according to the inoculum concentration of volume ratio 1-5%, condition of culture is 35-42 degree Celsius, ventilation is 0.5-1.5L/ minute, with 50-300rpm rotating speed when cultivate 360-450 hour, it is rinsed with the NaCl flushing liquor of 0.8-1.0%, filters after collection, after purification, sterilizing, namely obtain Mycobacterium phlei stock solution.
The formula of described fermentation medium is (unit: g/L): potassium dihydrogen phosphate 1.5, disodium hydrogen phosphate 1.5, ammonium sulfate 0.5, magnesium sulfate 0.055, copper sulfate 0.001, monosodium glutamate 0.5, sodium citrate 0.45, agar powder 15, glycerol 6, yeast extract 5, potato starch 15, glucose 5, then carrying out inactivation treatment by moist hear heat test again, concrete grammar is:
The fermentation medium prepared is carried out inactivation treatment 30 minutes under 121 degree celsius temperature.
The preparation of Mycobacterium phlei medicinal liquid and embedding
Mix with polyoxyethylene sorbitan monoleate and sodium chloride and water for injection, filtration is configured to diluent, in described diluent, the percent by volume of polyoxyethylene sorbitan monoleate is 0.05-0.4%, weight sodium chloride percentage ratio is 0.7-1.1%, diluent after wherein filtering mixes with Mycobacterium phlei stock solution, it is thus achieved that Mycobacterium phlei medicinal liquid. The proportioning of diluent and stock solution is different and select different proportionings according to the concentration of Mycobacterium phlei in medicinal liquid. It is said that in general, the dilution that diluent is to Mycobacterium phlei stock solution, carry out according to following four kinds of specifications:
0.172 �� g/ml (extremely low concentration), the F.U.36 clump count in every ml injection is 4.6 �� 105;
1.72 �� g/ml (low concentration), the F.U.36 clump count in every ml injection is 4.6 �� 106;
17.2 �� g/ml (middle concentration), the F.U.36 clump count in every ml injection is 4.6 �� 107;
172 �� g/ml (high concentration), the F.U.36 clump count in every ml injection is 4.6 �� 108��
Mycobacterium phlei medicine liquid irrigation is enclosed in the ampoule bottle after cleaning sterilizing, after terminal sterilization leak detection, is Mycobacterium phlei injection.
It should be understood that it is above only in order to illustrative not limiting technical scheme, although the present invention being described in detail with reference to above-described embodiment, it will be understood by those within the art that: still the present invention can be modified or equivalent replacement, without deviating from any modification or partial replacement of the spirit and scope of the present invention, all should be encompassed in the middle of scope of the presently claimed invention.
Claims (5)
1. a preparation method for Mycobacterium phlei injection, comprises the following steps:
A) just the original freeze-drying lactobacillus of Mycobacterium phlei dissolves, leads to culture medium dilution, subpackage with the Soviet Union of dilution, completes the foundation that primordial seed is criticized, and preserves under-70 degrees Celsius of environment;
B) primordial seed is criticized strain to check, and qualified primordial seed is criticized strain thaw, filter, dilute, be inoculated on modified Russell medium under the environment of 30-40 degree Celsius cultivate 120-150 hour;
C) it is inoculated on modified Russell medium after the thalline obtained in step b) being ground, dilutes, cultivates 168-196 hour under the environment of 30-40 degree Celsius, through eluting, grinding, subpackage, form main seed lot strain, and test;
D) by checking qualified main seed lot strain to be inoculated on modified Russell medium, cultivate 196-240 hour under the environment of 30-40 degree Celsius, through eluting, grinding, subpackage, form working seed lots strain, and check;
E) working seed lots strain is formed work seed liquor by recovering, expanding, work seed liquor is inoculated in the fermentation medium in fermentation tank by work seed liquor according to the inoculum concentration of volume ratio 1-5%, condition of culture is 35-42 degree Celsius, ventilation is 0.5-1.5L/ minute, with 50-300rpm rotating speed when cultivate 360-450 hour, it is rinsed with the NaCl flushing liquor of 0.8-1.0%, filters after collection, after purification, sterilizing, namely obtain Mycobacterium phlei stock solution;
F) mix with polyoxyethylene sorbitan monoleate and sodium chloride and water for injection, filtration is configured to diluent, in described diluent, the percent by volume of polyoxyethylene sorbitan monoleate is 0.05-0.4%, weight sodium chloride percentage ratio is 0.7-1.1%, diluent after wherein filtering mixes with Mycobacterium phlei stock solution, it is thus achieved that Mycobacterium phlei medicinal liquid;
G) Mycobacterium phlei medicine liquid irrigation is enclosed in the ampoule bottle after cleaning sterilizing, after terminal sterilization leak detection, is Mycobacterium phlei injection.
2. preparation method as claimed in claim 1, it is characterised in that:
The formula of the logical culture medium of described Soviet Union is (unit: g/L): asparagine 4g, citrate 2g, dipotassium hydrogen phosphate 0.5g, MgSO4.7H2O0.5g, ferric ammonium citrate 0.5g, glycerol-6ml;
Regulating pH value to alkalescence, then adopt moist hear heat test to carry out secondary inactivation, step is:
The logical culture medium of the Soviet Union prepared is carried out inactivation treatment 30 minutes under 121 degree celsius temperature;
Culture medium is led in Soviet Union after once having inactivated, stands 4 hours at normal temperatures, then again carries out inactivation treatment 35 minutes with the temperature of 121 degrees Celsius, complete secondary inactivation treatment.
3. the method as described in as arbitrary in claim 1-2, it is characterised in that:
The formula of described modified Russell medium is (unit: g/L):
Potassium dihydrogen phosphate 2.4, magnesium sulfate 0.24, magnesium citrate 0.6, L-sodium 7.2, peacock green 0.4, potato starch 30, and regulate pH value to alkalescence, then adopt moist hear heat test to carry out secondary inactivation, step is:
The modified Russell medium prepared is carried out inactivation treatment 30 minutes under 121 degree celsius temperature;
Modified Russell medium after once having inactivated, stands 4 hours at normal temperatures, then again carries out inactivation treatment 30 minutes with the temperature of 121 degrees Celsius, complete secondary inactivation treatment.
4. the method as described in as arbitrary in claim 1-3, it is characterised in that:
The formula of described fermentation medium is (unit: g/L): potassium dihydrogen phosphate 1.5, disodium hydrogen phosphate 1.5, ammonium sulfate 0.5, magnesium sulfate 0.055, copper sulfate 0.001, monosodium glutamate 0.5, sodium citrate 0.45, agar powder 15, glycerol 6, yeast extract 5, potato starch 15, glucose 5, then carrying out inactivation treatment by moist hear heat test again, concrete grammar is:
The fermentation medium prepared is carried out inactivation treatment 30 minutes under 121 degree celsius temperature.
5. a Mycobacterium phlei injection, described Mycobacterium phlei injection is to be prepared from by the method as described in as arbitrary in claim 1-4.
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| CN201610070768.5A CN105616447A (en) | 2016-01-28 | 2016-01-28 | Mycobacterium phlei injection and preparation method thereof |
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| CN201610070768.5A CN105616447A (en) | 2016-01-28 | 2016-01-28 | Mycobacterium phlei injection and preparation method thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3728367C1 (en) * | 1987-08-25 | 1988-09-01 | Burkhard Steglich | Means for immunizing the human body against HIV infections |
| CN103396958A (en) * | 2013-07-15 | 2013-11-20 | 广东温氏食品集团股份有限公司 | Preparation method of mycobacterium phlei and immune enhancer thereof |
| CN104560795A (en) * | 2014-12-19 | 2015-04-29 | 吉林大学 | Culture medium and culture method for in-vitro culture of mycobacterium tuberculosis biofilm body |
-
2016
- 2016-01-28 CN CN201610070768.5A patent/CN105616447A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3728367C1 (en) * | 1987-08-25 | 1988-09-01 | Burkhard Steglich | Means for immunizing the human body against HIV infections |
| CN103396958A (en) * | 2013-07-15 | 2013-11-20 | 广东温氏食品集团股份有限公司 | Preparation method of mycobacterium phlei and immune enhancer thereof |
| CN104560795A (en) * | 2014-12-19 | 2015-04-29 | 吉林大学 | Culture medium and culture method for in-vitro culture of mycobacterium tuberculosis biofilm body |
Non-Patent Citations (3)
| Title |
|---|
| 叶维民: "草分枝杆菌口服液的研制及其对断奶仔猪的临床应用研究", 《中国优秀硕士学位论文全文数据库》 * |
| 易美华: "《生物资源开发利用》", 30 June 2003 * |
| 胡熙明等: "《中国药物大全 西药卷 第2版》", 31 January 1998 * |
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Application publication date: 20160601 |