CN103074395B - Fermentation medium used for producing Safracin B - Google Patents
Fermentation medium used for producing Safracin B Download PDFInfo
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- CN103074395B CN103074395B CN201110330304.0A CN201110330304A CN103074395B CN 103074395 B CN103074395 B CN 103074395B CN 201110330304 A CN201110330304 A CN 201110330304A CN 103074395 B CN103074395 B CN 103074395B
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- safracin
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Abstract
The invention relates to the technical field of Safracin B production medium. The Safracin B production medium provided by the invention comprises a carbon source, an organic nitrogen source, and inorganic salt. The carbon source comprises glucose, sucrose, dextrin, corn starch, mannitol, and soluble starch. The organic nitrogen source comprises soybean peptone, fish meal peptone, soybean meal, soybean meal cake, cottonseed protein powder, soybean residue powder, peanut meal, and wheat germ powder. According to the Safracin B production medium provided by the invention, appropriate nitrogen source and carbon source are adopted, and dose and component compositions are optimized. A medium viscosity is appropriate. Safracin B production strains and mutant strain bacteria are prevented from excessive growth; fermentation product Safracin B yield is high; and titer is improved by 200-300% than that of media reported by literatures.
Description
Technical field
The present invention is specifically related to a kind of for cultivating Safracin B producing strains to produce fermention medium and the method and technology field thereof of Safracin B.
Background technology
Safracin B is separated a kind of non-ribosomal peptides obtained nineteen eighty-three from the fermentation culture of Pseudomonas fluorescens A2-2 (IFO14128), and its molecular formula is C
28h
36n
4o
7, a kind of antitumor drug ET-743 (Yondelis) can be obtained through structural modification transformation.Safracin B structural formula is as follows:
Safracin B experiment in vitro all has effect to most gram-positive microorganism and negative bacterium.Safracin B also has good anti-tumor activity except anti-microbial activity, and it can form covalent linkage with DNA.And ET-743 (Y ondelis) acts on the special DNA alkylating agent of DNA shallow ridges, guanine, it can be covalently bonded in the N 2 of guanine.Its identifies 325 base sequences in Ecolityr T-DNA promotor, then nonrecognition when the guanine in this sequence is changed into xanthoglobulin.
US4440752 discloses the process utilizing bacterium Pseudomonas fluorescens A2-2 fermentative production Safracin B: the producing strains getting a ring Safracin B from slant medium is inoculated in seed culture medium, be inoculated in after seed grows up to containing fermention medium (containing glucose 2.0%, N.F,USP MANNITOL 4.0%, dry yeast 2.0%, ammonium sulfate 1.0%, Repone K 0.4%, potassium primary phosphate 0.02%, calcium carbonate 0.8%, PH 7.0) 2L fermentor tank in, 24 DEG C-26 DEG C, 180rpm stirs, cultivate four days, fermentation PH in latter stage is at 7.5-8.0, Safracin B 4mg is obtained through separation and purification.
Summary of the invention
The object of this invention is to provide a kind of fermention medium for the production of Safracin B newly, this substratum is equally to cultivate Pseudomonas fluorescens A2-2 to produce Safracin B, but the productive rate of tunning SafracinB is high, tiring improves 200%-300% compared with the substratum of bibliographical information.
For reaching above-mentioned purpose, the technical scheme that the present invention takes is as follows:
For the production of the fermention medium of Safracin B, it comprises carbon source, organic nitrogen source and inorganic salt, said carbon source comprises glucose, sucrose, dextrin, W-Gum, N.F,USP MANNITOL and Zulkovsky starch, and said organic nitrogen source is the mixture of one or more in soy peptone, fish meal protein peptone, analysis for soybean powder, soybean cake powder, cottonseed flour, bean cake powder, peanut meal or wheat germ powder.
In above-mentioned fermention medium, inorganic salt can be inorganic potassium salt, inorganic magnesium salt etc., also can add the trace element such as VITAMIN, amino acid and natural fats and oils etc. in fermention medium.
Above-mentioned fermention medium, the mixture of the preferred glucose of said carbon source, N.F,USP MANNITOL or glucose and N.F,USP MANNITOL, the most preferably mixture of glucose and N.F,USP MANNITOL; Carbon source content is 1.0%-7.0%; The wherein preferred 1-2% of the content of glucose; The preferred 4-6% of content of N.F,USP MANNITOL, most preferably 5.0%.
Above-mentioned fermention medium, the preferred analysis for soybean powder of said organic nitrogen source and peanut meal can be one wherein or its mixture, the most preferably mixture of analysis for soybean powder, analysis for soybean powder and peanut meal.In fermention medium, the consumption of organic nitrogen source is 1.0%-6.0%, and optimum is 2%.
The bacterial strain that the present invention adopts is Pseudomonas fluorescens A2-2.
Beneficial effect of the present invention:
Fermention medium for the production of Safracin B provided by the invention, adopt suitable nitrogenous source and carbon source, consumption and component are all optimized and combinationally use, substratum viscosity is suitable, Safracin B producing strains and mutant strain thalline thereof can not over growths, the productive rate of tunning Safracin B is high, tires to improve 200%-300% compared with the substratum of bibliographical information.
Accompanying drawing explanation
Fig. 1 shows the impact that different carbon source generates Safracin B
Fig. 2 shows the impact that different organonitrogen generates Safracin B
Fig. 3 shows the impact that different grease generates Safracin B
Embodiment
With reference to experimental example: the formulation that fermentation culture method and standard are tired:
By Pseudomonas fluorescens A2-2 seed take out activation after inoculate slant medium (beef-protein medium:, 121 DEG C of sterilizing 20min), the inclined-plane of inoculation bacterial classification is placed in 28 DEG C of constant incubators, cultivate 2 days, obtain the thalline in vegetative period, then thalline is inoculated in and 30ml seed culture medium (glucose 1.5% is housed, peptone 0.5%, yeast extract 0.2%, dipotassium hydrogen phosphate 0.05%, magnesium sulfate heptahydrate 0.05%, calcium carbonate 0.4%) 250ml shaking flask in, at 28 DEG C, 220rpm shaking table is cultivated 1 day, obtain ripe seed, the seed obtained is equipped with 40ml fermention medium (glucose 2.0% with the inoculum size access of 2%, N.F,USP MANNITOL 4.0%, dry yeast 2.0%, ammonium sulfate 1.0%, Repone K 0.4%, potassium primary phosphate 0.02%, calcium carbonate 0.8%, PH 7.0) 250ml shaking flask in, be placed in 25 DEG C of shaking tables and continue cultivation 96 hours.The chromatographic instrument that HPLC adopts is Waters510 type high performance liquid chromatograph, and chromatographic column is C18 post, 4.6 × 250mm, 5 μm, determined wavelength 200nm, moving phase is acetonitrile: 1% diethanolamine (60: 40), flow velocity is 1ml/min, and sample size is 20 μ l, and column temperature is 30 DEG C.
Relative potency calculation formula is: relative potency=(peak area of the product of the standard that the improved culture medium peak areas ÷ that HPLC measures is above-mentioned) × 100%.
Embodiment 1
Slant medium and seed culture medium and working method, with experimental example 1, change fermention medium.
Fermention medium: glucose 2.0%, N.F,USP MANNITOL 4.0%, analysis for soybean powder 2.0%, ammonium sulfate 1.0%, Repone K 0.4%, potassium primary phosphate 0.02%, calcium carbonate 0.8%, PH 7.0.Relative potency is 180%.
Embodiment 2
Slant medium and seed culture medium and working method, with experimental example 1, change fermention medium.
Fermention medium: glucose 2.0%, N.F,USP MANNITOL 6.0%, analysis for soybean powder 2.0%, ammonium sulfate 1.0%, Repone K 0.4%, potassium primary phosphate 0.02%, calcium carbonate 0.8%, PH 7.0.Relative potency is 160%.
Embodiment 3
Slant medium and seed culture medium and working method, with experimental example 1, change fermention medium.
Fermention medium: glucose 1.0%, N.F,USP MANNITOL 5.0%, analysis for soybean powder 2.0%, ammonium sulfate 1.0%, Repone K 0.4%, potassium primary phosphate 0.02%, calcium carbonate 0.8%, PH 7.0.Relative potency is 220%.
Embodiment 4
Slant medium and seed culture medium and working method, with experimental example 1, change fermention medium.
Fermention medium: glucose 1.0%, N.F,USP MANNITOL 5.0%, analysis for soybean powder 2.0%, peanut meal 1%, ammonium sulfate 1.0%, Repone K 0.4%, potassium primary phosphate 0.02%, calcium carbonate 0.8%, PH 7.0.Relative potency is 160%.
Embodiment 5
Slant medium and seed culture medium and working method, with experimental example 1, change fermention medium.
Fermention medium: glucose 1.0%, N.F,USP MANNITOL 5.0%, analysis for soybean powder 1.0%, peanut meal 1%, ammonium sulfate 1.0%, Repone K 0.4%, potassium primary phosphate 0.02%, calcium carbonate 0.8%, PH 7.0.Relative potency is 280%.
Embodiment 6
Slant medium and seed culture medium and working method, with experimental example 1, change fermention medium.
Fermention medium: glucose 1.0%, N.F,USP MANNITOL 5.0%, analysis for soybean powder 1.0%, peanut meal 1%, ammonium sulfate 1.0%, Semen Maydis oil 0.5%, Repone K 0.4%, potassium primary phosphate 0.02%, calcium carbonate 0.8%, PH 7.0.Relative potency is 300%.
Carried out various comparing embodiment in addition, result is as follows:
Different carbon source produces Safracin B to fermentation obvious impact, the effects five kinds of carbon sources such as glucose, sucrose, Zulkovsky starch, N.F,USP MANNITOL and maltose, content is 5%, and fermentation termination HPLC detects product and tires, the results are shown in Figure shown in 1, the best results of N.F,USP MANNITOL.
Different nitrogen sources produces the impact had clearly on the fermentation of Safracin B, the effects fish meal protein peptone, cottonseed flour, soybean cake powder, Dried Corn Steep Liquor Powder, yeast extract, peanut meal seven kinds of conventional organic nitrogen sources, content is 2%, fermentation termination HPLC detects product and tires, the results are shown in Figure shown in 2, analysis for soybean powder and peanut meal effect best.
Add the growth that a small amount of natural fats and oils can promote thalline in the medium, better promote the conversion of substrate in fermented liquid simultaneously, experiment have chosen soya-bean oil, Semen Maydis oil, sunflower seed oil, the multiple natural fats and oils of rice wet goods add substratum respectively, adding concentration is 0.5%, not add the substratum of natural fats and oils in contrast, the results are shown in Figure shown in 3, the effect adding Semen Maydis oil is best.
Claims (7)
1. for the production of the fermention medium of Safracin B, it comprises carbon source, organic nitrogen source and inorganic salt, and said carbon source is the mixture of glucose and N.F,USP MANNITOL, and the content of glucose is 1-2%, and the content of N.F,USP MANNITOL is 4-6%; Said organic nitrogen source is the mixture of one or more in analysis for soybean powder, soybean cake powder, peanut meal, and the content of organic nitrogen source is 1.0%-6.0%.
2., as claimed in claim 1 for the production of the fermention medium of Safracin B, it is characterized in that: also comprise VITAMIN, amino acid and/or natural fats and oils.
3., as claimed in claim 2 for the production of the fermention medium of Safracin B, it is characterized in that: said natural fats and oils is Semen Maydis oil.
4., as claimed in claim 3 for the production of the fermention medium of Safracin B, it is characterized in that: said natural fats and oils Semen Maydis oil content is 0.5%.
5., as claimed in claim 1 for the production of the fermention medium of Safracin B, it is characterized in that: the content of said N.F,USP MANNITOL is 5%, and glucose content is 1%.
6. the arbitrary fermention medium for the production of Safracin B as described in claim 1-5, is characterized in that: said organic nitrogen source is the mixture of analysis for soybean powder and peanut meal.
7., as claimed in claim 6 for the production of the fermention medium of Safracin B, it is characterized in that: the content of analysis for soybean powder and peanut meal is respectively 1%.
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| CN201110330304.0A CN103074395B (en) | 2011-10-26 | 2011-10-26 | Fermentation medium used for producing Safracin B |
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| CN201110330304.0A CN103074395B (en) | 2011-10-26 | 2011-10-26 | Fermentation medium used for producing Safracin B |
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| CN103074395A CN103074395A (en) | 2013-05-01 |
| CN103074395B true CN103074395B (en) | 2015-01-07 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58113192A (en) * | 1981-12-26 | 1983-07-05 | Yoshitomi Pharmaceut Ind Ltd | Antibiotic substance safracin a and/or b |
| US4440752A (en) * | 1980-07-07 | 1984-04-03 | Yoshotomi Pharmaceutical Industries Ltd. | Antibiotics Y-16482 α and/or Y-16482 β, a process for production thereof, and a pharmaceutical composition containing either or both of them |
| CN1053446C (en) * | 1992-09-18 | 2000-06-14 | 三共株式会社 | Novel thiomarinol derivatives, and processes for their preparation |
-
2011
- 2011-10-26 CN CN201110330304.0A patent/CN103074395B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4440752A (en) * | 1980-07-07 | 1984-04-03 | Yoshotomi Pharmaceutical Industries Ltd. | Antibiotics Y-16482 α and/or Y-16482 β, a process for production thereof, and a pharmaceutical composition containing either or both of them |
| JPS58113192A (en) * | 1981-12-26 | 1983-07-05 | Yoshitomi Pharmaceut Ind Ltd | Antibiotic substance safracin a and/or b |
| CN1053446C (en) * | 1992-09-18 | 2000-06-14 | 三共株式会社 | Novel thiomarinol derivatives, and processes for their preparation |
Non-Patent Citations (2)
| Title |
|---|
| Ikeda Y, Idemoto H, Hirayama F, et al..Safracins, new antitumor antibiotics. I. Producing organism, fermentation and isolation.《J Antibiot (Tokyo)》.1983,第36卷(第10期), * |
| 陈天寿主编.第二节 微生物的营养物质.《微生物培养基的制造与应用》.1995, * |
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