CN108130285A - A kind of the sea micromonoad strain and its application of the production Rakicidin B that ferment - Google Patents

A kind of the sea micromonoad strain and its application of the production Rakicidin B that ferment Download PDF

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CN108130285A
CN108130285A CN201711187509.1A CN201711187509A CN108130285A CN 108130285 A CN108130285 A CN 108130285A CN 201711187509 A CN201711187509 A CN 201711187509A CN 108130285 A CN108130285 A CN 108130285A
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rakicidin
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周剑
林风
江红
连云阳
江宏磊
陈丽
赵薇
方志锴
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Fujian Institute of Microbiology
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Abstract

The invention belongs to technical field of microbial fermentation, and in particular to a kind of the sea micromonoad strain and its application of the production Rakicidin B that ferment.The present invention carries out mutation breeding by the sea micromonoad strain FIM02523 to existing production Rakicidins compounds, obtain the mutant strain sea micromonoad strain FIM R160708 of plant height production Rakicidin B, the bacterial strain can effectively improve the potency of Rakicidin B in zymotic fluid, in the fermenting experiment of 20 1000L fermentation tanks, the potency that sea micromonoad FIM R160708 fermentations generate Rakicidin B is up to 1000mg/L or so, and zymotic fluid component ratio B1：B≤3：97, the fermentation yield of target product Rakicidin B is not only greatly improved, but also effectively reduce the yield of other Rakicidins by-products, be conducive to the extraction purification work of follow-up Rakicidin B, industrialization demand can be met.

Description

A kind of the sea micromonoad strain and its application of the production Rakicidin B that ferment
Technical field
The invention belongs to technical field of microbial fermentation, and in particular to a kind of small list in ocean for the production Rakicidin B that ferment Spore bacterial strain and its application.
Background technology
Rakicidins classes compound is the tunning of sea micromonoad, current separated Rakicidins classes Compound mainly contains Rakicidin A, Rakicidin A1, Rakicidin B1 and Rakicidin B, chemical constitution And component is as follows：
Rakicidin s compounds are because contain 1 rare rare 4- amino -2,4- penta in its 15 yuan of cyclic lipopeptide structures Dienoic acid caprylamide simultaneously has Anti-tumor angiogenesis and attracts attention.Yamazaki (2007) is the study found that Rakicidin A With remarkable weary oxygen selective Anti-tumor angiogenesis, inhibitor against colon carcinoma cells HCT-8 cell activity is normal oxygen item under hypoxic condition 17.5 times under part；Takeuchi (2011) also reports Rakicidin A for the first time can induce marrow slow under hypoxic condition The apoptosis of property leukemic stem cells.Although the anti-hypoxic tumor cells of the Rakicidin A and the mechanism of action of anti-CSC are still not It is clear, but Rakicidin A are considered the anti-hypoxic tumor cells for being rich in development prospect and anti-CSC by many colleagues Drug.
Fujian Microorganism Inst. reported being separated to from microbial metabolic products at home for the first time in 2006 Object Rakicidin A and B (referring to Chinese patent CN 105709205A and CN 105753936A) are closed, and to Rakicidin B (FW523-3) related biological activity has carried out Primary Study, even more finds Rakicidins A and B compound tool for the first time Have the anaerobic bacterias such as anti-clinical pathogenic clostridium difficile, anti-vancomycin-resistant enterococcus activity, there are larger research and development to be worth.
But it studies and reports less about the fermentation manufacturing technique of Rakicidins class compounds in existing research. Although such as Kimberly D.Mcbrien report fermentation and the separation purifying technique of Rakicidins class compounds, but its The flask process stage is only limitted to, does not develop to suitable industrialized production, and its whole fermentation period is longer, and Key component in its zymotic fluid is mostly Rakicidin A, and the comparision contents of Rakicidin B are low.Fujian Province's microbe research Also reported in 2006 and a kind of extract Rakicidins from the zymotic fluid of ocean Micromonospora chalcea FIM 02-523 The method of class compound, the Rakicidins classes compound extracted include Rakicidin A and Rakicidin B.But institute The method stated also only is limited to the basic fermentation of shaking flask, and not only fermentation level is low, but also is dfficult to apply to large scale fermentation Production.
Chinese patent CN105779348A discloses a kind of fermentation process of Rakicidins classes compound, in this method, The total fermentation titer of highest of Rakicidins class compounds is 600mg/L or so, but is multi-components fermented, in zymotic fluid component B1：The ratio of B is approximately equal to 1：4, the yield of one side Rakicidins compounds is relatively low, on the other hand, due to Rakicidins The structure of B1 with Rakicidins B components is similar, further increases the difficulty isolated and purified, it is difficult to realize industrial metaplasia Production.
Invention content
For this purpose, the technical problems to be solved by the invention are to provide a kind of small list in ocean for the production Rakicidin B that ferment Spore bacterial strain, to solve the problems, such as that the fermentation yield of Rakicidin B compounds in the prior art is relatively low.
Second technical problem to be solved by this invention is to provide a kind of utilization above-mentioned bacterial strains fermenting and producing The method of Rakicidin B compounds.
In order to solve the above technical problems, a kind of sea micromonoad strain of the present invention, Classification And Nomenclature are Micromonospora sp.FIM-R160708 have been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms Center, deposit number are CGMCC NO.14824.Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation day Phase：On October 16th, 2017.
The invention also discloses the sea micromonoad strain answering in fermenting and producing Rakicidins compounds With.
The Rakicidins compounds are Rakicidin B.
The invention also discloses a kind of method of fermenting and producing Rakicidin B, including by the sea micromonoad The step of strain is inoculated in suitable for carrying out fermented and cultured in fermentation medium.
The fermentation medium includes the component of following mass content：Soluble starch 5.0-7.0%, sucrose 0.8- 1.2%, soybean cake powder 2.0-4.0%, dusty yeast 0.8-1.2%, MgSO4·7H2O 0.03-0.05%, FeSO4·7H2O 0.004-0.006%, CuSO4·5H2O 0.004-0.006%, CoCl2·6H2O 0.0004-0.0006%, CaCO3 0.4- 0.6%, tap water is prepared, and it is 7.5 to adjust pH value.
Preferably, the fermentation medium includes the component of following mass content：Soluble starch 6.0%, sucrose 1.0%, soybean cake powder 3.0%, dusty yeast 1.0%, MgSO4·7H2O 0.04%, FeSO4·7H2O 0.005%, CuSO4· 5H2O 0.005%, CoCl2·6H2O 0.0005%, CaCO30.5%, tap water is prepared, and it is 7.5 to adjust pH value.
The condition of the fermented and cultured step is：The bacterial strain is inoculated with the inoculum concentration of 3.0-12.0%, controls rotating speed 200-400rpm, ventilate 0.5-1.2vvm, and control fermentation process DO values are more than 20%, and fermented and cultured is carried out in 32 DEG C.
Preferably, the condition of the fermented and cultured step is：The good seed of seed tank culture is connect with 10% inoculum concentration In kind to fermentation tank, earlier fermentation control rotating speed 200rpm, ventilate 0.5vvm, and rotating speed is first stepped up with the growth of mycelia To 400rpm；According to the variation of fermentation tank DO parameters, then ventilation is stepped up to 1.2vvm, fermentation process controls DO values always More than 20%.
In the fermentation process, further include when concentration of reduced sugar is less than 10g/L in on-line period detection zymotic fluid, lead to The step of concentration of reduced sugar crossed in control of additive raw material zymotic fluid is 10-15g/L, the feed supplement matrix includes following mass content Composition：Glucose 25%, peptone 10%, ammonium sulfate 1%.
The method further includes the inoculation the step of seed liquor culture is carried out in seed culture medium, described Seed culture medium includes the component of following mass content：Soluble starch 1.5-2.5%, glucose 0.8-1.2%, dusty yeast 1.5-2.5%, peptone 0.8-1.2%, MgSO4·7H2O 0.04-0.06%, FeSO4·7H2O 0.004-0.006%, CuSO4·5H2O 0.004-0.006%, CoCl2·6H2O 0.0004-0.0006%, CaCO30.2-0.4%, tap water are matched System, tune pH value are 7.0-7.5, sterilize and carry out seed liquor culture after 30-35 DEG C.
Preferably, the seed culture medium includes the component of following mass content：Soluble starch 2.0%, glucose 1.0%, dusty yeast 2.0%, peptone 1.0%, MgSO4·7H2O 0.05%, FeSO4·7H2O 0.005%, CuSO4· 5H2O 0.005%, CoCl2·6H2O 0.0005%, CaCO30.3%, tap water is prepared, and tune pH value is 7.0-7.5, is sterilized Seed liquor culture is carried out after 32 DEG C.
The seed liquor incubation step includes shake-flask seed liquid culture and seed tank culture step.
The method is further included carries out activation culture by the inoculation in Gao Shi asparagines solid slope culture medium Step, the solid slope culture medium include the component of following mass content：Soluble starch 1-3%, L- asparagine 0.04- 0.06%, KNO30.08-0.12%, K2HPO4·3H2O 0.04-0.06%, NaCl 0.04-0.06%, MgSO4·7H2O 0.04-0.06%, CaCO30.08-0.12%, agar 1-2% adjust pH value 7.5.
Preferably, the solid slope culture medium includes the component of following mass content：Soluble starch 2%, L- asparagus ferns 0.05%, KNO of element30.1%, K2HPO4·3H2O 0.05%, NaCl 0.05%, MgSO4·7H2O 0.05%, CaCO3 0.1%, agar 1.5% adjusts pH value 7.5.
The present invention carries out mutagenesis by the sea micromonoad strain FIM02523 to existing production Rakicidins compounds and educates Kind, the mutant strain sea micromonoad strain FIM-R160708 of plant height production Rakicidin B is obtained, which can be effective Improve the potency of Rakicidin B in zymotic fluid, in the fermenting experiment of 20-1000L fermentation tanks, sea micromonoad FIM- The potency that R160708 fermentations generate Rakicidin B is up to 1000mg/L or so, and zymotic fluid component ratio B1：B≤3：97, The fermentation yield of target product Rakicidin B is not only greatly improved, but also effectively reduces other Rakicidins pairs The yield of product is conducive to the extraction purification work of follow-up Rakicidin B, can meet industrialization demand.
Description of the drawings
In order to make the content of the present invention more clearly understood, it below according to specific embodiments of the present invention and ties Attached drawing is closed, the present invention is described in further detail, wherein,
Fig. 1 is the electron-microscope scanning figure of mutant strain FIM-R160708 of the present invention；
Fig. 2 is original strain FIM02523 zymotic fluid HPLC spectrograms；Wherein, RT11.17 is Rakicidin B1 products, RT11.75 is Rakicidin B products；
Fig. 3 is mutant strain FIM-R160708 zymotic fluids HPLC spectrograms of the present invention；Wherein, RT11.20 is Rakicidin B1 products, RT11.79 are Rakicidin B；
Fig. 4 is mutant strain FIM-R160708 tanks top fermentation curve graph of the present invention.
Specific embodiment
The mutation breeding of 1 bacterial strain of embodiment
The method for mutation breeding of sea micromonoad FIM-R160708 includes the following steps described in the present embodiment：
(1) prepared by spore suspension：Ripe starting strain FIM02523 (deposit number CGMCC NO.12132) will be cultivated Fresh inclined-plane adds in appropriate sterile saline, is gently scraped with inoculation shovel, pours into the oscillation of the sterile flasks with bead and beats It dissipates, filters mycelia afterwards, it is spare to leave spore suspension；
(2) ultraviolet mutagenesis：Bacterium solution prepared by 2-4mL is taken to be added in diameter 9cm culture dishes, is put into sterilized magnetic stirring Then son is put magnetic force and is mixed on device, under 30W ultraviolet lights at 30cm；In formal pre-irradiation, ultraviolet light 10min should be first opened, is allowed ultraviolet Lamp preheats, and is then turned on ware lid and formally distinguishes treatment with irradiation under stiring：30S, 60S, 90S, 120S, 180S, 240S, 300S, Then it is diluted to suitable multiple in the dark, then 0.1mL is taken to be applied on Gao Shi asparagine tablets respectively, every grade of dilution is smeared 3 tablets, to be compared without irradiation, ultraviolet light irradiates later operation all to carry out in the dark, prevents light reparation；It will The tablet smoothened, is wrapped with black cloth, is placed in 32 DEG C of constant incubators and is protected from light culture 12d, and cultured plate causes for calculating Dead rate and bacterial screening；
(3) ARTP (atmospheric pressure at room plasma) mutagenesis：The operating power 120W of ARTP mutation breeding instrument, air source are set For argon gas, throughput 10SLM, the irradiation distance between plasma emission source and sample is 2mm, irradiation time is set as 0,60, 120、180、240、360S；The spore suspension prepared 10ul is taken uniformly to be applied on sterile metal slide glass, then put it into Sample stage in ARTP instrument；Plasma irradiating is carried out by program, is put slide glass with aseptic nipper after sample treatment Into the EP pipes equipped with 1ml physiological saline, EP pipes are shaken into 1min on the oscillator, the micro- life being attached on fungus slide glass Object is eluted in liquid, forms new bacteria suspension；New bacteria suspension is suitably diluted, takes the coating of 0.1ml dilutions flat Plate is placed in 32 DEG C of constant incubators and cultivates 12d, and cultured plate is used to calculate lethality and bacterial screening；
(4) the continuous mutagenesis of the ultraviolet-ARTP of ultraviolet-ARTP- and the screening of Rakicidin B enhanced variants：According to upper It states method and carries out the ultraviolet and mutagenic obtained preferable mutagenic conditions of ARTP, reach 75% for standard by lethality, setting is ultraviolet to lure Change condition into 30W ultraviolet lights under distance 30cm mutagenic treatment time 180S, ARTP mutagenic conditions be ARTP instrument operating powers 120W, air source are argon gas, throughput 10L/min, and the irradiation distance between plasma emission source and sample is 2mm, during irradiation Between be set as 60S；The single bacterium colony that ultraviolet mutagenesis is obtained carries out superior strain that shake flask fermentation HPLC verification obtains as setting out Bacterial strain carries out ARTP mutagenesis, then the mutagenic obtained superior strains of ARTP are repeated ultraviolet and ARTP mutagenesis；By a large amount of Shaking flask selection and breeding obtain the mutant strain of plant height production Rakicidin B, and number is FIM-R160708, is named as The electron-microscope scanning figure of Micromonospora sp.FIM-R160708, mutant strain FIM R160708 are shown in Fig. 1.
The morphological feature of gained sea micromonoad FIM R160708 is：Without aerial hyphae, in Gao Shi-asparagus fern element agar On tablet, bacterium colony is round, and fold protrusion, regular edges are neat, and initial stage is orange-yellow, dark brown (spore and mycelia) after maturation, training Base non-pigment is supported to generate；On Ban Shi agar plates, bacterium colony is round, fold protrusion, neat in edge, substrate mycelium reddish orange, spore Sublayer taupe, dry, part mycelia curls protrusion, and non-pigment generates；Micro- sem observation, substrate mycelium is elongated, straight or soft Song has branch, can be broken, and 0.3 μm of hyphal diameter, single spore is born on base silk sporophore, and spore has handle, round or ellipse Circle, spore size (0.6-0.7) × (0.6-0.9) μm, epispore is hair-like.
6 generation of superior strain FIM-R160708 inclined-plane continuous passages that will be obtained after mutagenesis, Rakicidin B fermentation titers And component ratio does not find significant change.The bacterium is stored in 20 degree of conservation cabinets took former strain passage one time fermentation to test every 2 months Card, as a result bacterium Rakicidin B fermentations potency and component ratio in 12 months does not find significant change.Show above Mutant strain FIM-R160708 has high yield and inheritance stability character, is suitble to industrialized production.By the small list in obtained strains ocean It is commonly micro- that spore bacterial strain Micromonospora sp.FIM-R160708 are preserved in China Committee for Culture Collection of Microorganisms Bio-Centers, deposit number are CGMCC NO.14824, and preservation date is on October 16th, 2017.
2 starting strain FIM02523 of embodiment and the comparison of mutant strain FIM-R160708 shake flask fermentations
Sea micromonoad FIM02523 and mutant strain the FIM-R160708 slant pore of fresh cultured is taken to scrape respectively After be made in bacterial suspension inoculation to shake-flask seed culture, 32 DEG C, 250rpm cultivate 48 hours, after be inoculated by 5% inoculum concentration In Medium of shaking flask fermentation, 30 DEG C, 250rpm put bottle after cultivating 120 hours, HPLC measures tunning.
Prepare seed culture based formulas (mass fraction)：Soluble starch 2.0%, glucose 1.0%, dusty yeast 2.0%, peptone 1.0%, MgSO4·7H2O 0.05%, FeSO4·7H2O 0.005%, CuSO4·5H2O 0.005%, CoCl2·6H2O 0.0005%, CaCO30.3%, tap water is prepared, and adjusts pH value 7.0-7.5；Seed liquor training is carried out in 32 DEG C It supports.
Prepare fermentative medium formula (mass fraction)：Soluble starch 6.0%, sucrose 1.0%, soybean cake powder 3.0%, dusty yeast 1.0%, MgSO4·7H2O 0.04%, FeSO4·7H2O 0.005%, CuSO4·5H2O 0.005%, CoCl2·6H2O 0.0005%, CaCO30.5%, tap water is prepared, and adjusts pH value 7.5；Zymotic fluid culture is carried out in 32 DEG C.
After fermentation, the content of Rakicidin B and B1 in zymotic fluid are detected by HPLC, detection gained goes out bacterium germination The HPLC of strain FIM02523 and mutant strain FIM-R160708 zymotic fluids figures are shown in Fig. 2 and 3 respectively, calculate product in zymotic fluid and form And content, fermentation results are shown in Table 1.
Rakicidin B potency and component ratio in 1 FIM02325 of table and mutant strain FIM-R160708 zymotic fluids
In bacterial strain FIM02523 zymotic fluids, Rakicidin B contents are 115.03mg/L, Rakicidin B1： Rakicidin B components ratio is 28.8：71.2；The content of Rakicidin B is in bacterial strain FIM-R160708 zymotic fluids 668.5mg/L Rakicidin B1：Rakicidin B components ratio is 2.4：97.6.As it can be seen that mutant strain can effectively improve hair The content of Rakicidin B in zymotic fluid, while reduce the adverse effect of Rakicidin B1.
3 mutant strain FIM-R160708 of embodiment is in 20L fermentation tank top fermentations
Seed culture medium and fermentation medium are prepared according to formula in embodiment 2.
Seed culture medium is shake-flask seed, 500ml shaking flasks per bottled liquid measure 100ml, in 32 DEG C, 250rpm cultures 48 it is small When；It is inoculated in 20L fermentation tanks (practical liquid amount 13L) and ferments according to 5% inoculum concentration later, 30 DEG C of cultures, control Tank presses 0.03-0.05Mpa, and starting rotating speed is 200rpm, and 350rpm is gradually adjusted to according to fermentation parameter DO variations after 48 hours, Fermentation process controls DO values to be more than 20% always, ventilatory capacity 1：1vvm, until fermentation ends about 96-120 hours.
Fermentation process detects the content of Rakicidins class compounds in zymotic fluid by HPLC, and final fermentation titer is： Rakicidin B1 contents are 19.5mg/L, and Rakicidin B contents are 763.2mg/L, B1：B component ratio is 2.5： 97.5。
Embodiment 4
The fermentation process of the present embodiment differs only in, with embodiment 3 according to sampling after fermentation starts 48 hours Content of reducing sugar in zymotic fluid is detected, starts flow feeding：It is less than 10g/L when sampling detects concentration of reduced sugar in zymotic fluid When, by the concentration of reduced sugar 10-15g/L in control of additive raw material zymotic fluid, the increase with product is increased with the concentration for keeping thalline It matches；The matrix composition of flow feeding includes：Glucose 25%, peptone 10%, ammonium sulfate 1%.
The content of Rakicidins class compounds in zymotic fluid, final fermentation titer are detected in fermentation process by HPLC For：Rakicidin B1 contents are 26.9mg/L, and Rakicidin B contents are 985.8mg/L, calculate B1：B component ratio is 2.7：97.3.
Embodiment 5
The fermentation process of the present embodiment is differed only in embodiment 3, in 100L fermentation tanks liquid amount be 70L, Inoculum concentration is 10%, detects content of reducing sugar in zymotic fluid according to sampling after fermentation starts 48 hours, starts according to zymotic fluid Middle content of reducing sugar sets flow feeding technique：I.e. when concentration of reduced sugar is less than 10g/L in sampling detection zymotic fluid, pass through Concentration of reduced sugar 10-15g/L in control of additive raw material zymotic fluid is matched with the increase that the concentration for keeping thalline increases with product； The matrix composition of flow feeding includes：Glucose 25%, peptone 10%, ammonium sulfate 1%.
Fermentation process detects the content of Rakicidins class compounds in zymotic fluid by HPLC, and final fermentation titer is： Rakicidin B1 contents are 33.2mg/L, and Rakicidin B contents are 1108.6mg/L, B1：B component ratio is 2.9： 97.1。
Embodiment 6
The fermentation process of the present embodiment differs only in, liquid amount is in 1000L fermentation tanks with embodiment 3 720L detects content of reducing sugar in zymotic fluid according to sampling after fermentation starts 48 hours, starts flow feeding, that is, work as sampling When detecting that concentration of reduced sugar is less than 10g/L in zymotic fluid, by the concentration of reduced sugar 10-15g/L in control of additive raw material zymotic fluid, It is matched with the increase that the concentration for keeping thalline increases with product；The matrix of flow feeding forms：Glucose 25%, albumen Peptone 10%, ammonium sulfate 1%.
Fermentation process detects the content of Rakicidins class compounds in zymotic fluid by HPLC, and final fermentation titer is： Rakicidin B1 contents are 31.2mg/L, and Rakicidin B contents are 1068.5mg/L, calculate B1：B component ratio is 2.8：97.2, fermentation process curve is as shown in Figure 4.
Obviously, the above embodiments are merely examples for clarifying the description, and is not intended to limit the embodiments. For those of ordinary skill in the art, other various forms of changes can also be made on the basis of the above description Change or change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out Among changing or changing still in the protection domain of the invention.

Claims (10)

1. a kind of sea micromonoad strain, Classification And Nomenclature is Micromonospora sp.FIM-R160708, in being preserved in State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC NO.14824.
2. application of the sea micromonoad strain described in claim 1 in fermenting and producing Rakicidins compounds.
3. application according to claim 2, which is characterized in that the Rakicidins compounds are Rakicidin B.
A kind of 4. method of fermenting and producing Rakicidin B, which is characterized in that including by the small list in ocean described in claim 1 The step of spore inoculation carries out fermented and cultured in suitable fermentation medium.
5. the method for fermenting and producing Rakicidin B according to claim 4, which is characterized in that the fermentation medium Include the component of following mass content：Soluble starch 5.0-7.0%, sucrose 0.8-1.2%, soybean cake powder 2.0-4.0%, ferment Female powder 0.8-1.2%, MgSO4·7H2O 0.03-0.05%, FeSO4·7H2O 0.004-0.006%, CuSO4·5H2O 0.004-0.006%, CoCl2·6H2O 0.0004-0.0006%, CaCO30.4-0.6%, tap water are prepared, and tune pH value is 7.5。
6. the method for fermenting and producing Rakicidin B according to claim 4 or 5, which is characterized in that the fermented and cultured The condition of step is：The bacterial strain is inoculated with the inoculum concentration of 3.0-12.0%, controls rotating speed 200-400rpm, ventilate 0.05- 1.2vvm, control fermentation process DO value are more than 20%, and fermented and cultured is carried out in 30-35 DEG C.
7. according to the method for claim 4-6 any one of them fermenting and producing Rakicidin B, which is characterized in that the hair During ferment, further include when concentration of reduced sugar is less than 10g/L in on-line period detection zymotic fluid, pass through control of additive raw material zymotic fluid In concentration of reduced sugar be 10-15g/L the step of, the feed supplement matrix includes the composition of following mass content：Glucose 25%, Peptone 10%, ammonium sulfate 1%.
8. according to the method for claim 4-7 any one of them fermenting and producing Rakicidin B, which is characterized in that the side Method further includes the inoculation the step of seed liquor culture is carried out in seed culture medium, and the seed culture medium is included such as The component of lower mass content：Soluble starch 1.5-2.5%, glucose 0.8-1.2%, dusty yeast 1.5-2.5%, peptone 0.8-1.2%, MgSO4·7H2O 0.04-0.06%, FeSO4·7H2O 0.004-0.006%, CuSO4·5H2O 0.004- 0.006%, CoCl2·6H2O 0.0004-0.0006%, CaCO30.2-0.4%, tap water are prepared, and tune pH value is 7.0- 7.5, it sterilizes and carries out seed liquor culture after 30-35 DEG C.
9. the method for fermenting and producing Rakicidin B according to claim 8, which is characterized in that the seed liquor culture Step includes shake-flask seed liquid culture and seed tank culture step.
10. according to the method for claim 4-9 any one of them fermenting and producing Rakicidin B, which is characterized in that the side Method further include by the inoculation in Gao Shi asparagines solid slope culture medium carry out activation culture the step of, the solid is oblique Face culture medium includes the component of following mass content：Soluble starch 1-3%, L- asparagine 0.04-0.06%, KNO3 0.08- 0.12%, K2HPO4·3H2O 0.04-0.06%, NaCl 0.04-0.06%, MgSO4·7H2O 0.04-0.06%, CaCO3 0.08-0.12%, agar 1-2% adjust pH value 7.5.
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