CN104946708A - Fermentation medium producing fidaxomicin and fermentation method - Google Patents
Fermentation medium producing fidaxomicin and fermentation method Download PDFInfo
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Abstract
The invention discloses a fermentation medium producing an antibiotic fidaxomicin by using fermentation production of dactylosporangium aurantiacum. The fermentation medium comprises the following raw material components: 5-13 percent of an organic carbon source, 1-3.5 percent of an organic nitrogen source and an inorganic salt, wherein the percentage refers to the mass percentage relative to the total mass of the fermentation medium; the organic carbon source comprises malt sugar and glycerin; the organic nitrogen source comprises one or more of cottonseed cake powder, soybean cake powder, polypeptone, corn gluten, yeast extraction powder and yeast powder; and the inorganic salt comprises CaCO3 and/or MgSO4.7H2O. The invention also discloses a fermentation method for producing fidaxomicin by using fermentation production of dactylosporangium aurantiacum. The method comprises the following steps: activating bacteria and inoculating (respectively primary inoculation and secondary inoculation in the two methods); and inoculating the obtained seed solution in the fermentation medium for performing fermentation cultivation, thereby obtaining the product. According to the fermentation medium and fermentation method disclosed by the invention, the fermentation unit of fidaxomicin is greatly improved, and the industrial production is promoted.
Description
Technical field
The present invention relates to industrial microbial technology field, particularly relate to a kind of fermention medium of dactylosporangium aurantiacum fermentative production microbiotic feldamycin and corresponding fermentation process.
Background technology
Feldamycin (Fidaxomicin) is a kind of macrolide antibiotics of eighteen ring, there is unique structure and biological activity, be mainly used in treating the diarrhoea caused by clostridium difficile (Clostridium difficile) infection, have a good application prospect.The chemosynthesis of current feldamycin has no report, is mainly produced by microbial fermentation processes.Feldamycin can be produced by dactylosporangium aurantiacum (Dactylosporangium aurantiacum) or strains A ctinoplanes deccanesis (ATCC21983) metabolism.In US Patent No. 7507564, feldamycin is produced by the subspecies hamdenensis metabolism of dactylosporangium aurantiacum, and the fermentation unit disclosed in it only reaches 250mg/l.Therefore, be necessary to improve yeasting, exploitation can improve feldamycin output thus reduce costs, adapts to fermention medium and the fermentation process of suitability for industrialized production.
Summary of the invention
The technical problem to be solved in the present invention is for the low defect of the method fermentation unit of existing microbial metabolism production feldamycin, a kind of fermention medium of dactylosporangium aurantiacum fermentative production microbiotic feldamycin and corresponding fermentation process are provided, it can improve the fermentation unit of feldamycin greatly, enhances productivity.
The present invention solves the problems of the technologies described above one of adopted technical scheme: a kind of fermention medium of dactylosporangium aurantiacum fermentative production microbiotic feldamycin, and its feed composition comprises organic carbon source, organic nitrogen source and inorganic salt; Wherein, organic carbon source comprises maltose and glycerine, and the consumption of organic carbon source is 5 ~ 13%; Organic nitrogen source comprise in cottonseed meal, soybean cake powder, polyprotein peptone, seitan powder, extraction from yeast powder and yeast powder one or more, the consumption of organic nitrogen source is 1 ~ 3.5%; Inorganic salt comprise CaCO
3and/or MgSO
47H
2o; Per-cent is the mass percent relative to fermention medium total mass.
Described organic carbon source can also comprise various organic carbon sources conventional in fermention medium.
The consumption of described organic carbon source preferably 8.0 ~ 13.0%, more preferably 10.0 ~ 13.0%, per-cent is the mass percent relative to fermention medium total mass.
The consumption of described maltose preferably 4.0 ~ 9.0%, more preferably 6.0 ~ 9.0%, per-cent is the mass percent relative to fermention medium total mass.
The consumption of described glycerine preferably 1.0 ~ 4.0%, more preferably 2.0 ~ 4.0%, per-cent is the mass percent relative to fermention medium total mass.
Described organic nitrogen source preferably comprises cottonseed meal, soybean cake powder and yeast powder, be more preferably comprise the cottonseed meal of 1.0 ~ 2.0%, the soybean cake powder of 1.0 ~ 2.0% and 1.0 ~ 2.0% yeast powder, per-cent is the mass percent relative to fermention medium total mass.
Described organic nitrogen source preferably comprises the one in 2.0% cottonseed meal, 2.0% soybean cake powder, 2.0% polyprotein peptone, 2.0% seitan powder, 2.0% yeast powder or 2.0% extraction from yeast powder, and per-cent is the mass percent relative to fermention medium total mass.
The consumption of described inorganic salt preferably 0.2 ~ 0.6%, more preferably 0.4 ~ 0.6%, per-cent is the mass percent relative to fermention medium total mass.
Described inorganic salt preferably comprise CaCO
3and MgSO
47H
2o.Wherein, CaCO
3consumption is preferably 0.3 ~ 0.5%, is more preferably 0.35 ~ 0.45%, most preferably is 0.4%; MgSO
47H
2the consumption of O is preferably 0.1 ~ 0.4%, is more preferably 0.15 ~ 0.25%, most preferably is 0.2%; Per-cent is the mass percent relative to fermention medium total mass.
In fermention medium of the present invention, the same with the fermention medium of routine, preferably also can comprise CaCO
3and MgSO
47H
2this area conventional kind outside O and the various inorganic salt of consumption.
Fermention medium of the present invention, as the fermention medium of routine, preferably can also comprise the defoamer of this area conventional kind and consumption.Wherein, defoamer is preferably bubble enemy, and bubble enemy consumption is preferably 0.05 ~ 0.2%, and better is 0.1%, and per-cent is the mass percent relative to fermention medium total mass.
The pH of fermention medium of the present invention is preferably 6.8 ~ 7.5, is more preferably 7.1 ~ 7.4, most preferably is 7.3.
In fermention medium of the present invention, preferably do not comprise polymeric adsorbent.
In fermention medium of the present invention, preferably do not comprise emulsifying agent.
The present invention solves the problems of the technologies described above two of adopted technical scheme: a kind of fermentation process of dactylosporangium aurantiacum fermentative production microbiotic feldamycin, comprise the following steps: dactylosporangium aurantiacum is activated, then be inoculated in seed culture medium and obtain seed liquor, seed liquor is seeded in aforesaid fermention medium, fermentation culture.
In described fermentation process, the inoculum size of seed liquor preferably 5 ~ 25%, more preferably 5 ~ 15%, most preferably 10%, per-cent is the volume percent relative to fermention medium cumulative volume.
In described fermentation process, the leavening temperature of fermentation culture preferably 25 ~ 32 DEG C, more preferably 27 ~ 32 DEG C, most preferably 28 DEG C.
In described fermentation process, the fermentation period of fermentation culture preferably 8 ~ 15 days, more preferably 10 ~ 15 days, most preferably 12 days.
In described fermentation process, the container of fermentation culture is preferably fermentation flask or fermentor tank, is more preferably fermentation flask.
In described fermentation process, fermentation culture is preferably carried out on shaking table, and the rotating speed preferably 100 ~ 400rpm of shaking table, more preferably 150 ~ 300rpm, most preferably is 200rpm.
In described fermentation process, seed liquor can obtain by this area routine operation, preferably according to step generation below:
(1) dactylosporangium aurantiacum producing feldamycin is inoculated in slant medium postactivated, the feed composition of slant medium comprises extraction from yeast powder 0.1 ~ 0.4%, Fructus Hordei Germinatus leaching powder 0.1 ~ 1%, glucose 0.1 ~ 0.4% and agar 0.1 ~ 2.0%, per-cent is the mass percent relative to slant medium total mass, and the pH of described slant medium is 7.0 ~ 7.3;
(2) obtain seed liquor by activating the colony inoculation obtained in seed culture medium, the feed composition of seed culture medium comprises glucose 0.05 ~ 0.1%, starch 1 ~ 2.4%, extraction from yeast powder 01 ~ 0.5%, Tryptones 0.1 ~ 0.5%, beef extract 0.1 ~ 0.3% and CaCO
30.1 ~ 0.4%, per-cent is the mass percent relative to seed culture medium total mass, and the pH of described seed culture medium is 7.0 ~ 7.3.
The present invention solves the problems of the technologies described above three of adopted technical scheme: a kind of fermentation process of dactylosporangium aurantiacum fermentative production microbiotic feldamycin, comprise the following steps: dactylosporangium aurantiacum is activated, then be inoculated in seed culture medium and obtain primary seed solution, secondary seed solution is obtained after primary seed solution being inoculated in seed culture medium, secondary seed solution is seeded in aforesaid fermention medium, fermentation culture.
In described fermentation process, the inoculum size of primary seed solution preferably 1 ~ 10%, more preferably 3 ~ 10%, most preferably 5%, per-cent is the volume percent relative to seed culture medium cumulative volume.
In described fermentation process, the inoculum size of secondary seed solution preferably 5 ~ 25%, more preferably 5 ~ 15%, most preferably 10%, per-cent is the volume percent relative to fermention medium cumulative volume.
In described fermentation process, the leavening temperature of fermentation culture preferably 25 ~ 32 DEG C, more preferably 27 ~ 32 DEG C, most preferably 28 DEG C.
In described fermentation process, the fermentation period of fermentation culture preferably 8 ~ 15 days, more preferably 10 ~ 15 days, most preferably 12 days.
In described fermentation process, the container of fermentation culture is preferably fermentation flask or fermentor tank, is more preferably fermentor tank.
In described fermentation process, the control dissolved oxygen of fermentation culture is preferably 1 ~ 30%, and more preferably 10 ~ 30%, most preferably 20 ~ 30%, per-cent is the volume percent relative to fermention medium cumulative volume.
In described fermentation process, fermentation culture is preferably carried out on shaking table, and shaking speed is 100 ~ 400rpm preferably, more preferably 150 ~ 300rpm, most preferably is 200rpm.
In described fermentation process, secondary seed solution can obtain by this area routine operation, preferably according to step generation below:
(1) dactylosporangium aurantiacum producing feldamycin is inoculated in slant medium postactivated, the feed composition of slant medium comprises extraction from yeast powder 0.1 ~ 0.4%, Fructus Hordei Germinatus leaching powder 0.1 ~ 1%, glucose 0.1 ~ 0.4% and agar 0.1 ~ 2.0%, per-cent is the mass percent relative to slant medium total mass, and the pH of described slant medium is 7.0 ~ 7.3;
(2) primary seed solution is obtained activating the colony inoculation obtained in seed culture medium, the feed composition of seed culture medium comprises glucose 0.05 ~ 0.1%, starch 1 ~ 2.4%, extraction from yeast powder 01 ~ 0.5%, Tryptones 0.1 ~ 0.5%, beef extract 0.1 ~ 0.3% and CaCO
30.1 ~ 0.4%, per-cent is the mass percent relative to seed culture medium total mass, and the pH of described seed culture medium is 7.0 ~ 7.3;
(3) primary seed solution is inoculated in seed culture medium and obtains secondary seed solution, the feed composition of seed culture medium comprises glucose 0.05 ~ 0.1%, starch 1 ~ 2.4%, extraction from yeast powder 01 ~ 0.5%, Tryptones 0.1 ~ 0.5%, beef extract 0.1 ~ 0.3% and CaCO
30.1 ~ 0.4%, per-cent is the mass percent relative to seed culture medium total mass, and the pH of described seed culture medium is 7.0 ~ 7.3.
In described in the present invention two kind of fermentation process, the feldamycin that fermentation culture obtains can adopt the method for conventional results tunning to carry out aftertreatment, preferably by ultrasonic after the fermention medium containing feldamycin product and methanol mixed, gets supernatant and get final product after centrifugal.Described methyl alcohol volume is preferably 2 ~ 4 times relative to the fermention medium volume containing feldamycin product, most preferably is 3 times; Fermention medium containing feldamycin product and methanol mixed mode preferably add methyl alcohol in the fermention medium containing feldamycin product; Ultrasonic time is preferably 20 ~ 40min, most preferably is 30min.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is: fermention medium cost of the present invention is low, substantially increases the fermentation unit of feldamycin, improves production efficiency, is suitable for carrying out suitability for industrialized production.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
In following embodiment, described per-cent if no special instructions, all refers to the mass percent relative to fermention medium total mass.
In following embodiment, the dactylosporangium aurantiacum used is for studying DSMZ purchased from Agricultural Research Service Culture Collection(american agriculture, ARS), be numbered the dactylosporangium aurantiacum bacterial strain of NRRL18085.
Embodiment 1
Get a strain dactylosporangium aurantiacum and be inoculated in slant medium, 30 DEG C cultivate activation in 4 days after, be inoculated in seed culture medium, liquid amount is 20ml/250ml, 28 DEG C, the rotary shaker of 200rmp cultivates 48 hours to obtain seed liquor.Seed liquor with 10% inoculum size (per-cent is the volume percent relative to fermention medium cumulative volume) to be inoculated in liquid amount be ferment in the 250ml fermentation shake flask of 30ml, leavening temperature 28 DEG C, fermentation period 12 days.To add the long-pending methyl alcohol of triploid in fermention medium containing product feldamycin, centrifuging and taking supernatant after ultrasonic 30 minutes after fermentation ends, the output that HPLC measures feldamycin reaches 276mg/l.
The feed composition of slant medium used comprises extraction from yeast powder 0.4%, and Fructus Hordei Germinatus leaching powder 1%, glucose 0.4% and agar 2.0%, adjust pH to 7.2, per-cent is the mass percent relative to slant medium total mass.
The feed composition of seed culture medium used comprises glucose 0.1%, starch 2.4%, extraction from yeast powder 0.5%, Tryptones 0.5%, beef extract 0.3% and CaCO
30.4%, adjust pH to 7.3, per-cent is the mass percent relative to seed culture medium total mass.
The feed composition of fermention medium used comprises maltose 4.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Embodiment 2
The feed composition of fermention medium comprises maltose 6.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size is inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 530mg/l.
Embodiment 3
The feed composition of fermention medium comprises maltose 7.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size is inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 440mg/l.
Embodiment 4
The feed composition of fermention medium comprises maltose 8.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size is inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 678mg/l.
Embodiment 5
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size is inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 854mg/l.
Embodiment 6
The feed composition of fermention medium comprises maltose 9.0%, glycerine 1.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size is inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 384mg/l.
Embodiment 7
The feed composition of fermention medium comprises maltose 9.0%, glycerine 3.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size is inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 1126mg/l.
Embodiment 8
The feed composition of fermention medium composition comprises maltose 9.0%, glycerine 4.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size is inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 1234mg/l.
Embodiment 9
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 2.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size is inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 811mg/l.
Embodiment 10
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, seitan powder 2.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size is inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 380mg/l.
Embodiment 11
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, polyprotein peptone 2.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size is inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 554mg/l.
Embodiment 12
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, soybean cake powder 2.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size is inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 508mg/l.
Embodiment 13
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, extraction from yeast powder 2.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size is inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 394mg/l.
Embodiment 14
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, yeast powder 2.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size is inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 489mg/l.
Embodiment 15
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 5%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 832mg/l.
Embodiment 16
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 15%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 889mg/l.
Embodiment 17
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 20%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 789mg/l.
Embodiment 18
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 25%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 736mg/l.
Embodiment 19
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 6.8.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 816mg/l.
Embodiment 20
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.5.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 788mg/l.
Embodiment 21
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 25 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 547mg/l.
Embodiment 22
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 32 DEG C, fermentation period 12 days, other condition is with embodiment 1, and the output of feldamycin reaches 768mg/l.
Embodiment 23
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 8 days, other condition is with embodiment 1, and the output of feldamycin reaches 320mg/l.
Embodiment 24
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 9 days, other condition is with embodiment 1, and the output of feldamycin reaches 345mg/l.
Embodiment 25
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 10 days, other condition is with embodiment 1, and the output of feldamycin reaches 648mg/l.
Embodiment 26
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 11 days, other condition is with embodiment 1, and the output of feldamycin reaches 835mg/l.
Embodiment 27
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 13 days, other condition is with embodiment 1, and the output of feldamycin reaches 902mg/l.
Embodiment 28
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 14 days, other condition is with embodiment 1, and the output of feldamycin reaches 1050mg/l.
Embodiment 29
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, adjusts pH to 7.3.
Be volume percent relative to fermention medium cumulative volume according to the obtained seed liquor of embodiment 1 with 10%(per-cent) inoculum size be inoculated in fermention medium, leavening temperature 28 DEG C, fermentation period 15 days, other condition is with embodiment 1, and the output of feldamycin reaches 782mg/l.
Embodiment 30
Get a strain dactylosporangium aurantiacum (Dactylosporangium aurantiacum) NRRL18085 and be inoculated in slant medium, seed culture medium is inoculated in after 30 DEG C of cultivations activation in 4 days, liquid amount is 20ml/250ml, 28 DEG C, the rotary shaker of 200rmp cultivates 48 hours to obtain primary seed solution.Primary seed solution is the volume percent relative to seed culture medium cumulative volume with 5%(per-cent) to be inoculated in liquid amount be in the 750ml seed culture medium of 100ml, secondary seed solution cultivated 48 hours to obtain by 28 DEG C of shaking tables.Secondary seed solution is the volume percent relative to fermention medium cumulative volume with 10%(per-cent) inoculum size be inoculated in liquid amount be 2L 5L fermentation cylinder for fermentation cultivate, leavening temperature 28 DEG C, fermentation period 10 days, controlling dissolved oxygen in fermenting process at 20%(per-cent is volume percent relative to fermention medium cumulative volume).To add the long-pending methyl alcohol of triploid in fermention medium, centrifuging and taking supernatant after ultrasonic 30 minutes after fermentation ends, the output that HPLC measures feldamycin reaches 1222mg/l.
The feed composition of slant medium used comprises extraction from yeast powder 0.4%, and Fructus Hordei Germinatus leaching powder 1%, glucose 0.4% and agar 2.0%, adjust pH to 7.2.
The feed composition of seed culture medium used comprises glucose 0.1%, starch 2.4%, extraction from yeast powder 0.5%, Tryptones 0.5%, beef extract 0.3% and CaCO
30.4%, adjust pH to 7.3.
The feed composition of fermention medium comprises maltose 6.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, bubble enemy 0.1%, adjusts pH to 7.3.
Embodiment 31
The feed composition of fermention medium comprises maltose 7.5%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, bubble enemy 0.1%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained secondary seed solution of embodiment 30 with 10%(per-cent) inoculum size is inoculated in fermention medium and carries out 5L ferment tank, leavening temperature 28 DEG C, fermentation period 10 days, controlling dissolved oxygen 20%(per-cent in fermenting process is volume percent relative to fermention medium cumulative volume), other condition is with embodiment 30, and the output of feldamycin reaches 1684mg/l.
Embodiment 32
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, bubble enemy 0.1%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained secondary seed solution of embodiment 30 with 10%(per-cent) inoculum size is inoculated in fermention medium and carries out 5L ferment tank, leavening temperature 28 DEG C, fermentation period 10 days, controlling dissolved oxygen 20%(per-cent in fermenting process is volume percent relative to fermention medium cumulative volume), other condition is with embodiment 30, and the output of feldamycin reaches 1897mg/l.
Embodiment 33
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, bubble enemy 0.1%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained secondary seed solution of embodiment 30 with 10%(per-cent) inoculum size is inoculated in fermention medium and carries out 5L ferment tank, leavening temperature 28 DEG C, fermentation period 10 days, controlling dissolved oxygen 30%(per-cent in fermenting process is volume percent relative to fermention medium cumulative volume), other condition is with embodiment 30, and the output of feldamycin reaches 2219mg/l.
Comparative example 1
The feed composition of fermention medium comprises maltose 9.0%, glycerine 2.0%, cottonseed meal 1.0%, soybean cake powder 1.5%, yeast powder 1.0%, CaCO
30.4% and MgSO
47H
2o0.2%, bubble enemy 0.1%, adjusts pH to 7.3.
Being volume percent relative to fermention medium cumulative volume according to the obtained secondary seed solution of embodiment 30 with 10%(per-cent) inoculum size is inoculated in fermention medium and carries out 5L ferment tank, leavening temperature 28 DEG C, fermentation period 10 days, controlling dissolved oxygen 0.5%(per-cent in fermenting process is volume percent relative to fermention medium cumulative volume), other condition is with embodiment 30, and the output of feldamycin reaches 175mg/l.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. the fermention medium with dactylosporangium aurantiacum fermentative production microbiotic feldamycin, it is characterized in that: its feed composition comprises the organic carbon source of 5 ~ 13%, the organic nitrogen source of 1 ~ 3.5% and inorganic salt, per-cent is the mass percent relative to fermention medium total mass; Wherein, organic carbon source comprises maltose and glycerine, organic nitrogen source comprise in cottonseed meal, soybean cake powder, polyprotein peptone, seitan powder, extraction from yeast powder and yeast powder one or more, inorganic salt comprise CaCO
3and/or MgSO
47H
2o.
2. fermention medium as claimed in claim 1, is characterized in that: the consumption of described organic carbon source is 8.0 ~ 13.0%, preferably 10.0 ~ 13.0%, and per-cent is the mass percent relative to fermention medium total mass.
3. fermention medium as claimed in claim 1 or 2, is characterized in that: the consumption of described maltose is 4.0 ~ 9.0%, preferably 6.0 ~ 9.0%; The consumption of described glycerine is 1.0 ~ 4.0%, preferably 2.0 ~ 4.0%; Per-cent is the mass percent relative to fermention medium total mass.
4. fermention medium as claimed in claim 1 or 2, it is characterized in that: described organic nitrogen source comprises cottonseed meal, soybean cake powder and yeast powder, preferably comprise the cottonseed meal of 1.0 ~ 2.0%, the soybean cake powder of 1.0 ~ 2.0% and 1.0 ~ 2.0% yeast powder, per-cent is the mass percent relative to fermention medium total mass.
5. fermention medium as claimed in claim 1 or 2, it is characterized in that: described organic nitrogen source is 2.0 ~ 3.5% cottonseed meals, 2.0 ~ 3.5% soybean cake powders, 2.0 ~ 3.5% polyprotein peptones, 2.0 ~ 3.5% seitan powder, 2.0% ~ 3.5 yeast powder or 2.0% ~ 3.5 extraction from yeast powder, and per-cent is the mass percent relative to fermention medium total mass; The pH of described fermention medium is 6.8 ~ 7.5, preferably 7.1 ~ 7.4.
6. the fermentation process with dactylosporangium aurantiacum fermentative production microbiotic feldamycin, it is characterized in that comprising the following steps: be inoculated in seed culture medium after being activated by dactylosporangium aurantiacum and obtain seed liquor, seed liquor is seeded in the fermention medium as described in any one of Claims 1 to 5, fermentation culture.
7. the fermentation process with dactylosporangium aurantiacum fermentative production microbiotic feldamycin, it is characterized in that comprising the following steps: be inoculated in seed culture medium after being activated by dactylosporangium aurantiacum and obtain primary seed solution, primary seed solution obtains secondary seed solution after being inoculated in seed culture medium, secondary seed solution is seeded in the fermention medium as described in any one of Claims 1 to 5, controlling dissolved oxygen is 1 ~ 30%, per-cent is the volume percent relative to fermention medium cumulative volume, fermentation culture.
8. fermentation process as claimed in claims 6 or 7, is characterized in that: the inoculum size when seed liquor of described fermentation culture or secondary seed solution inoculation is 5 ~ 25%, preferably 5 ~ 15%, and per-cent is the volume percent relative to fermention medium cumulative volume.
9. fermentation process as claimed in claims 6 or 7, is characterized in that: the leavening temperature of described fermentation culture is 25 ~ 32 DEG C, preferably 27 ~ 32 DEG C; The fermentation period of described fermentation culture is 8 ~ 15 days, preferably 10 ~ 15 days.
10. fermentation process as claimed in claim 7, is characterized in that: the control dissolved oxygen of described fermentation culture is 10 ~ 30%, preferably 20 ~ 30%, and per-cent is the volume percent relative to fermention medium cumulative volume.
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| CN109136286A (en) * | 2017-06-27 | 2019-01-04 | 淡江大学 | culture medium composition for producing α -glucosidase inhibitor by fermentation of paenibacillus |
| CN107236686B (en) * | 2017-06-21 | 2019-10-15 | 杭州华东医药集团新药研究院有限公司 | A kind of dactylosporangium aurantiacum and its application in regulating microorganism metabolism object feldamycin |
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| CN107236686B (en) * | 2017-06-21 | 2019-10-15 | 杭州华东医药集团新药研究院有限公司 | A kind of dactylosporangium aurantiacum and its application in regulating microorganism metabolism object feldamycin |
| CN109136286A (en) * | 2017-06-27 | 2019-01-04 | 淡江大学 | culture medium composition for producing α -glucosidase inhibitor by fermentation of paenibacillus |
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