CN104846044A - Fermentation culture medium for increasing fidaxomicin yield - Google Patents

Fermentation culture medium for increasing fidaxomicin yield Download PDF

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CN104846044A
CN104846044A CN201410053563.7A CN201410053563A CN104846044A CN 104846044 A CN104846044 A CN 104846044A CN 201410053563 A CN201410053563 A CN 201410053563A CN 104846044 A CN104846044 A CN 104846044A
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fermention medium
polymeric adsorbent
medium
feldamycin
percent
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CN104846044B (en
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陈少欣
张柯
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Shanghai Institute of Pharmaceutical Industry
China State Institute of Pharmaceutical Industry
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Shanghai Institute of Pharmaceutical Industry
China State Institute of Pharmaceutical Industry
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Abstract

The present invention discloses a fermentation culture medium for increasing the fidaxomicin yield, wherein a conventional fermentation culture medium for Dactylosporangium aurantiacum culture is added with an adsorption resin with a volume percentage of 1-6% to obtain the fermentation culture medium of the present invention, the adsorption resin is any one or a plurality of materials selected HP20, HZ830, XAD-16, and XAD1600, and the pH value of the fermentation culture medium is 7.2-7.4. With the application of the fermentation culture medium of the present invention to culture the Dactylosporangium aurantiacum, the fidaxomicin yield can be substantially increased so as to substantially reduce the production cost and provide good industrial application prospects. In addition, the used adsorption resins such as HP20 and HZ830 have ideal mechanical strength and can be repeatedly recycled so as to further reduce the production cost while improve the fidaxomicin yield.

Description

A kind of fermention medium improving feldamycin output
Technical field
The present invention relates to a kind of substratum, be specifically related to a kind of fermention medium improving feldamycin output.
Background technology
Feldamycin (Fidaxomicin) is the macrolide antibiotics of a kind of eighteen ring produced by dactylosporangium aurantiacum (Dactylosporangiumaurantiacum), 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.At present industrial by fermentation process produce feldamycin time, the output reported only has 400mg/l, can't meet the demand of application.Therefore, be necessary to improve yeasting, exploitation can improve feldamycin output thus reduce costs, adapts to the substratum of suitability for industrialized production.
Summary of the invention
Technical problem to be solved by this invention cultivates dactylosporangium aurantiacum, the present situation that its feldamycin output is not high for current fermention medium, and provide a kind of substratum that can improve feldamycin output newly.Utilize fermention medium of the present invention to cultivate dactylosporangium aurantiacum, greatly can improve the fermentation unit of feldamycin, enhance productivity.
One of technical scheme provided by the invention is: a kind of fermention medium improving feldamycin output, the polymeric adsorbent gained that percent by volume is 1 ~ 6% is added in its fermention medium by the cultivation dactylosporangium aurantiacum of routine, described polymeric adsorbent is selected from HP20, HZ830, XAD-16, any one or more in XAD1600, the pH value of described fermention medium is 7.2 ~ 7.4.
In the present invention, the fermention medium of the cultivation dactylosporangium aurantiacum of described routine be this area routine for cultivating dactylosporangium aurantiacum to obtain the fermention medium of corresponding tunning, as document " RobertJ.Theriault, James P.Karwowski, et al.TIacumicin, A novel complex of18-membered macrolide antibiotics.I.Taxonomy, fermentation and antibacterialactivity.The Journal of Antibiotics.1987, 5:567-574. " in the substratum that uses of cultivation dactylosporangium aurantiacum that discloses.
In the present invention, described dactylosporangium aurantiacum, for described in the routine of this area, is preferably dactylosporangium aurantiacum NRRL18085.
In the present invention, it is the glucose of 1.5 ~ 2.5%, the bean powder of 0.5 ~ 1.5% that described fermention medium preferably includes mass percent, the beef extract of 0.2 ~ 0.4%, the soya-bean oil of 0.05 ~ 0.15%, the MgSO of 0.04 ~ 0.06% 47H 2o, the KCl of 0.02 ~ 0.04%, the K of 0.04 ~ 0.06% 2hPO 4, the CaCO of 0.2 ~ 0.4% 3, and percent by volume is the polymeric adsorbent of 3 ~ 5%, and described polymeric adsorbent is selected from HP20, HZ830, XAD-16, any one or more in XAD1600, and the pH value of described fermention medium is 7.2 ~ 7.4.
In the present invention, it is the glucose of 2%, the bean powder of 1% that described fermention medium more preferably comprises mass percent, the beef extract of 0.3%, the soya-bean oil of 0.1%, the MgSO of 0.05% 47H 2o, the KCl of 0.03%, the K of 0.05% 2hPO 4, the CaCO of 0.3% 3, and percent by volume is the polymeric adsorbent of 3 ~ 5%, described polymeric adsorbent is selected from HP20, HZ830, XAD-16, any one or more (more preferably HP20 or HZ830) in XAD1600, and the pH value of described fermention medium is 7.2 ~ 7.4.
In the present invention, it is the glucose of 2%, the bean powder of 1% that described fermention medium most preferably comprises mass percent, the beef extract of 0.3%, the soya-bean oil of 0.1%, the MgSO of 0.05% 47H 2o, the KCl of 0.03%, the K of 0.05% 2hPO 4, the CaCO of 0.3% 3, and percent by volume is the polymeric adsorbent of 4%, and described polymeric adsorbent is selected from HP20, HZ830, XAD-16, any one in XAD1600, and the pH value of described fermention medium is 7.3.
In the present invention, described polymeric adsorbent more preferably HP20 or HZ830, when polymeric adsorbent selects HP20 or HZ830, can not only improve the fermentation yield of feldamycin greatly, more polymeric adsorbent can be carried out reclaiming after fermentation ends and use with iterative cycles, thus reduce costs further.
The preparation method of fermention medium of the present invention is conventional, as long as by each component with water, is preferably distilled water and dissolves, then with high-temperature sterilization.
Two of technical scheme provided by the invention is: a kind of method of fermentative production feldamycin, and it comprises the step of producing feldamycin with prior culture media fermentation culture dactylosporangium aurantiacum.
In the present invention, when producing feldamycin with prior culture media fermentation culture dactylosporangium aurantiacum, use conventional method, be inoculated in prior culture media by dactylosporangium aurantiacum, then carry out normal fermentation.
Preferably, described method comprises step: by dactylosporangium aurantiacum seed liquor in mass ratio 5 ~ 15% inoculum size be inoculated in foregoing fermention medium, then by fermentation flask 25 ~ 30 DEG C, the shaking table of rotating speed 150 ~ 220rpm is cultivated 8 ~ 15 days.
Described method preferably also comprises the step preparing described seed liquor:
(1) be inoculated on slant medium activate producing the dactylosporangium aurantiacum of feldamycin;
(2) seed liquor is obtained activating the colony inoculation obtained in seed culture medium cultivation.
Wherein, the step preparing described seed liquor more preferably comprises:
(1) be inoculated on slant medium activate producing the dactylosporangium aurantiacum of feldamycin;
(2) seed liquor is obtained activating the colony inoculation obtained in seed culture medium cultivation; Described seed culture medium by 0.05 ~ 0.15% glucose, 2 ~ 3% starch, 0.3 ~ 0.6% extraction from yeast powder, 0.3 ~ 0.6% Tryptones, 0.2 ~ 0.4% beef extract and 0.3 ~ 0.5%CaCO 3composition, described per-cent is mass percent, and the pH of described seed culture medium is 7.2 ~ 7.4.
Best, described method comprises the steps:
(1) be inoculated in slant medium cultivates in 30 DEG C of incubators activate producing the dactylosporangium aurantiacum of feldamycin for 4-5 days, described slant medium is by 0.4% extraction from yeast powder, 1% Fructus Hordei Germinatus leaching powder, 0.4% glucose and 2.0% agar composition, described per-cent is mass percent, and the pH of described slant medium is 7.0-7.2;
(2) by activating the colony inoculation that obtains in seed culture medium, cultivating in 28 DEG C of shaking tables and obtaining seed liquor in 48 hours, described seed culture medium by 0.1% glucose, 2.4% starch, 0.5% extraction from yeast powder, 0.5% Tryptones, 0.3% beef extract and 0.4%CaCO 3composition, described per-cent is mass percent, and the pH of described seed culture medium is 7.3;
(3) by the seed liquor of (2) gained inoculation aforesaid fermentation substratum, then by fermentation flask 28 DEG C, the shaking table of rotating speed 200rpm is cultivated 12 days.
In the present invention, after step (3), also preferably include the step of results feldamycin.Described results feldamycin adopts the method for general results tunning, in fermented liquid, more preferably adds the methyl alcohol of 2 ~ 4 times of volumes, ultrasonic 20 ~ 35min, gets supernatant and get final product after centrifugal; In fermented liquid, most preferably add the methyl alcohol of 3 times of volumes, ultrasonic 30min, get supernatant after centrifugal and get final product.
The compositions such as carbon source, nitrogenous source and the inorganic salt that can be utilized can also be added in fermention medium of the present invention, as long as there is not mutual antagonistic action between each component in those compositions and prior culture media of the present invention, these compositions can add in substratum in advance once, and the high yield realizing feldamycin quantizes.
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:
Compare existing substratum, fermention medium of the present invention can improve the output of feldamycin greatly for cultivating dactylosporangium aurantiacum, the highlyest promotes 114%, thus greatly reduces production cost, has extraordinary industrial applications prospect.In addition, polymeric adsorbent HP20 and HZ830 used in the present invention can recycle and reuse repeatedly, thus reduce further production cost while raising feldamycin output.
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 mass percent.
In following embodiment, the dactylosporangium aurantiacum used is for studying DSMZ purchased from Agricultural ResearchService Culture Collection(american agriculture, ARS), be numbered the dactylosporangium aurantiacum bacterial strain of NRRL18085.
Embodiment 1
Consisting of of the fermention medium that the present embodiment uses: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil 0.1%, MgSO 47H 2o0.05%, KCl0.03%, K 2hPO 40.05%, CaCO 30.3%, HP20 resin 4%(v/v), NaOH adjusts pH to 7.3.
Before inoculation fermentation substratum, first frozen dactylosporangium aurantiacum is activated on slant medium and in seed culture medium cultivate obtain seed liquor.Described slant medium consist of extraction from yeast powder 0.4%, Fructus Hordei Germinatus leaching powder 1%, glucose 0.4%, agar 2.0%, with NaOH adjust pH to 7.0-7.2, in 121 DEG C of sterilizings 30 minutes, paved one-tenth solid slope.Bacterial classification is connected on this inclined-plane, cultivates 4-5 days at 30 DEG C of incubators, well-grown slant strains is inoculated in seed culture medium.Described seed culture medium consist of glucose 0.1%, starch 2.4%, extraction from yeast powder 0.5%, Tryptones 0.5%, beef extract 0.3%, CaCO 30.4%, NaOH adjusts pH to 7.3.Seed culture cools for subsequent use based on 121 DEG C of sterilizings after 20 minutes.Be inoculated at 28 DEG C after in seed culture medium, after the shaking table of rotating speed 200rpm cultivates 48 hours, the seed liquor obtained is for inoculation fermentation substratum.
Seed liquor is connected to fermention medium by the inoculum size of 10%, after inoculation fermentation substratum, by fermentation flask 28 DEG C, the shaking table of rotating speed 200rpm is cultivated 12 days, in fermented liquid, the methyl alcohol of 3 times of volumes is added after fermentation ends, ultrasonic 30min, get supernatant after centrifugal, HPLC measures the content of feldamycin, and its result is 1090mg/l.
Comparative example 1
The fermention medium of feldamycin is produced by prior art preparation, it discloses in document " Robert J.Theriault; James P.Karwowski; et al.TIacumicin; A novel complex of18-membered macrolide antibiotics.I.Taxonomy; fermentation and antibacterialactivity.The Journal of Antibiotics.1987; 5:567-574. ", it consists of glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil 0.1%, MgSO 47H 2o0.05%, KCl0.03%, K 2hPO 40.05%, CaCO 30.3%, adjust pH to 7.3 with NaOH.
Seed liquor embodiment 1 obtained is inoculated in fermention medium with the inoculum size of 10%, and the control condition in fermenting process is all with embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, and its result is 779mg/l.
This comparative example is as blank, and each embodiment compares.
Can find out, the output of embodiment 1 feldamycin comparatively blank improves 40%.
Embodiment 2
Consisting of of the fermention medium that the present embodiment uses: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil 0.1%, MgSO 47H 2o0.05%, KCl0.03%, K 2hPO 40.05%, CaCO 30.3%, XAD-1600 resin 4%(v/v), NaOH adjusts pH to 7.3.
Seed liquor embodiment 1 obtained is inoculated in fermention medium with the inoculum size of 10%, and the control condition in fermenting process is all with embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, and its result is 1288mg/, and comparatively blank improves 65%.
Embodiment 3
Consisting of of the fermention medium that the present embodiment uses: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil 0.1%, MgSO 47H 2o0.05%, KCl0.03%, K 2hPO 40.05%, CaCO 30.3%, HZ830 resin 4%(v/v), NaOH adjusts pH to 7.3.
Seed liquor embodiment 1 obtained is inoculated in fermention medium with the inoculum size of 10%, and the control condition in fermenting process is all with embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, and its result is 974mg/l, and comparatively blank improves 25%.
Embodiment 4
Consisting of of the fermention medium that the present embodiment uses: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil 0.1%, MgSO 47H 2o0.05%, KCl0.03%, K 2hPO 40.05%, CaCO 30.3%, XAD-16 resin 4%(v/v), NaOH adjusts pH to 7.3.
Seed liquor embodiment 1 obtained is inoculated in fermention medium with the inoculum size of 10%, and the control condition in fermenting process is all with embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, and its result is 822mg/, and comparatively blank improves 5%.
Embodiment 5 ~ 10
Consisting of of the fermention medium that embodiment 5 ~ 10 uses: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil 0.1%, MgSO 47H 2o0.05%, KCl0.03%, K 2hPO 40.05%, CaCO 30.3%, and HP20 resin, wherein, be followed successively by 1%(v/v respectively from the addition of embodiment 5 ~ 10, HP20 resin), 2%(v/v), 3%(v/v) and, 4%(v/v), 5%(v/v) and 6%(v/v), NaOH adjusts pH to 7.3.
Seed liquor embodiment 1 obtained is inoculated in fermention medium with the inoculum size of 10%, and the control condition in fermenting process is all with embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, and its result is followed successively by 806mg/l respectively from embodiment 5 ~ 10,1054mg/l, 1086mg/l, 1090mg/l, 1615mg/l and 1669mg/l, comparatively blank improves 3% respectively, 35%, 39%, 40%, 107% and 114%.
Embodiment 11
Consisting of of the fermention medium that the present embodiment uses: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil 0.1%, MgSO 47H 2o0.05%, KCl0.03%, K 2hPO 40.05%, CaCO 30.3%, embodiment 1 uses the HP20 resin 4%(v/v reclaiming gained), NaOH adjusts pH to 7.3.Wherein, the process that HP20 resin reclaims is: by the filtering fermentation liquor containing HP20 resin, collect HP20 resin, clean with the thalline of deionized water by resin surface, then three times are washed with 95% alcohol immersion HP20 resin, with deionized water, ethanol is cleaned afterwards, obtain can recirculation use HP20 resin.
Seed liquor embodiment 1 obtained is inoculated in fermention medium with the inoculum size of 10%, and the control condition in fermenting process is all with embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, and its result is 1216mg/l, and comparatively blank improves 56%.
Embodiment 12
Consisting of of the fermention medium that the present embodiment uses: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil 0.1%, MgSO 47H 2o0.05%, KCl0.03%, K 2hPO 40.05%, CaCO 30.3%, embodiment 3 uses the HZ830 resin 4%(v/v reclaiming gained), NaOH adjusts pH to 7.3.Wherein, the process that HZ830 resin reclaims is: by the filtering fermentation liquor containing HZ830 resin, collect HZ830 resin, clean with the thalline of deionized water by resin surface, then three times are washed with 95% alcohol immersion HZ830 resin, with deionized water, ethanol is cleaned afterwards, obtain can recirculation use HZ830 resin.
Seed liquor embodiment 1 obtained is inoculated in fermention medium with the inoculum size of 10%, and the control condition in fermenting process is all with embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, and its result is 1015mg/l, and comparatively blank improves 30%.
Comparative example 2
Consisting of of the fermention medium that the present embodiment uses: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil 0.1%, MgSO 47H 2o0.05%, KCl0.03%, K 2hPO 40.05%, CaCO 30.3%, SP850 resin 4%(v/v), NaOH adjusts pH to 7.3.
Seed liquor embodiment 1 obtained is inoculated in fermention medium with the inoculum size of 10%, and the control condition in fermenting process is all with embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, and its result is 510mg/l, and comparatively blank reduces 34%.
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. one kind is improved the fermention medium of feldamycin output, it is characterized in that, it adds by the fermention medium of the cultivation dactylosporangium aurantiacum (Dactylosporangium aurantiacum) of routine the polymeric adsorbent gained that percent by volume is 1 ~ 6%, described polymeric adsorbent is selected from HP20, HZ830, XAD-16, any one or more in XAD1600, the pH value of described fermention medium is 7.2 ~ 7.4.
2. fermention medium as claimed in claim 1, it is characterized in that, described dactylosporangium aurantiacum is dactylosporangium aurantiacum NRRL18085.
3. fermention medium as claimed in claim 1, it is characterized in that, it is the glucose of 1.5 ~ 2.5%, the bean powder of 0.5 ~ 1.5% that described fermention medium comprises mass percent, the beef extract of 0.2 ~ 0.4%, the soya-bean oil of 0.05 ~ 0.15%, the MgSO of 0.04 ~ 0.06% 47H 2o, the KCl of 0.02 ~ 0.04%, the K of 0.04 ~ 0.06% 2hPO 4, the CaCO of 0.2 ~ 0.4% 3, and percent by volume is the polymeric adsorbent of 1 ~ 6%, and described polymeric adsorbent is selected from HP20, HZ830, XAD-16, any one or more in XAD1600, and the pH value of described fermention medium is 7.2 ~ 7.4.
4. fermention medium as claimed in claim 1, it is characterized in that, it is the glucose of 2%, the bean powder of 1% that described fermention medium comprises mass percent, the beef extract of 0.3%, the soya-bean oil of 0.1%, the MgSO of 0.05% 47H 2o, the KCl of 0.03%, the K of 0.05% 2hPO 4, the CaCO of 0.3% 3, and percent by volume is the polymeric adsorbent of 3 ~ 5%, and described polymeric adsorbent is selected from HP20, HZ830, XAD-16, any one or more in XAD1600, and the pH value of described fermention medium is 7.2 ~ 7.4.
5. fermention medium as claimed in claim 1, it is characterized in that, it is the glucose of 2%, the bean powder of 1% that described fermention medium comprises mass percent, the beef extract of 0.3%, the soya-bean oil of 0.1%, the MgSO of 0.05% 47H 2o, the KCl of 0.03%, the K of 0.05% 2hPO 4, the CaCO of 0.3% 3, and percent by volume is the polymeric adsorbent of 4%, and described polymeric adsorbent is selected from HP20, HZ830, XAD-16, any one in XAD1600, and the pH value of described fermention medium is 7.3.
6. fermention medium as claimed in claim 1, it is characterized in that, described fermention medium is the glucose of 2%, the bean powder of 1% by mass percent, the beef extract of 0.3%, the soya-bean oil of 0.1%, the MgSO of 0.05% 47H 2o, the KCl of 0.03%, the K of 0.05% 2hPO 4, the CaCO of 0.3% 3, and percent by volume is the polymeric adsorbent composition of 4%, described polymeric adsorbent is selected from HP20, HZ830, XAD-16, any one in XAD1600, and the pH value of described fermention medium is 7.3.
7. fermention medium as claimed in claim 1, it is characterized in that, described polymeric adsorbent is HP20 or HZ830.
8. a method for fermentative production feldamycin, is characterized in that, it comprises the step of producing feldamycin with the fermention medium fermentation culture dactylosporangium aurantiacum described in any one of claim 1 ~ 7.
9. method as claimed in claim 8, it is characterized in that, it comprises step: by dactylosporangium aurantiacum seed liquor in mass ratio 5 ~ 15% inoculum size be inoculated in fermention medium as described in any one of claim 1 ~ 7, then by fermentation flask 25 ~ 30 DEG C, the shaking table of rotating speed 150 ~ 220rpm is cultivated 8 ~ 15 days.
10. method as claimed in claim 9, it is characterized in that, described method also comprises the step preparing described seed liquor:
(1) be inoculated on slant medium activate producing the dactylosporangium aurantiacum of feldamycin;
(2) seed liquor is obtained activating the colony inoculation obtained in seed culture medium cultivation; Described seed culture medium by 0.05 ~ 0.15% glucose, 2 ~ 3% starch, 0.3 ~ 0.6% extraction from yeast powder, 0.3 ~ 0.6% Tryptones, 0.2 ~ 0.4% beef extract and 0.3 ~ 0.5%CaCO 3composition, described per-cent is mass percent, and the pH of described seed culture medium is 7.2 ~ 7.4.
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Publication number Priority date Publication date Assignee Title
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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