CN104846044B - A kind of fermentation medium improving feldamycin yield - Google Patents
A kind of fermentation medium improving feldamycin yield Download PDFInfo
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- CN104846044B CN104846044B CN201410053563.7A CN201410053563A CN104846044B CN 104846044 B CN104846044 B CN 104846044B CN 201410053563 A CN201410053563 A CN 201410053563A CN 104846044 B CN104846044 B CN 104846044B
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Abstract
The invention discloses a kind of fermentation mediums for improving feldamycin yield, it is added obtained by the absorption resin that percentage by volume is 1~6% in fermentation medium by conventional culture dactylosporangium aurantiacum, the absorption resin is selected from HP20, HZ830, XAD-16, XAD1600 is any one or more of, and the pH value of the fermentation medium is 7.2~7.4.Fermentation medium of the invention can greatly improve the yield of feldamycin for cultivating dactylosporangium aurantiacum, thus greatly reduce production cost, have extraordinary industrial applications prospect.In addition, absorption resin HP20 and HZ830 used in the present invention have ideal mechanical strength, can recycle repeatedly, to further reduced production cost while improving feldamycin yield.
Description
Technical field
The present invention relates to a kind of culture mediums, and in particular to a kind of fermentation medium for improving feldamycin yield.
Background technique
Feldamycin (Fidaxomicin) is by dactylosporangium aurantiacum (Dactylosporangium aurantiacum)
A kind of macrolide antibiotics of the eighteen membered ring generated has unique structure and bioactivity, is mainly used for treating difficult
The caused diarrhea of difficult clostridium (Clostridium difficile) infection, has a good application prospect.It is industrial at present logical
When everfermentation method produces feldamycin, reported yield only has 400mg/l, is not met by the demand of application.Therefore, have
Necessity improves yeasting, and exploitation can be improved feldamycin yield, the culture to reduce cost, adapt to industrialized production
Base.
Summary of the invention
It is non-to reach the technical problem to be solved by the present invention is to be directed to current fermentation medium culture dactylosporangium aurantiacum
The not high status of mycin yield, and a kind of new culture medium that can be improved feldamycin yield is provided.Utilize hair of the invention
Ferment culture medium culture dactylosporangium aurantiacum can greatly improve the fermentation unit of feldamycin, improve production efficiency.
Technical solution provided by the invention first is that: it is a kind of improve feldamycin yield fermentation medium, by routine
Culture dactylosporangium aurantiacum fermentation medium in the absorption obtained by the absorption resin that percentage by volume is 1~6% is added
Resin is selected from HP20, HZ830, XAD-16, and XAD1600 is any one or more of, and the pH value of the fermentation medium is 7.2~
7.4。
In the present invention, the fermentation medium of the conventional culture dactylosporangium aurantiacum is this field routine for training
Dactylosporangium aurantiacum is supported to obtain the fermentation medium of corresponding tunning, such as document " Robert J.Theriault, James
P.Karwowski,et al.TIacumicin,A novel complex of18-membered macrolide
antibiotics.I.Taxonomy,fermentation and antibacterial activity.The Journal of
Antibiotics.1987,5:567-574. culture medium used in the culture dactylosporangium aurantiacum disclosed in ".
In the present invention, the dactylosporangium aurantiacum is preferably dactylosporangium aurantiacum described in the routine of this field
NRRL18085。
In the present invention, the fermentation medium preferably include mass percent be 1.5~2.5% glucose, 0.5~
1.5% bean powder, 0.2~0.4% beef extract, 0.05~0.15% soya-bean oil, 0.04~0.06% MgSO4·7H2O, 0.02
~0.04% KCl, 0.04~0.06% K2HPO4, 0.2~0.4% CaCO3And the absorption tree that percentage by volume is 3~5%
Rouge, the absorption resin are selected from HP20, HZ830, XAD-16, and XAD1600 is any one or more of, the fermentation medium
PH value is 7.2~7.4.
In the present invention, the fermentation medium more preferably include mass percent be 2% glucose, 1% bean powder,
0.3% beef extract, 0.1% soya-bean oil, 0.05% MgSO4·7H2O, 0.03% KCl, 0.05% K2HPO4, 0.3%
CaCO3And the absorption resin that percentage by volume is 3~5%, the absorption resin are selected from HP20, HZ830, XAD-16, XAD1600
Any one or more of (more preferable HP20 or HZ830), the pH value of the fermentation medium are 7.2~7.4.
In the present invention, the fermentation medium most preferably include mass percent be 2% glucose, 1% bean powder,
0.3% beef extract, 0.1% soya-bean oil, 0.05% MgSO4·7H2O, 0.03% KCl, 0.05% K2HPO4, 0.3%
CaCO3And the absorption resin that percentage by volume is 4%, the absorption resin is in HP20, HZ830, XAD-16, XAD1600
It is any, the pH value of the fermentation medium is 7.3.
In the present invention, the more preferable HP20 or HZ830 of absorption resin, when adsorbing Choice of Resin HP20 or HZ830, no
The fermentation yield of feldamycin can be only greatly improved, more can after fermentation recycled absorption resin to follow repeatedly
Ring uses, to further reduce the cost.
The preparation method of fermentation medium of the present invention is routine, as long as each component is preferably distilled with water
Water dissolution, then with high-temperature sterilization.
Technical solution provided by the invention second is that: a kind of method of fermenting and producing feldamycin comprising with aforementioned training
The step of supporting base fermented and cultured dactylosporangium aurantiacum production feldamycin.
In the present invention, when producing feldamycin with prior culture media fermented and cultured dactylosporangium aurantiacum, conventional side is used
Dactylosporangium aurantiacum is inoculated in prior culture media by method, then carry out normal fermentation.
Preferably, the method is comprising steps of by the inoculum concentration of dactylosporangium aurantiacum seed liquor in mass ratio 5~15%
It is inoculated in foregoing fermentation medium, then cultivates fermentation flask on 25~30 DEG C, the shaking table of 150~220rpm of revolving speed
8~15 days.
The method preferably further includes the steps that preparing the seed liquor:
(1) dactylosporangium aurantiacum for producing feldamycin is inoculated on slant medium and is activated；
(2) colony inoculation that activation obtains is obtained into seed liquor in seed culture medium culture.
Wherein, the step of preparing the seed liquor more preferably include:
(1) dactylosporangium aurantiacum for producing feldamycin is inoculated on slant medium and is activated；
(2) colony inoculation that activation obtains is obtained into seed liquor in seed culture medium culture；The seed culture medium by
0.05~0.15% glucose, 2~3% starch, 0.3~0.6% extraction from yeast powder, 0.3~0.6% tryptone, 0.2~0.4% N
Meat medicinal extract and 0.3~0.5%CaCO3Composition, the percentage are mass percent, the pH of the seed culture medium is 7.2~
7.4。
Most preferably, the method includes the following steps:
(1) dactylosporangium aurantiacum for producing feldamycin is inoculated on slant medium and cultivates 4-5 in 30 DEG C of incubators
It is activated, and the slant medium is by 0.4% extraction from yeast powder, 1% fructus hordei germinatus leaching powder, 0.4% glucose and 2.0% agar group
At the percentage is mass percent, and the pH of the slant medium is 7.0-7.2；
(2) colony inoculation for obtaining activation obtains seed liquor in shaking table culture 48 hours in 28 DEG C, institute in seed culture medium
Seed culture medium is stated by 0.1% glucose, 2.4% starch, 0.5% extraction from yeast powder, 0.5% tryptone, 0.3% beef extract and
0.4%CaCO3Composition, the percentage are mass percent, and the pH of the seed culture medium is 7.3；
(3) (2) resulting seed liquor is inoculated with aforesaid fermentation culture medium, then by fermentation flask in 28 DEG C, revolving speed 200rpm
Shaking table on cultivate 12 days.
In the present invention, further preferably include the steps that harvesting feldamycin after step (3).The harvest feldamycin uses
The method of general harvest tunning, the methanol of 2~4 times of volumes of addition more preferably into fermentation liquid, 20~35min of ultrasound, from
Supernatant is taken after the heart to obtain the final product；The methanol of 3 times of volumes is added most preferably into fermentation liquid, ultrasonic 30min takes supernatant after centrifugation to obtain the final product.
The ingredients such as the carbon source, nitrogen source and inorganic salts that can be utilized can also be added in fermentation medium of the present invention,
As long as mutual antagonism is not present between each component in those ingredients and prior culture media of the present invention, these ingredients can
It is disposably added in culture medium in advance, realizes the high yield quantization of feldamycin.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that:
Compared to existing culture medium, fermentation medium of the invention for cultivate dactylosporangium aurantiacum can greatly improve it is non-
Up to the yield of mycin, highest can promote 114%, thus greatly reduce production cost, before having extraordinary industrial applications
Scape.In addition, absorption resin HP20 and HZ830 used in the present invention can be recycled repeatedly, thus improving non-reach
It further reduced production cost while mycin yield.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
In following embodiments, the percentage unless otherwise instructed, refers both to mass percent.
In following embodiments, used dactylosporangium aurantiacum is purchased from Agricultural Research Service
Culture Collection(american agriculture studies Culture Collection Center, ARS), the tangerine orange that number is NRRL18085 refers to sporangiocyst
Bacteria strain.
Embodiment 1
The composition of fermentation medium used in the present embodiment are as follows: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil
0.1%, MgSO4·7H2O0.05%, KCl0.03%, K2HPO40.05%, CaCO30.3%, HP20 resin 4%(v/v), NaOH tune pH is extremely
7.3。
Before inoculation fermentation culture medium, first by the dactylosporangium aurantiacum frozen in activation on slant medium and in kind
Culture obtains seed liquor in sub- culture medium.The group of the slant medium becomes extraction from yeast powder 0.4%, fructus hordei germinatus leaching powder 1%, Portugal
Grape sugar 0.4%, agar 2.0% is sterilized 30 minutes with NaOH tune pH to 7.0-7.2 in 121 DEG C, paved at solid slope.By strain
Be connected on this inclined-plane, 30 DEG C incubator culture 4-5 days, well-grown slant strains are inoculated in seed culture medium.Institute
The group for the seed culture medium stated becomes glucose 0.1%, starch 2.4%, extraction from yeast powder 0.5%, tryptone 0.5%, beef leaching
Cream 0.3%, CaCO30.4%, NaOH tune pH to 7.3.Seed culture is cooled down spare after twenty minutes based on 121 DEG C of sterilizings.It is inoculated in kind
After in sub- culture medium after 28 DEG C, shaking table culture 48 hours of revolving speed 200rpm, obtained seed liquor is for inoculation fermentation culture
Base.
Seed liquor is connected to fermentation medium by 10% inoculum concentration, after inoculation fermentation culture medium, by fermentation flask in 28 DEG C, turns
It is cultivated 12 days on the shaking table of fast 200rpm, the methanol of 3 times of volumes, ultrasonic 30min, centrifugation is added after fermentation ends into fermentation liquid
After take supernatant, HPLC measures the content of feldamycin, as a result 1090mg/l.
Comparative example 1
By the fermentation medium of prior art preparation production feldamycin, in document " Robert
J.Theriault,James P.Karwowski,et al.TIacumicin,A novel complex of18-membered
macrolide antibiotics.I.Taxonomy,fermentation and antibacterial activity.The
It is disclosed in Journal of Antibiotics.1987,5:567-574. ", consisting of glucose 2%, bean powder 1%, beef leaching
Cream 0.3%, soya-bean oil 0.1%, MgSO4·7H2O0.05%, KCl0.03%, K2HPO40.05%, CaCO30.3%, extremely with NaOH tune pH
7.3。
Seed liquor made from embodiment 1 is inoculated in fermentation medium with 10% inoculum concentration, the control in fermentation process
Condition is the same as embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, as a result 779mg/l.
This comparative example is compared as blank control and each embodiment.
As can be seen that the yield of 1 feldamycin of embodiment improves 40% compared with blank control.
Embodiment 2
The composition of fermentation medium used in the present embodiment are as follows: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil
0.1%, MgSO4·7H2O0.05%, KCl0.03%, K2HPO40.05%, CaCO30.3%, XAD-1600 resin 4%(v/v), NaOH tune
PH to 7.3.
Seed liquor made from embodiment 1 is inoculated in fermentation medium with 10% inoculum concentration, the control in fermentation process
Condition is the same as embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, as a result 1288mg/, mentions compared with blank control
It is high by 65%.
Embodiment 3
The composition of fermentation medium used in the present embodiment are as follows: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil
0.1%, MgSO4·7H2O0.05%, KCl0.03%, K2HPO40.05%, CaCO30.3%, HZ830 resin 4%(v/v), NaOH tune pH
To 7.3.
Seed liquor made from embodiment 1 is inoculated in fermentation medium with 10% inoculum concentration, the control in fermentation process
Condition is the same as embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, as a result 974mg/l, mentions compared with blank control
It is high by 25%.
Embodiment 4
The composition of fermentation medium used in the present embodiment are as follows: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil
0.1%, MgSO4·7H2O0.05%, KCl0.03%, K2HPO40.05%, CaCO30.3%, XAD-16 resin 4%(v/v), NaOH tune pH
To 7.3.
Seed liquor made from embodiment 1 is inoculated in fermentation medium with 10% inoculum concentration, the control in fermentation process
Condition is the same as embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, as a result 822mg/, is improved compared with blank control
5%.
Embodiment 5~10
The composition of fermentation medium used in embodiment 5~10 are as follows: glucose 2%, bean powder 1%, beef extract 0.3%, beans
0.1%, MgSO of oil4·7H2O0.05%, KCl0.03%, K2HPO40.05%, CaCO30.3% and HP20 resin, wherein from implementation
The additive amount of example 5~10, HP20 resin is followed successively by 1%(v/v respectively), 2%(v/v), 3%(v/v) and, 4%(v/v), 5%(v/v) and 6%
(v/v), NaOH tune pH to 7.3.
Seed liquor made from embodiment 1 is inoculated in fermentation medium with 10% inoculum concentration, the control in fermentation process
Condition is the same as embodiment 1.Fermentation ends, HPLC measure the content of feldamycin, and result is followed successively by respectively from embodiment 5~10
806mg/l, 1054mg/l, 1086mg/l, 1090mg/l, 1615mg/l and 1669mg/l have been respectively increased 3% compared with blank control,
35%, 39%, 40%, 107% and 114%.
Embodiment 11
The composition of fermentation medium used in the present embodiment are as follows: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil
0.1%, MgSO4·7H2O0.05%, KCl0.03%, K2HPO40.05%, CaCO30.3%, embodiment 1 has used recycling resulting
HP20 resin 4%(v/v), NaOH tune pH to 7.3.Wherein, the process of HP20 resin recycling are as follows: by the fermentation containing HP20 resin
Liquid filtering, collects HP20 resin, is cleaned the thallus of resin surface with deionized water, then impregnates HP20 resin with 95% ethyl alcohol
And wash three times, after ethyl alcohol is cleaned to get can be with HP20 resin that repetitive cycling uses with deionized water.
Seed liquor made from embodiment 1 is inoculated in fermentation medium with 10% inoculum concentration, the control in fermentation process
Condition is the same as embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, as a result 1216mg/l, mentions compared with blank control
It is high by 56%.
Embodiment 12
The composition of fermentation medium used in the present embodiment are as follows: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil
0.1%, MgSO4·7H2O0.05%, KCl0.03%, K2HPO40.05%, CaCO30.3%, embodiment 3 has used recycling resulting
HZ830 resin 4%(v/v), NaOH tune pH to 7.3.Wherein, the process of HZ830 resin recycling are as follows: by the hair containing HZ830 resin
Zymotic fluid filtering, collects HZ830 resin, is cleaned the thallus of resin surface with deionized water, then impregnates HZ830 with 95% ethyl alcohol
Resin simultaneously washs three times, after ethyl alcohol is cleaned to get can be with HZ830 resin that repetitive cycling uses with deionized water.
Seed liquor made from embodiment 1 is inoculated in fermentation medium with 10% inoculum concentration, the control in fermentation process
Condition is the same as embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, as a result 1015mg/l, mentions compared with blank control
It is high by 30%.
Comparative example 2
The composition of fermentation medium used in the present embodiment are as follows: glucose 2%, bean powder 1%, beef extract 0.3%, soya-bean oil
0.1%, MgSO4·7H2O0.05%, KCl0.03%, K2HPO40.05%, CaCO30.3%, SP850 resin 4%(v/v), NaOH tune pH
To 7.3.
Seed liquor made from embodiment 1 is inoculated in fermentation medium with 10% inoculum concentration, the control in fermentation process
Condition is the same as embodiment 1.Fermentation ends, HPLC measures the content of feldamycin, as a result 510mg/l, drops compared with blank control
Low 34%.
It should be understood that those skilled in the art can make the present invention various after having read above content of the invention
Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.
Claims (7)
1. a kind of fermentation medium for improving feldamycin yield, which is characterized in that the fermentation medium is by quality percentage
Number is 1.5~2.5% glucose, 0.5~1.5% bean powder, 0.2~0.4% beef extract, 0.05~0.15% beans
Oil, 0.04~0.06% MgSO4·7H2O, 0.02~0.04% KCl, 0.04~0.06% K2HPO4, 0.2~0.4%
CaCO3And the absorption resin that percentage by volume is 1~6% forms, the absorption resin is HP20 or HZ830, the fermentation
The pH value of culture medium is 7.2~7.4, and the strain of the fermentation medium culture is dactylosporangium aurantiacum
(Dactylosporangium aurantiacum)。
2. fermentation medium as described in claim 1, which is characterized in that the dactylosporangium aurantiacum is dactylosporangium aurantiacum
NRRL18085。
3. fermentation medium as described in claim 1, which is characterized in that the fermentation medium is by mass percent
2% glucose, 1% bean powder, 0.3% beef extract, 0.1% soya-bean oil, 0.05% MgSO4·7H2O, 0.03%
KCl, 0.05% K2HPO4, 0.3% CaCO3And the absorption resin that percentage by volume is 3~5% forms, the absorption tree
Rouge is selected from HP20 or HZ830, and the pH value of the fermentation medium is 7.2~7.4.
4. fermentation medium as described in claim 1, which is characterized in that the fermentation medium is by mass percent
2% glucose, 1% bean powder, 0.3% beef extract, 0.1% soya-bean oil, 0.05% MgSO4·7H2O, 0.03%
KCl, 0.05% K2HPO4, 0.3% CaCO3And the absorption resin that percentage by volume is 4% forms, the absorption resin choosing
From HP20 or HZ830, the pH value of the fermentation medium is 7.3.
5. a kind of method of fermenting and producing feldamycin, which is characterized in that it includes described in any item with Claims 1 to 4
Fermentation medium fermented and cultured dactylosporangium aurantiacum produces the step of feldamycin.
6. method as claimed in claim 5, which is characterized in that it is comprising steps of press quality for dactylosporangium aurantiacum seed liquor
It is inoculated in than 5~15% inoculum concentration such as the described in any item fermentation mediums of Claims 1 to 4, then by fermentation flask 25
~30 DEG C, cultivate 8~15 days on the shaking table of 150~220rpm of revolving speed.
7. method as claimed in claim 6, which is characterized in that the method further includes the steps that preparing the seed liquor:
(1) dactylosporangium aurantiacum for producing feldamycin is inoculated on slant medium and is activated；
(2) colony inoculation that activation obtains is obtained into seed liquor in seed culture medium culture；The seed culture medium by 0.05~
0.15% glucose, 2~3% starch, 0.3~0.6% extraction from yeast powder, 0.3~0.6% tryptone, 0.2~0.4% N
Meat medicinal extract and 0.3~0.5%CaCO3Composition, the percentage are mass percent, and the pH of the seed culture medium is 7.2
~7.4.
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| CN1688707A (en) * | 2002-07-29 | 2005-10-26 | 浩鼎生技公司 | Tiacumicin production |
| CN101126073A (en) * | 2007-07-02 | 2008-02-20 | 江南大学 | Sorangium cellulosum, preparation method and application for Phoxalone fermented by the same |
| CN102465154A (en) * | 2010-11-04 | 2012-05-23 | 苏州大学 | Method for improving hypocrellin yield in shiraia bambusicola |
| CN102503994A (en) * | 2005-10-21 | 2012-06-20 | 奥普蒂姆药物公司 | Method of treating clostridium difficile-associated diarrhea |
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|---|---|---|---|---|
| CN1688707A (en) * | 2002-07-29 | 2005-10-26 | 浩鼎生技公司 | Tiacumicin production |
| CN102503994A (en) * | 2005-10-21 | 2012-06-20 | 奥普蒂姆药物公司 | Method of treating clostridium difficile-associated diarrhea |
| CN101126073A (en) * | 2007-07-02 | 2008-02-20 | 江南大学 | Sorangium cellulosum, preparation method and application for Phoxalone fermented by the same |
| CN102465154A (en) * | 2010-11-04 | 2012-05-23 | 苏州大学 | Method for improving hypocrellin yield in shiraia bambusicola |
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