CN104774892B - A kind of fermentation medium and its preparation method and application for producing Polymyxin B sulfate - Google Patents
A kind of fermentation medium and its preparation method and application for producing Polymyxin B sulfate Download PDFInfo
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- CN104774892B CN104774892B CN201410014183.2A CN201410014183A CN104774892B CN 104774892 B CN104774892 B CN 104774892B CN 201410014183 A CN201410014183 A CN 201410014183A CN 104774892 B CN104774892 B CN 104774892B
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- 230000004151 fermentation Effects 0.000 title claims abstract description 39
- 108010093965 Polymyxin B Proteins 0.000 title claims abstract description 37
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Abstract
The invention discloses a kind of fermentation mediums and its preparation method and application for producing Polymyxin B sulfate.With the total weight of culture medium, the culture medium contains 0.1-1.5w/w% organic nitrogen source, 5.0-15.0w/w% wholemeal and 0.3-1.2w/w% inorganic salts.
Description
Technical field
The present invention relates to field of biotechnology；More particularly to a kind of fermentation medium and its preparation method and application.
Background technique
Polymyxin B sulfate（Polymyxin B, PMB）It is the one kind generated by Bacillus polymyxa or more glutinous series bacillus
Alkaline cyclic peptide antibiotic, there is good anti-Gram-negative bacteria activity.The drug of Polymyxin B sulfate includes the more slime moulds of sulfuric acid
The compound external-use preparation of plain B injection and sulfuric acid Polymyxin B sulfate and other antibiotic, causes for treating Gram-negative bacteria
The positions such as skin wound, peritonitis, urinary system and respiratory system infection；PMB can also significantly inhibit endotoxin and lure
The acute phase response led, therefore can also be used for septicemia.
The preparation of Polymyxin B sulfate at present uses fermentation method, and Bacillus polymyxa or mostly glutinous series bacillus are trained by seed
After supporting, Polymyxin B sulfate is cultivated in the fermenter and is generated in inoculation, then through the subsequent product for isolating and purifying to obtain high-purity.Hair
Culture medium prescription has a great impact to yield in ferment incubation.It is existing to report that the related culture medium prescription being related to has fermentation
The not high problem of yield.
Therefore, there is an urgent need in the art to provide a kind of fermentation medium of high yield Polymyxin B sulfate.
Summary of the invention
The present invention is intended to provide a kind of fermentation medium.
It is a further object to provide the preparation methods of above-mentioned fermentation medium.
It is also another object of the present invention to provide the purposes of above-mentioned fermentation medium.
In the first aspect of the present invention, provide it is a kind of for producing the fermentation medium of Polymyxin B sulfate, with culture medium
Total weight, the culture medium contain 0.1-1.5w/w% organic nitrogen source, 5.0-15.0w/w% wholemeal and 0.3-1.2w/w%
Inorganic salts.
In another preferred example, the organic nitrogen source is selected from following one or more：Peptone, extraction from yeast
Object, soybean powder or corn starch；More preferably, the organic nitrogen source is selected from soybean powder or corn starch.
The inorganic salts are selected from following one or more：Potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate, zinc sulfate or
Calcium carbonate；More preferably, the inorganic salts are ammonium sulfate or ammonium sulfate and the mixing selected from following one or more kinds of substances：
Potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate or calcium carbonate.
In another preferred example, the content of the wholemeal is 7.0-13.0w/w%.
In the second aspect of the present invention, a kind of preparation side of fermentation medium provided present invention as described above is provided
Method, the method include step：
（1）With the total weight of culture medium, by 0.1-1.5w/w% organic nitrogen source, 5.0-15.0w/w% wholemeal and 0.3-
1.2w/w% inorganic salts are mixed to get mixture 1；
（2）By diastatic action in through gelatinization or the mixture 1 without gelatinization（It is preferred that being gelatinized）, obtain sheet as described above
The fermentation medium provided is provided.
In another preferred example, the amylase is selected from alpha-amylase, beta amylase, glucoamylase or different starch
Enzyme；More preferable alpha-amylase.
In another preferred example, in terms of the weight of wholemeal, the dosage of the amylase is 2-1500U/g；More preferably
2-1000U/g。
In another preferred example, the operative temperature of the amylase is 50-80 DEG C.
In another preferred example, the action time of the amylase is 2-40 minutes；More preferably 5-20 minutes.
In the third aspect of the present invention, a kind of purposes of fermentation medium provided present invention as described above is provided,
For cultivating Bacillus polymyxa (Bacillus polymyxa) or mostly glutinous series bacillus (Paenibacillus
Polymyxa) to produce Polymyxin B sulfate.
Accordingly, the present invention provides a kind of fermentation mediums for keeping tunning Polymyxin B sulfate yield high.
Specific embodiment
Inventor is after extensive and in-depth study, it has unexpectedly been found that if using the wholemeal of higher concentration as culture
Base component, and before sterilization with the fermentation medium after being gelatinized of starch enzymatic treatment, it can biggish raising Polymyxin B sulfate
Yield.On this basis, the present invention is completed.
Definition
As used herein, " wholemeal " refers to that Intact wheat contains the powder that wheat bran and plumule are all worn into.I.e. whole grain is small
Wheat only passes through and pulverizes in milling, without by removing wheat bran program.
As used herein, after " gelatinization " refers to that starch granules is heated, in certain temperature range, amylum body starts to destroy,
Crystal structure disappears, and enlarged volume, viscosity steeply rises, in sticky paste.
In the present invention, the culture medium after diastatic action, starch therein become dextrin and maltose, sugar chain
It is shortened by length, maltose will increase, the decline of culture medium viscosity.
As used herein, " soybean powder " can be used method well known in the art and produce through milling process, such as but unlimited
In soya bean（Soybean）Residual cake after squeezing de-oiling is round pie after Hydraulic oil press, is flake after screw oil-extracting；It is hard
Block, crushed again after generally first smashing.It can also be obtained by commercial channel, be such as, but not limited to purchased from（Shijiazhuang is emerging
Cypress bioengineering Co., Ltd）.
As used herein, " corn starch ", which refers to, is spray-dried using fresh corn slurry as raw material through low temperature transient heating,
Its water soluble protein is intact, maintains all characteristics of corn slurries.Method preparation well known in the art can be used
It obtains, can also be obtained by commercial channel, be such as, but not limited to purchased from（Zouping Xi Wang bioengineering Co., Ltd）.
Its structure of Polymyxin B sulfate of the present invention is shown in formula I：
The architectural difference for each component being directed to is referring to table 1
| Polymyxin B sulfate each component | FA | X |
| B1 | 6-MOA | L-Leu |
| B1-I | 6-MOA | L-Ile |
| B2 | 6-MHA | L-Leu |
| B3 | OA | L-Leu |
Note：MOA-6 methyloctanoic acid, MHA- isooctyl acid（Or methyl enanthic acid）, OA- octanoic acid, Leu- leucine, the different bright ammonia of Ile-
Acid.
Fermentation medium
Fermentation medium provided by the invention is the culture for cultivating more glutinous series bacillus or more glutinous series bacillus
Base, with the total weight of the culture medium, wherein including 0.1-1.5w/w% organic nitrogen source, 5.0-15.0w/w% wholemeal
With 0.3-1.2w/w% inorganic salts.
Organic nitrogen source is selected from following one or more in the culture medium：Peptone, yeast extract, soya bean
Powder, corn starch, wherein preferably soybean powder or corn starch, the dosage of organic nitrogen source is preferably 0.3-1.2w/ in culture medium
W%, most preferably 0.5-1.2w/w%；Inorganic salts include ammonium sulfate in the culture medium, it is other selected from following one kind or it is a kind of with
On：Potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate, calcium carbonate, wherein it is preferred that potassium dihydrogen phosphate and calcium carbonate, preferably, in culture medium
The content of inorganic salts is respectively 0.3-1.0w/w% ammonium sulfate, 0.01-0.08w/w% potassium dihydrogen phosphate and 0.01-0.1w/w% carbonic acid
Calcium；The content of ammonium sulfate is more preferably 0.7-1.0% in culture medium；The content of the potassium dihydrogen phosphate of culture medium is more preferably
0.01-0.04w/w%；The content of wholemeal in culture medium is preferably 7.0-13.0w/w%, most preferably 8.0-11.0w/w%.
The culture bacterial strain that the present invention uses is ATCC10401's for more glutinous series bacillus, preferably deposit number
Paenibacillus polymyxa；Bacillus polymyxa deposit number is the Bacillus polymyxa of NRRL B-719.
The preparation method of fermentation medium
Culture medium provided by the invention uses starch enzymatic treatment before sterilization.
Specifically, the preparation method of fermentation medium provided by the invention includes the following steps：
Organic nitrogen source, wholemeal, inorganic salts and water are mixed according to above-mentioned formula, obtain mixture 1 by the first step；
Second step obtains fermentation training provided by the invention by diastatic action in through gelatinization or the mixture 1 without gelatinization
Support base.
Amylase involved in the above method is selected from alpha-amylase, beta amylase, glucoamylase or isoamylase；It is excellent
Select alpha-amylase；In terms of the weight of the wholemeal put into the first step, the dosage of the amylase is 2-1500U/g；Preferably
2-1000U/g, more preferably 100-1000U/g；The operative temperature of the amylase is 50-80 DEG C, and action time is 10-60 points
Clock；Preferably 20-40 minutes.
In the second step of the above method, preferably by diastatic action in the mixture 1 through being gelatinized.
In an embodiment of the present invention, the culture medium after diastatic action 121 DEG C high-temperature sterilization 20 minutes.
The feature that the features described above or embodiment that the present invention mentions are mentioned can be in any combination.Disclosed in this case specification
All features can be used in combination with any composition form, each feature disclosed in specification, any can provide it is identical,
The alternative characteristics of impartial or similar purpose replace.Therefore except there is special instruction, revealed feature is only impartial or similar spy
The general example of sign.
Main advantages of the present invention are：
1, culture medium provided by the invention improves Polymyxin B sulfate fermentation yield, and reduces the viscosity of fermentation liquid, convenient
Post-processing.
2, using culture medium provided by the invention, Polymyxin B sulfate fermentation yield is higher than 1.90g/L.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise all percentage, ratio, ratio or number is pressed
Poidometer.
The unit in percent weight in volume in the present invention is well-known to those skilled in the art, such as is referred to
The weight of solute in 100 milliliters of solution.
Unless otherwise defined, it anticipates known to all professional and scientific terms as used herein and one skilled in the art
Justice is identical.In addition, any method similar to or equal to what is recorded and material can be applied to the method for the present invention.Wen Zhong
The preferred implement methods and materials are for illustrative purposes only.
In following embodiment, the measuring method reference literature of Polymyxin B sulfate yield（Tiandong County, Yuan Qipeng, Sun Xinxiao wait to ring
Answer fermentation condition [J] China's agronomy notification of face method optimization polymyxin B, 2010,26 (15):53-57）
Wholemeal used in the following embodiment is purchased from（Weifang kite flour Co., Ltd）
Corn starch used in the following embodiment is purchased from（Zouping Xi Wang bioengineering Co., Ltd）
Soybean powder used in the following embodiment is purchased from（Shijiazhuang Xing Bai bioengineering Co., Ltd）
Bacillus polymyxa used in the following embodiment（Bacillus polymyxa NRRL B-719）Purchased from the U.S.
Agricultural research Culture Collection Center.
More glutinous series bacillus used in the following embodiment（Paenibacillus polymyxa ATCC10401）Purchase
From American Type Culture Collecti.
BF7658 alpha-amylase used in the following embodiment is purchased from Xingtai Wanda bioengineering Co., Ltd
Beta amylase used in the following embodiment is purchased from Shandong Anke Bioengineering Co., Ltd.
Glucoamylase used in the following embodiment is purchased from Shandong Long great bioengineering Co., Ltd
Isoamylase used in the following embodiment is purchased from Denmark Novo company
Embodiment 1
Cultivate bacterial strain：Mostly glutinous series bacillus（Paenibacillus polymyxa ATCC10401）
Seed culture medium/%：Corn pulp 2.0；Glucose 5.0；Ammonium sulfate 0.4；Sodium chloride 0.1；Calcium carbonate 0.3；pH6.8
~7.0.
Fermentation medium/%：Wholemeal 8.0；Corn starch 0.4；Ammonium sulfate 0.9；Potassium dihydrogen phosphate 0.03；Calcium carbonate
0.03。
Fermentation medium is gelatinized, BF7658 alpha-amylase is added（Xingtai Wanda's bioengineering Co., Ltd）, form sediment
Powder enzyme dosage 100U/g acts on 30min at 50 DEG C -80 DEG C；121 DEG C of high-temperature sterilization 20min later.
Cultural method：The strain of slant preservation is seeded in seed culture medium, 28 DEG C of culture 18h.It is connect with 4% inoculum concentration
Enter in the above-mentioned fermentation medium of 18L（Fermentor total volume 35L）, mixing speed 400r/min, in the pressure of 50kPa tank, ventilatory capacity
For 1.2vvm, 30 DEG C of culture 48h.
Polymyxin B sulfate assay, reference literature（Tiandong County, Yuan Qipeng, Sun Xinxiao wait response phase method to optimize polymyxins
Fermentation condition [J] China's agronomy of B is notified to, 2010,26 (15):53-57.）
Through detecting, Polymyxin B sulfate yield is 3.22g/L under above-mentioned culture medium condition.
Embodiment 2
Other operations are with embodiment 1, the difference is that starting strain is NRRL B-719.It is detected, is cultivated
Polymyxin B sulfate yield is 3.14g/L.
Embodiment 3-14
Other operations are with embodiment 1, the difference is that whole wheat powder content.It is detected, the Polymyxin B sulfate cultivated produces
Amount is as shown in table 2 respectively：
| Embodiment | Whole wheat powder content（w/w%） | Polymyxin B sulfate yield（g/L） |
| 3 | 5 | 1.90 |
| 4 | 6 | 2.73 |
| 5 | 6.6 | 2.82 |
| 6 | 7 | 2.90 |
| 7 | 8 | 3.22 |
| 8 | 9 | 3.18 |
| 9 | 10 | 3.14 |
| 10 | 11 | 3.11 |
| 11 | 12 | 3.07 |
| 12 | 13 | 3.04 |
| 13 | 14 | 2.95 |
| 14 | 15 | 2.56 |
Embodiment 15
Other operations are with embodiment 1, the difference is that fermentation medium is wholemeal 8.0%, soybean powder 1.0%, sulfuric acid
Ammonium 0.3%, potassium dihydrogen phosphate 0.06% and calcium carbonate 0.1%, are detected, and the Polymyxin B sulfate yield cultivated is 2.90g/L.
Embodiment 16
Other operations are added beta amylase, are detected with embodiment 1 the difference is that whole wheat powder content is 8.0%, train
Supporting obtained Polymyxin B sulfate yield is 2.43g/L.
Embodiment 17
With embodiment 1 glucoamylase is added, through examining the difference is that whole wheat powder content is 8.0% in other operations
It surveys, the Polymyxin B sulfate yield cultivated is 2.21g/L.
Embodiment 18
Other operations are added isoamylase, are detected with embodiment 1 the difference is that whole wheat powder content is 8.0%, train
Supporting obtained Polymyxin B sulfate yield is 1.97g/L.
Embodiment 19
Other operations are with embodiment 1, the difference is that whole wheat powder content is 8.0%, addition α-amylasecontent is 2U/
G is detected, and the Polymyxin B sulfate yield cultivated is 1.99g/L.
Embodiment 20
α-amylasecontent 1000U/ is added the difference is that whole wheat powder content is 8.0% with embodiment 1 in other operations
G is detected, and the Polymyxin B sulfate yield cultivated is 3.08g/L.
Embodiment 21
Other operations are with embodiment 1, the difference is that culture medium is directly added into amylase sterilization without gelatinization.Through
Detection, the Polymyxin B sulfate yield cultivated are 2.27g/L.
Embodiment 22
Other operations are with embodiment 1, the difference is that culture medium does not add potassium dihydrogen phosphate, magnesium sulfate and zinc sulfate.
It is detected, the Polymyxin B sulfate yield cultivated is 2.83g/L.
Comparative example 1
According to document（Anand, H., Guduri, B, et al.Process for the preparation of
Polymyxin B employing Bacillus polymyxa【P】US8,119,371,B2Feb.21,2012.）Middle preparation training
Support base and cultural method.After fermentation, fermentation liquid 200mL is taken, is filtered under diminished pressure with Medium speed filter paper, used time 18min.
Comparative example 2
Operation takes fermentation liquid 200ml identical as the filter condition of comparative example 1 with embodiment 1, filters used time 10min.
Comparative example 1 and comparative example 2 illustrate, can reduce the viscosity of fermentation liquid after fermentation in culture medium of the present invention, make to train
It supports base and is separated by solid-liquid separation speed faster.
Comparative example 3
According to document（Anand, H., Guduri, B et al.Process for the preparation of
Polymyxin B employing Bacillus polymyxa【P】US8,119,371,B2Feb.21,2012.）Middle preparation training
Support base and cultural method（Referring to this document embodiment 1）.Polymyxin B sulfate fermentation yield 1.77g/L.
The foregoing is merely illustrative of the preferred embodiments of the present invention, the substantial technological content model being not intended to limit the invention
It encloses, substantial technological content of the invention is broadly defined in the scope of the claims of application, any technology that other people complete
Entity or method also or a kind of equivalent change, will if identical with defined in the scope of the claims of application
It is considered as being covered by among the scope of the claims.
Claims (5)
1. a kind of purposes of fermentation medium, which is characterized in that for cultivating Bacillus polymyxa (Bacillus polymyxa)
Or stick series bacillus (Paenibacillus polymyxa) to produce Polymyxin B sulfate more；
The fermentation medium is prepared by following step：
(1) with the total weight of culture medium, by 0.1-1.5w/w% organic nitrogen source, 7.0-13.0w/w% wholemeal and 0.3-
1.2w/w% inorganic salts are mixed to get mixture 1；The organic nitrogen source is selected from soybean powder or corn starch；The inorganic salts are sulphur
Sour ammonium or ammonium sulfate and mixing selected from following one or more kinds of substances：Potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate or carbon
Sour calcium；
(2) alpha-amylase is acted on into the mixture 1 through being gelatinized, obtains fermentation medium；
In terms of the weight of wholemeal, the dosage of the alpha-amylase is 100-1500U/g.
2. purposes as described in claim 1, which is characterized in that in terms of the weight of wholemeal, the dosage of the amylase is
100-1000U/g。
3. purposes as described in claim 1, which is characterized in that the operative temperature of the alpha-amylase is 50-80 DEG C.
4. purposes as described in claim 1, which is characterized in that the action time of the alpha-amylase is 2-40 minutes.
5. purposes as described in claim 1, which is characterized in that the action time of the alpha-amylase is 5-20 minutes.
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