CN104774892A - Polymyxin B production fermentation culture medium, preparation method and uses thereof - Google Patents
Polymyxin B production fermentation culture medium, preparation method and uses thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 17
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- 238000000034 method Methods 0.000 claims description 22
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Abstract
The present invention discloses a polymyxin B production fermentation culture medium, a preparation method and uses thereof, wherein the culture medium comprises, by weight, 0.1-1.5 w/w% of an organic nitrogen source, 5.0-15.0 w/w% of whole wheat powder, and 0.3-1.2 w/w% of an inorganic salt.
Description
Technical field
The present invention relates to biological technical field; Particularly relate to a kind of fermention medium and its production and use.
Background technology
Polymyxin B sulfate (Polymyxin B, PMB) is a kind of alkaline cyclic peptide microbiotic produced by Bacillus polymyxa or many glutinous series bacillus, has good against gram-negative bacteria active.The medicine of Polymyxin B sulfate comprises the antibiotic compound external-use preparation of sulfuric acid Polymyxin B sulfate injection and sulfuric acid Polymyxin B sulfate and other, is used for treating the infection at the positions such as the Gram-negative bacteria skin wound, peritonitis, urinary system and the respiratory system that cause; PMB significantly can also suppress the acute phase reaction of endotaxin induction, therefore also can be used for septicemia.
The preparation of current Polymyxin B sulfate adopts fermentation method, and Bacillus polymyxa or many glutinous series bacillus, after seed culture, are seeded in fermentor tank and cultivate and produce Polymyxin B sulfate, then obtain highly purified product through follow-up separation and purification.In fermentation culture process, culture medium prescription has a great impact output.There is the not high problem of fermentation yield in the relevant culture medium prescription that existing report relates to.
Therefore, this area is in the urgent need to providing a kind of fermention medium of high yield Polymyxin B sulfate.
Summary of the invention
The present invention aims to provide a kind of fermention medium.
Another object of the present invention is to provide the preparation method of above-mentioned fermention medium.
Another object of the present invention is to provide the purposes of above-mentioned fermention medium.
In a first aspect of the present invention, provide a kind of fermention medium for the production of Polymyxin B sulfate, with the total weight of substratum, described substratum contains 0.1-1.5w/w% organic nitrogen source, 5.0-15.0w/w% whole wheat flour and 0.3-1.2w/w% inorganic salt.
In another preference, described organic nitrogen source is selected from following one or more: peptone, yeast extract, analysis for soybean powder or corn starch; More preferably, described organic nitrogen source is selected from analysis for soybean powder or corn starch.
Described inorganic salt are selected from following one or more: potassium primary phosphate, ammonium sulfate, magnesium sulfate, zinc sulfate or calcium carbonate; More preferably, described inorganic salt are ammonium sulfate or ammonium sulfate and the mixing being selected from one or more following materials: potassium primary phosphate, magnesium sulfate, zinc sulfate or calcium carbonate.
In another preference, the content of described whole wheat flour is 7.0-13.0w/w%.
In a second aspect of the present invention, provide a kind of preparation method of fermention medium provided by the invention as above, described method comprises step:
(1) with the total weight of substratum, 0.1-1.5w/w% organic nitrogen source, 5.0-15.0w/w% whole wheat flour and 0.3-1.2w/w% inorganic salt are mixed to get mixture 1;
(2) by diastatic action in through gelatinization or the preferred gelatinization of mixture 1(without gelatinization), obtain fermention medium provided by the invention as above.
In another preference, described amylase is selected from α-amylase, beta-amylase, glucoamylase or isoamylase; More preferably α-amylase.
In another preference, with the weighing scale of whole wheat flour, described diastatic consumption is 2-1500U/g; Be more preferably 2-1000U/g.
In another preference, described diastatic operative temperature is 50-80 DEG C.
In another preference, described diastatic action time is 2-40 minute; Be more preferably 5-20 minute.
In a third aspect of the present invention, provide a kind of purposes of fermention medium provided by the invention as above, for cultivating Bacillus polymyxa (Bacillus polymyxa) or sticking series bacillus (Paenibacilluspolymyxa) to produce Polymyxin B sulfate more.
Accordingly, the invention provides and a kind ofly make the fermention medium that tunning Polymyxin B sulfate output is high.
Embodiment
Contriver, through extensive and deep research, is surprised to find that if adopt the whole wheat flour of higher concentration as nutrient media components, and before sterilization with the fermention medium after the gelatinization of amylase process, the output of raising Polymyxin B sulfate that can be larger.On this basis, the present invention is completed.
Definition
As used herein, " whole wheat flour " refers to the powder that Intact wheat contains wheat bran and plumule and all wears into.Namely Intact wheat is when abrasive dust, only passes through and pulverizes, and does not need through removing wheat bran program.
As used herein, after " gelatinization " refers to that starch granules is heated, in certain temperature range, starch small grain starts to destroy, and crystalline structure disappears, and enlarged volume, viscosity sharply rises, the pasty state in thickness.
In the present invention, the substratum after diastatic action, starch wherein becomes dextrin and maltose, and sugar chain is shortened by long, and maltose can increase, substratum viscosity degradation.
As used herein, " analysis for soybean powder " can use method well known in the art to produce through milling process, such as but not limited to, the residual cake of soya bean (soybean) after squeezing de-oiling is round pie after Hydraulic oil press, is flake after screw oil-extracting; For hard block, pulverize again after generally first smashing.Also can be obtained by commercial channel, such as but not limited to purchased from (Shijiazhuang Xing Bai biotechnology company limited).
As used herein, " corn starch " refers to that its water soluble protein is intact with fresh corn steep liquor for raw material forms through low temperature instantaneously heating spraying dry, maintains all characteristics of corn slurries.Method well known in the art can be used to prepare, also can be obtained by commercial channel, such as but not limited to purchased from (Zouping Xi Wang biotechnology company limited).
Its structure of the Polymyxin B sulfate that the present invention relates to is such as formula shown in I:
The textural difference of each component wherein related to is see table 1
| The each component of Polymyxin B sulfate | 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-isocaprylic acid (or methyl enanthic acid), OA-are sad, Leu-leucine, Ile-Isoleucine.
Fermention medium
Fermention medium provided by the invention is the substratum for cultivating many glutinous series bacillus or many glutinous series bacillus, with the total weight of described substratum, wherein comprise 0.1-1.5w/w% organic nitrogen source, 5.0-15.0w/w% whole wheat flour and 0.3-1.2w/w% inorganic salt.
In described substratum, organic nitrogen source is selected from following one or more: peptone, yeast extract, analysis for soybean powder, corn starch, wherein be preferably analysis for soybean powder or corn starch, in substratum, the consumption of organic nitrogen source is preferably 0.3-1.2w/w%, and the best is 0.5-1.2w/w%; In described substratum, inorganic salt comprise ammonium sulfate, other is selected from following one or more: potassium primary phosphate, magnesium sulfate, zinc sulfate, calcium carbonate, wherein preferably phosphoric acid potassium dihydrogen and calcium carbonate, preferably, in substratum, the content of inorganic salt is respectively 0.3-1.0w/w% ammonium sulfate, 0.01-0.08w/w% potassium primary phosphate and 0.01-0.1w/w% calcium carbonate; In substratum, the content of ammonium sulfate is more preferably 0.7-1.0%; The content of the potassium primary phosphate of substratum is more preferably 0.01-0.04w/w%; The content of the whole wheat flour in substratum is preferably 7.0-13.0w/w%, and the best is 8.0-11.0w/w%.
The cultivation bacterial strain that the present invention adopts is many glutinous series bacillus, and preferred preserving number is the Paenibacillus polymyxa of ATCC10401; Bacillus polymyxa preserving number is the Bacilluspolymyxa of NRRL B-719.
The preparation method of fermention medium
Substratum provided by the invention, uses amylase process before sterilization.
Particularly, the preparation method of fermention medium provided by the invention comprises the following steps:
The first step, mixes organic nitrogen source, whole wheat flour, inorganic salt and water according to above-mentioned formula, obtains mixture 1;
Second step, by diastatic action in through gelatinization or the mixture 1 without gelatinization, obtains fermention medium provided by the invention.
The amylase related in aforesaid method is selected from α-amylase, beta-amylase, glucoamylase or isoamylase; Preferred α-amylase; With the weighing scale of the whole wheat flour dropped in the first step, described diastatic consumption is 2-1500U/g; Be preferably 2-1000U/g, be more preferably 100-1000U/g; Described diastatic operative temperature is 50-80 DEG C, and action time is 10-60 minute; Be preferably 20-40 minute.
In the second step of aforesaid method, preferably by diastatic action in the mixture 1 through gelatinization.
In an embodiment of the present invention, the substratum after diastatic action was 121 DEG C of high-temperature sterilizations 20 minutes.
The above-mentioned feature that the present invention mentions, or the feature that embodiment is mentioned can arbitrary combination.All features that this case specification sheets discloses can with any composition forms and use, each feature disclosed in specification sheets, anyly can provide identical, alternative characteristics that is impartial or similar object replaces.Therefore apart from special instruction, the feature disclosed is only general example that is impartial or similar features.
Major advantage of the present invention is:
1, substratum provided by the invention improves Polymyxin B sulfate fermentation yield, and reduces the viscosity of fermented liquid, facilitates aftertreatment.
2, adopt substratum provided by the invention, Polymyxin B sulfate fermentation yield is higher than 1.90g/L.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, the usually conveniently conditioned disjunction condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage ratio, ratio, ratio or number by weight.
Unit in percent weight in volume in the present invention is well-known to those skilled in the art, such as, refer to the weight of solute in the solution of 100 milliliters.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.
In following examples, the measuring method reference literature of Polymyxin B sulfate output (Tiandong County, Yuan Qipeng, Sun Xinxiao, etc. response phase method optimizes the fermentation condition [J] of PXB. Chinese agronomy circular, 2010,26 (15): 53-57)
The whole wheat flour used in following examples is purchased from (Weifang kite flour limited liability company)
The corn starch used in following examples is purchased from (Zouping Xi Wang biotechnology company limited)
The analysis for soybean powder used in following examples is purchased from (Shijiazhuang Xing Bai biotechnology company limited)
Bacillus polymyxa (Bacillus polymyxa NRRL B-719) the purchased from American farming research DSMZ used in following examples.
How glutinous series bacillus (Paenibacillus polymyxaATCC10401) the purchased from American DSMZ used in following examples.
The BF7658 α-amylase used in following examples is purchased from Xingtai Wanda's biotechnology company limited
The beta-amylase used in following examples is purchased from Shandong Anke Bioengineering Co., Ltd.
The glucoamylase used in following examples is purchased from the grand mcroorganism Engineering Co., Ltd in Shandong
The isoamylase used in following examples is purchased from Novo company of Denmark
Embodiment 1
Cultivate bacterial strain: stick series bacillus (Paenibacillus polymyxa ATCC10401) more
Seed culture medium/%: corn steep liquor 2.0; Glucose 5.0; Ammonium sulfate 0.4; Sodium-chlor 0.1; Calcium carbonate 0.3; PH6.8 ~ 7.0.
Fermention medium/%: whole wheat flour 8.0; Corn starch 0.4; Ammonium sulfate 0.9; Potassium primary phosphate 0.03; Calcium carbonate 0.03.
Fermention medium is carried out gelatinization, adds BF7658 α-amylase (Xingtai Wanda's biotechnology company limited), amylase dosage 100U/g, at 50 DEG C-80 DEG C, effect 30min; 121 DEG C of high-temperature sterilization 20min afterwards.
Cultural method: by the strain inoculation of slant preservation in seed culture medium, cultivates 18h for 28 DEG C.With in the above-mentioned fermention medium of 4% inoculum size access 18L (fermentor tank total volume 35L), stirring velocity 400r/min, under 50kPa tank pressure, air flow is 1.2vvm, cultivates 48h for 30 DEG C.
Polymyxin B sulfate assay, reference literature (Tiandong County, Yuan Qipeng, Sun Xinxiao, etc. response phase method optimizes the fermentation condition [J] of PXB. Chinese agronomy circular, 2010,26 (15): 53-57.)
After testing, under above-mentioned culture medium condition, Polymyxin B sulfate output is 3.22g/L.
Embodiment 2
Other operation is with embodiment 1, and difference is that starting strain is NRRL B-719.After testing, cultivating the Polymyxin B sulfate output obtained is 3.14g/L.
Embodiment 3-14
Other operation is with embodiment 1, and difference is whole wheat flour content.After testing, the Polymyxin B sulfate output obtained is cultivated as shown in table 2 respectively:
| Embodiment | Whole wheat flour content (w/w%) | Polymyxin B sulfate output (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 operation is with embodiment 1, and difference is that fermention medium is whole wheat flour 8.0%, analysis for soybean powder 1.0%, ammonium sulfate 0.3%, potassium primary phosphate 0.06% and calcium carbonate 0.1%, and after testing, cultivating the Polymyxin B sulfate output obtained is 2.90g/L.
Embodiment 16
Other operation is with embodiment 1, and difference is that whole wheat flour content is 8.0%, adds beta-amylase, and after testing, cultivating the Polymyxin B sulfate output obtained is 2.43g/L.
Embodiment 17
Other operation is with embodiment 1, and difference is that whole wheat flour content is 8.0%, adds glucoamylase, and after testing, cultivating the Polymyxin B sulfate output obtained is 2.21g/L.
Embodiment 18
Other operation is with embodiment 1, and difference is that whole wheat flour content is 8.0%, adds isoamylase, and after testing, cultivating the Polymyxin B sulfate output obtained is 1.97g/L.
Embodiment 19
Other operation is with embodiment 1, and difference is that whole wheat flour content is 8.0%, and adding α-amylasecontent is 2U/g, and after testing, cultivating the Polymyxin B sulfate output obtained is 1.99g/L.
Embodiment 20
Other operation is with embodiment 1, and difference is that whole wheat flour content is 8.0%, adds α-amylasecontent 1000U/g, and after testing, cultivating the Polymyxin B sulfate output obtained is 3.08g/L.
Embodiment 21
Other operation is with embodiment 1, and difference is that substratum directly adds amylase sterilization without gelatinization.After testing, cultivating the Polymyxin B sulfate output obtained is 2.27g/L.
Embodiment 22
Other operation is with embodiment 1, and difference is that substratum does not add potassium primary phosphate, magnesium sulfate and zinc sulfate.After testing, cultivating the Polymyxin B sulfate output obtained is 2.83g/L.
Comparative example 1
According to document (Anand, H., Guduri, B, et al.Process for the preparation ofPolymyxin B employing Bacillus polymyxa[P] US8,119,371, B2Feb.21,2012.) in preparation substratum and cultural method.After fermentation ends, get fermented liquid 200mL, use Medium speed filter paper filtration under diminished pressure, used time 18min.
Comparative example 2
Operation, with embodiment 1, gets fermented liquid 200ml identical with the filtration condition of comparative example 1, filters used time 10min.
Comparative example 1 and comparative example 2 illustrate, can reduce the viscosity of fermentation ends secondary fermentation liquid, make substratum solid-liquid separation speed faster at substratum of the present invention.
Comparative example 3
According to document (Anand, H., Guduri, B et al.Process for the preparation ofPolymyxin B employing Bacillus polymyxa[P] US8,119,371, B2Feb.21,2012.) in preparation substratum and cultural method (see this document embodiment 1).Polymyxin B sulfate fermentation yield 1.77g/L.
The foregoing is only preferred embodiment of the present invention, and be not used to limit substantial technological context of the present invention, substantial technological content of the present invention is broadly defined in the right of application, any technology entities that other people complete or method, if with application right define identical, also or a kind of change of equivalence, be all covered by being regarded as among this right.
Claims (11)
1. for the production of a fermention medium for Polymyxin B sulfate, it is characterized in that, with the total weight of substratum, described substratum contains 0.1-1.5w/w% organic nitrogen source, 5.0-15.0w/w% whole wheat flour and 0.3-1.2w/w% inorganic salt.
2. fermention medium as claimed in claim 1, is characterized in that, described organic nitrogen source is selected from following one or more: peptone, yeast extract, analysis for soybean powder or corn starch;
Described inorganic salt are selected from following one or more: potassium primary phosphate, ammonium sulfate, magnesium sulfate, zinc sulfate or calcium carbonate.
3. fermention medium as claimed in claim 1, is characterized in that, described inorganic salt are ammonium sulfate or ammonium sulfate and the mixing being selected from one or more following materials: potassium primary phosphate, magnesium sulfate, zinc sulfate or calcium carbonate.
4. fermention medium as claimed in claim 2, it is characterized in that, described organic nitrogen source is selected from analysis for soybean powder or corn starch.
5. the fermention medium as described in any one of claim 1-4, is characterized in that, the content of described whole wheat flour is 7.0-13.0w/w%.
6. a preparation method for the fermention medium as described in any one of claim 1-5, is characterized in that, described method comprises step:
(1) with the total weight of substratum, 0.1-1.5w/w% organic nitrogen source, 5.0-15.0w/w% whole wheat flour and 0.3-1.2w/w% inorganic salt are mixed to get mixture 1;
(2) by diastatic action in through gelatinization or the preferred gelatinization of mixture 1(without gelatinization), obtain the fermention medium as described in any one of claim 1-5.
7. preparation method as claimed in claim 6, it is characterized in that, described amylase is selected from α-amylase, beta-amylase, glucoamylase or isoamylase; Preferred α-amylase.
8. preparation method as claimed in claim 6, it is characterized in that, with the weighing scale of whole wheat flour, described diastatic consumption is 2-1500U/g; Be preferably 2-1000U/g.
9. preparation method as claimed in claim 6, it is characterized in that, described diastatic operative temperature is 50-80 DEG C.
10. preparation method as claimed in claim 6, it is characterized in that, described diastatic action time is 2-40 minute; Be preferably 5-20 minute.
The purposes of 11. fermention mediums as described in any one of claim 1-5, it is characterized in that, for cultivating Bacillus polymyxa (Bacillus polymyxa) or sticking series bacillus (Paenibacillus polymyxa) to produce Polymyxin B sulfate more.
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| CN110438048A (en) * | 2019-08-23 | 2019-11-12 | 华中农业大学 | Utilize the method and its application of spirit stillage production Bacillus polymyxa additive |
| CN111100823A (en) * | 2020-01-17 | 2020-05-05 | 黄石曼菲特生物科技有限公司 | Polymyxin B sulfate production strain, preparation method and application of polymyxin B sulfate |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106868079A (en) * | 2017-04-26 | 2017-06-20 | 山东鲁抗医药股份有限公司 | The method of fermentation aerosporin culture medium and fermenting and producing aerosporin |
| CN106868079B (en) * | 2017-04-26 | 2020-12-08 | 山东鲁抗医药股份有限公司 | Culture medium for fermenting polymyxin B sulfate and method for producing polymyxin B sulfate through fermentation |
| CN110438048A (en) * | 2019-08-23 | 2019-11-12 | 华中农业大学 | Utilize the method and its application of spirit stillage production Bacillus polymyxa additive |
| CN111100823A (en) * | 2020-01-17 | 2020-05-05 | 黄石曼菲特生物科技有限公司 | Polymyxin B sulfate production strain, preparation method and application of polymyxin B sulfate |
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