CN102002469B - Bacterial strain for producing prodigiosin and method thereof - Google Patents
Bacterial strain for producing prodigiosin and method thereof Download PDFInfo
- Publication number
- CN102002469B CN102002469B CN 201010295552 CN201010295552A CN102002469B CN 102002469 B CN102002469 B CN 102002469B CN 201010295552 CN201010295552 CN 201010295552 CN 201010295552 A CN201010295552 A CN 201010295552A CN 102002469 B CN102002469 B CN 102002469B
- Authority
- CN
- China
- Prior art keywords
- prodigiosin
- fermentative production
- bacterial strain
- liquid
- production prodigiosin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
The invention provides a bacterial strain for producing prodigiosin, belonging to Serratia marcescens, wherein the preservation number of the bacterial strain is CGMCC No.4074. The bacterial strain provided by the invention has the advantages that the haematochrome can be efficiently synthesized within a liquid fermentation culture medium by using the bacterial strain and the production efficiency is high. The bacterial strain provided by the invention has low requirements to the carbon source and nitrogen source, the carbon source can be a single carbon source or a mixed carbon source and the nitrogen source can be a single nitrogen source or a mixed nitrogen source. The bacterial strain provided by the invention is continuously reproduced for more than ten generations, the genetic stability of the bacterial strain is good and the operation is simple.
Description
Technical field
The present invention relates to a kind of Serratia and uses thereof, relate in particular to a kind of Serratia strain and method thereof of producing prodigiosin.
Background technology
Prodigiosin (Prodigiosins) has structure shown below:
It is the secondary metabolite that has the important biomolecule activity by the class that multiple-microorganism produces.Usually contain the methoxyl group pyrrole skeleton structure that 3 pyrrole rings form.It has antibacterium, anti-malarial, antimycotic, protozoacide and autoimmunity depression is active as: can suppress the biological activitys such as host's rejection after delayed type hypersensitivity and the organ transplantation.The discovered in recent years prodigiosin is (part per billion concentration) under extremely low concentration, can kill fast the most of planktonic organism that causes red tide, is demonstrating huge power aspect the improvement of water pollution.Research also find prodigiosin in that the various human cancer cells such as lung cancer, mammary cancer, prostate cancer are had the resistance effect, under identical Dosages, to normal cell without any toxic action.But the other prodigiosin apoptosis of inducing cancer cell also.Therefore, prodigiosin becomes a kind of PTS that has development potentiality.
The prodigiosin preparation method mainly comprises chemical synthesis, and from nineteen sixties, just relevant for the report of prodigiosin chemosynthesis, the investigator had reported the different process route successively afterwards, and has synthesized a series of new prodigiosin analogues.Chemosynthesis is mainly opened up its derivative as main to study its molecular structure, and production and productivity is all lower, and environment is had larger pollution.
Because that the synthetic prodigiosin of biological process has is environmentally friendly, process is easy to control, cost is low, is easy to the advantages such as industrialization, receives much concern.So far the prodigiosin of finding produces the different strains that bacterium mainly concentrates on serratia marcescens, comprise Serratia marcescens Nima, Serratia marcescens R-2, Serratia marcescens W 0206B-20, Serratia marcescens RZ21, but bacterial strain is different, and prodigiosin synthesis capability difference is very large, and overall productivity is lower.A kind of endophyte serratia marcescens is disclosed among the patent CN101392227A, produce that to contain haematochrome output be 7.85g/L, fermentation time is 5 days.
Because prodigiosin has broad application prospects in fields such as medicine, foodstuff additive, water pollution improvement, ecological dyestuffs.Therefore, screening a strain prodigiosin superior strain has very important significance.
Summary of the invention
One object of the present invention is to provide a kind of serratia marcescens, and it can efficiently synthesize prodigiosin in the liquid nutrient medium cheaply, and method is simple, and is workable.
Another object of the present invention is to provide a kind of method of producing prodigiosin with the serratia marcescens bacterial strain.
A kind of bacterial strain that produces prodigiosin of the present invention is characterized in that described bacterial strain is Serratia marcescens, is serratia marcescens, is deposited in Chinese common micro-organisms DSMZ, and preserving number is: CGMCC 4074.
Bacterial strain provided by the invention is 37 ℃ of cultivation 24h on the LB solid medium, and bacterium colony takes on a red color, and is opaque, and rat is moistening, than thickness, easily provokes, and the edge is irregular.Utilize transmission electron microscope observing, peritrichous, without pod membrane, without gemma, size is (0.5～1.0) μ m * (0.8～1.2) μ m.Physiological and biochemical property sees Table 1.
The physiological and biochemical property of table 1 bacterial strain S.marcescens
Annotate: "+" represents positive, and "-" represents negative.
This bacterial strain 16sRNA gene is checked order, and the sequence of surveying is signed in among the Genbank, sequence number is: FJ919562.Resulting sequence is carried out homology relatively with existing 16SrDNA sequence among Blast software and the Genbank, utilize software MEGA4.0 to make up evolutionary tree, the result shows that it is one that bacterial strain of the present invention and Pseudomonas fluorescens (EU543578) and Serratia sp.SB 16S (EU816383) gather, sequence similarity is 99%, the results are shown in Figure 1.Comprehensive morphological is observed, Physiology and biochemistry is identified and the 16SrDNA sequential analysis, finally is accredited as the serratia serratia marcescens.
A kind of method of fermentative production prodigiosin comprises the steps:
1) spawn culture: with the bacterial classification of serratia marcescens (CGMCC 4074) in the LB solid culture based on 25-40 ℃ of constant temperature culture;
2) preparation of seed liquor: with step 1) the single bacterium colony access on the solid medium is equipped with in the container of liquid nutrient medium, and liquid amount is container volume 1/3-1/4, and in 20-40 ℃ of lower constant temperature culture 10-24 hour, rotating speed was 150-220rpm.
3) shaking flask liquid fermenting: with step 2) seed liquor changes in the liquid fermentation and culture after the sterilization, and inoculum size is 1-10v/v%, and liquid amount 50ml/250ml shaking flask is in 28-38 ℃ of constant temperature culture 48-72h, rotating speed 150-220rmp.
4) extract prodigiosin: fermented liquid centrifugal treating step 3), supernatant liquor adds isopyknic ethyl acetate extraction, vibration 20min, leave standstill the 30min layering, the centrifugal 10min of 8000rpm, phase ethyl acetate in the collection, the centrifugal residue of fermented liquid adds and the isopyknic acetone of original fermented solution, vibration 10min leaves standstill 10min, the centrifugal 10min of 8000rpm, residue continues with repeatedly extracting 3 times of acetone, mix mutually with ethyl acetate on the gained, concentrated through rotary evaporation, obtain haematochrome prodigiosin crude product.
Step 1) bacteria culture fluid that makes can place 2-4 ℃ of short term storage before use.The composition of employed LB solid medium, in percent weight in volume, unit is g/100ml, wherein peptone 0.6-1.5%, yeast powder 0.5-1.05%, NaCl 1.0-2.0%, the pH value is 5.0-9.0.
The composition of the liquid nutrient medium step 2), in percent weight in volume, the g/100ml of unit, glycerine 0.1-2.0% wherein, peptone 0.8-1.2%, yeast powder 0.5-1.0%, NaCl 1.0-2.0%, the pH value is 5.0-9.0.
The composition of the fermention medium step 3), in percent weight in volume, the g/100ml of unit, carbon source 1-10% wherein, nitrogenous source 0.5-3%, amino acid 0.04-4%, NaCl 0-5%, MgCl
20.01-0.1%, the pH value is 5.0-9.0.
One or more of carbon source preferably glycerine, N.F,USP MANNITOL, sucrose, glucose, fructose, soya-bean oil and sesame oil.
Nitrogenous source optimization protein peptone, yeast extract paste, corn steep liquor, soybean cake powder, black sesame powder, peanut powder, (NH
4)
2SO
4With one or more of nitrate, the preferred 0.5-2% of its content.
The preferred glycine of amino acid, proline(Pro) or aspartic acid, more preferably glycine 2%-4%, proline(Pro) or aspartate content 0.04-0.2%.
Serratia marcescens bacterial strain provided by the present invention has the following advantages when being used for prodigiosin production:
1) the serratia marcescens bacterial strain of the present invention's use can efficiently synthesize haematochrome in the liquid state fermentation substratum, and production efficiency is higher.Bacterial strain of the present invention in the shaking flask of 250ml, fermentation period 36-60h, haematochrome output can reach 9.10g/L.
2) bacterial strain of the present invention is lower to the Carbon and nitrogen sources requirement, and carbon source can be single carbon source or mixed carbon source, and nitrogenous source can be single nitrogenous source or mixed nitrogen.
3) this bacterial strain genetic stability is good.This bacterial strain is simple to operate, and more than 10 generations of continuous passage, the synthesis capability of haematochrome prodigiosin is substantially constant.
Embodiment
Below describe technical scheme of the present invention in detail.Only for the explanation concrete grammar, its scale of the method is not subjected to the restriction of embodiment to the embodiment of the invention.
Producing of embodiment 1 prodigiosin
The bacterial classification (CGMCC 4074) of serratia marcescens (Serratia marcescens) is lined 25-40 ℃ of constant temperature culture at the LB solid medium, then in 2-4 ℃ of short term storage: the composition of described solid medium, in percent weight in volume, unit is g/100ml, wherein peptone 1.0%, yeast powder 0.5%, NaCl 1.0%, agar powder 2.0%, pH value are 6.5.
Single bacterium colony of above-mentioned activation is accessed in the triangular flask of the 250ml that liquid nutrient medium is housed, liquid amount 50ml/250ml shaking flask, with 37 ℃ of lower constant temperature culture 12h, rotating speed is 180rpm; The composition of described liquid nutrient medium, in percent weight in volume, the g/100ml of unit, wherein peptone 1.0%, yeast powder 0.5%, NaCl 1.0%, the pH value is 6.5.
Seed liquor is changed in the liquid fermentation and culture after the sterilization, and inoculum size is 1%, and liquid amount 50ml/250ml shaking flask is in 28 ℃ of constant temperature culture 48h, rotating speed 180rpm; The composition of described fermention medium, in percent weight in volume, the g/100ml of unit, wherein glycerine 2.0%, peptone 1.3%, NaCl 0.5.0%, the pH value is 6.5.
Fermented liquid centrifugal treating, supernatant liquor add isopyknic ethyl acetate extraction, and vibration 20min leaves standstill the 30min layering, the centrifugal 10min of 8000rpm, phase ethyl acetate in the collection.The centrifugal residue of fermented liquid adds and the isopyknic acetone of original fermented solution, and vibration 10min leaves standstill 10min, the centrifugal 10min of 8000rpm, residue continue to mix mutually with ethyl acetate on the gained with repeatedly extracting 3 times of acetone, concentrated through rotary evaporation, obtain haematochrome prodigiosin crude product.
Obtain crude product and analyze through liquid matter, instrument and the condition of employing are respectively: Waters ZMD 4000 LC-MS instrument; Chromatographic column: Lichrospher C18 column (416mm * 250mm), moving phase: 80% methyl alcohol and 20% 10mmol/L ammonium formate solution, volumetric flow rate: 0.3ml/min, column temperature: 30 ℃; MS condition: ESI2MS taper hole voltage 60V, capillary voltage 3188KV, 120 ℃ of ion source temperatures, 300 ℃ of desolventizing temperature.
Above-mentionedly obtain the LC-MS analysis of haematochrome sample, the results are shown in Figure 2.In the ESI collection of illustrative plates, m/z324.26[M+H is arranged], the relative molecular mass of judging accordingly this pigment is 323.26.Can also obtain fragment ion m/z 309, m/z292, m/z 267, m/z 252, m/z 236, m/z 161 and m/z 149 etc. by LC/MS, with the mass spectrum of prodigiosin standard substance and consistent (the J Bacteriol of prodigiosin crumb data of document record, 1974,118,756-757; Microbio Biotech, 2000,2,251-257).
The Plactet-Burman optimum experimental of embodiment 2 plain prodigiosins
Rule of thumb and document, choose 9 wherein more important factors and carry out the Plactet-Burman experiment.Each factor and level see Table 2, Plactet-Burman and test concrete experimental design and see Table 3 in the PLACKETT-BURMAN experiment.
Factor and level that table 2PLACKETT-BURMAN experiment relates to
The design of table 3PLACKETT-BURMAN experiment
Press embodiment 1 preparation seed liquor, and seed culture medium is inoculated in the above-mentioned fermention medium 28 ℃ of constant temperature culture 48h, rotating speed 180rpm by inoculum size 5%.Press embodiment 1 after the fermentation ends, the measuring method of haematochrome prodigiosin, the content of detection prodigiosin, experimental data calculates by Design-Expert 7.0, and experimental result is as shown in table 4, and result is as shown in table 5.Model significantly (p=0.0256) has statistical significance.
Table 4PLACKETT-BURMAN experimental result
Table 5PLACKETT-BURMAN experiment is processed
Affecting as can be known prodigiosin output principal element by analytical results is the content of peptone content and NaCl, and prodigiosin output has reached 9.10g/L after reducing NaCl concentration and improving peptone concentration as can be seen from Table 5.
Claims (10)
1. the method for a fermentative production prodigiosin comprises the steps:
1) spawn culture: with preserving number be the serratia marcescens (Serratia marcescens) of CGMCC4074 in the LB solid culture based on 25-40 ℃ of constant temperature culture;
2) preparation of seed liquor: with step 1) the single bacterium colony access on the solid medium is equipped with in the container of liquid nutrient medium, and liquid amount is container volume 1/3-1/4, and in 20-40 ℃ of lower constant temperature culture 10-24 hour, rotating speed was 150-220rpm;
3) shaking flask liquid fermenting: with step 2) seed liquor changes in the liquid fermentation medium after the sterilization, and inoculum size is 1-10v/v%, and liquid amount 50ml/250ml shaking flask is in 28-38 ℃ of constant temperature culture 48-72h, rotating speed 150-220rmp;
4) extract prodigiosin.
2. the method for fermentative production prodigiosin according to claim 1, it is characterized in that described step 3) the fermented liquid centrifugal treating, supernatant liquor adds isopyknic ethyl acetate extraction, vibration 20min, leave standstill the 30min layering, the centrifugal 10min of 8000rpm, phase ethyl acetate in the collection, the centrifugal residue of fermented liquid add and the isopyknic acetone of original fermented solution, vibration 10min, leave standstill 10min, the centrifugal 10min of 8000rpm, residue continue to mix mutually with ethyl acetate on the gained with repeatedly extracting 3 times of acetone, concentrated through rotary evaporation, obtain haematochrome prodigiosin crude product.
3. the method for fermentative production prodigiosin according to claim 1 is characterized in that the composition of described liquid nutrient medium, in percent weight in volume, glycerine 0.1-2.0%, peptone 0.8-1.2%, yeast powder 0.5-1.0%, NaCl 1.0-2.0%, the pH value is 5.0-9.0.
4. the method for fermentative production prodigiosin according to claim 1 is characterized in that the composition of described fermention medium, in percent weight in volume, and carbon source 1-10%, nitrogenous source 0.5-3%, amino acid 0.04-4%, NaCl 0-5%, MgCl
20.01-0.1%, the pH value is 5.0-9.0.
5. the method for fermentative production prodigiosin according to claim 4 is characterized in that described carbon source is one or more of glycerine, N.F,USP MANNITOL, sucrose, glucose, fructose, soya-bean oil and sesame oil.
6. the method for fermentative production prodigiosin according to claim 4 is characterized in that described nitrogenous source is peptone, yeast extract paste, corn steep liquor, soybean cake powder, black sesame powder, peanut powder, (NH
4)
2SO
4With one or more of nitrate.
7. the method for fermentative production prodigiosin according to claim 4 is characterized in that described nitrogenous source content is 0.5-2%.
8. the method for fermentative production prodigiosin according to claim 4 is characterized in that described amino acid is glycine, proline(Pro) or aspartic acid.
9. the method for fermentative production prodigiosin according to claim 8 is characterized in that described glycine content is 0.2%-0.4%.
10. the method for fermentative production prodigiosin according to claim 8 is characterized in that described proline(Pro) or described aspartate content are 0.04-0.2%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010295552 CN102002469B (en) | 2010-09-28 | 2010-09-28 | Bacterial strain for producing prodigiosin and method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010295552 CN102002469B (en) | 2010-09-28 | 2010-09-28 | Bacterial strain for producing prodigiosin and method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102002469A CN102002469A (en) | 2011-04-06 |
| CN102002469B true CN102002469B (en) | 2013-05-01 |
Family
ID=43810173
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010295552 Expired - Fee Related CN102002469B (en) | 2010-09-28 | 2010-09-28 | Bacterial strain for producing prodigiosin and method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102002469B (en) |
Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337234A (en) * | 2011-07-29 | 2012-02-01 | 东南大学 | Serratia marcescens LTH-2 as well as screening method and application thereof |
| CN102277323B (en) * | 2011-08-08 | 2013-02-06 | 浙江师范大学 | High-prodigiosin-yield serratia marcescens strain Sm-128 and use thereof |
| CN102888351B (en) * | 2012-09-03 | 2014-06-11 | 广东药学院 | Prodigiosin high-producing strain and production method thereof |
| CN103467352B (en) * | 2013-08-15 | 2016-04-20 | 上海理工大学 | A kind of method of prodigiosin extraction purification |
| CN103757069A (en) * | 2014-01-09 | 2014-04-30 | 嘉兴学院 | Method for producing prodigiosin by coupling resin adsorption in-situ separation with fermentation |
| CN103755619B (en) * | 2014-01-09 | 2016-06-08 | 嘉兴学院 | A kind of extraction separation method of prodigiosin |
| CN103923958B (en) * | 2014-05-06 | 2016-05-04 | 合肥工业大学 | A kind of method of the zymotic fluid of producing prodigiosin |
| CN104789614A (en) * | 2015-04-08 | 2015-07-22 | 嘉兴学院 | Preparation methods of prodigiosin and biological stain as well as application of biological stain in fabric |
| CN108720032A (en) * | 2017-04-19 | 2018-11-02 | 嘉兴学院 | A kind of natural pigment pure cellulose food and preparation method thereof |
| CN109164082B (en) * | 2018-11-06 | 2019-09-24 | 益善生物技术股份有限公司 | Fluorescence-enhancing agent and the preparation method and application thereof |
| CN109679873A (en) * | 2019-01-14 | 2019-04-26 | 中国水产科学研究院长江水产研究所 | A kind of serratia marcescens metabolite and the application as aquatic immune reinforcing agent |
| CN111575221B (en) * | 2020-05-26 | 2021-12-28 | 江南大学 | Method for producing prodigiosin based on PNTs |
| CN111621436B (en) * | 2020-06-02 | 2022-05-13 | 四川省农业科学院土壤肥料研究所 | Culture solution suitable for producing prodigiosin by Serratia marcescens XD1-B-1 |
| CN111778298B (en) * | 2020-07-27 | 2023-06-27 | 中国热带农业科学院热带生物技术研究所 | Application of Serratia marcescens ITBB B5-1 in prodigiosin production |
| CN113046270A (en) * | 2021-04-06 | 2021-06-29 | 广东轻工职业技术学院 | Method for improving fermentation level of prodigiosin and application thereof |
| CN113564183A (en) * | 2021-07-15 | 2021-10-29 | 江南大学 | Method for improving synthesis of prodigiosin by serratia marcescens through overexpression gene psrA |
| CN113549643B (en) * | 2021-07-15 | 2023-08-08 | 江南大学 | Method for improving synthesis of prodigiosin by Serratia marcescens through overexpression of gene psrB |
| CN116555369B (en) * | 2023-05-10 | 2024-02-09 | 芝诺(苏州)生物科技有限公司 | Method for producing prodigiosin by fermentation of waste corn steep liquor |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101392227A (en) * | 2008-10-23 | 2009-03-25 | 新疆大学 | Bacillus prodigiosus and prodigiosin producted thereby |
-
2010
- 2010-09-28 CN CN 201010295552 patent/CN102002469B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101392227A (en) * | 2008-10-23 | 2009-03-25 | 新疆大学 | Bacillus prodigiosus and prodigiosin producted thereby |
Non-Patent Citations (1)
| Title |
|---|
| Liu,X.X et al.FJ919562.《Genbank》.2009, * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102002469A (en) | 2011-04-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102002469B (en) | Bacterial strain for producing prodigiosin and method thereof | |
| CN102965311B (en) | Bacillus subtilis and application thereof in preparation of gamma-D-polyglutamic acid | |
| CN106350469B (en) | High-temperature-resistant bacillus with cellulose degradation capability and application thereof | |
| CN104328064A (en) | Bacillus natto and application of Bacillus natto in fermentation production of vitamin K2 | |
| CN103614323B (en) | A kind of substratum of bacillus amyloliquefaciens and application | |
| CN103882080A (en) | Effective method for preparing avermectin | |
| CN102311981A (en) | Method for preparing and purifying prodigiosin | |
| CN102533612A (en) | Clostridium beijerinckii strain and screening method and use thereof | |
| CN105925503A (en) | Salt-tolerant rhizosphere growth-promoting enterobacter cloacae and application thereof | |
| CN105543147A (en) | Pseudomonas aeruginosa strain and application thereof in producing proteinase | |
| CN104726369A (en) | Raoultella ornithinolytica and applications thereof | |
| CN101338291B (en) | Method for preparing coronatine and special strain thereof | |
| CN102839130B (en) | Bacterial strain for producing gliotoxin and method for producing gliotoxin by adopting bacterial strain | |
| CN104560731B (en) | A kind of yeast-like fungi of high yield squalene and its application | |
| CN107446868B (en) | One plant of Methylotrophic bacillus and its degradation of feather produce the application of oligopeptides | |
| CN103695482A (en) | Method for producing microalgae grease by utilizing algae residues generated after oil extraction | |
| CN109797121A (en) | The microbial strains LM-1801 of one plant of degraded cellulose and its application | |
| CN101851591B (en) | Fermentation method for producing epothilone B by sorangium cellulosum and fermentation medium | |
| Bai et al. | Study on the Characteristics of Straw Fermentation by Bacillus megaterium MYB3 | |
| Xia et al. | Kitchen waste as a novel available substrate for prodigiosin production by Serratia marcescense | |
| Mohd Zakria et al. | Magnesium sulphate and β-alanine enhanced the ability of Kluyveromyces marxianus producing bioethanol using oil palm trunk sap | |
| CN107164258A (en) | Move coccus and its application in a kind of cave | |
| CN111154679B (en) | Efficient fermentation method of aflatoxin degradation bacteria | |
| CN109706100B (en) | Staphylococcus pasteuri mutant strain and application thereof in preparation of 5-aminolevulinic acid | |
| CN103074283A (en) | Bacillussp., microbial agent and applications of Bacillussp. and microbial agent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130501 Termination date: 20130928 |