CN110672541A - Method for quantifying lipopeptide in complex system - Google Patents
Method for quantifying lipopeptide in complex system Download PDFInfo
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- CN110672541A CN110672541A CN201910883387.2A CN201910883387A CN110672541A CN 110672541 A CN110672541 A CN 110672541A CN 201910883387 A CN201910883387 A CN 201910883387A CN 110672541 A CN110672541 A CN 110672541A
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- lipopeptide
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- complex system
- aromatic hydrocarbon
- polycyclic aromatic
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- 108010028921 Lipopeptides Proteins 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 18
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 claims abstract description 14
- MWPLVEDNUUSJAV-UHFFFAOYSA-N anthracene Chemical compound C1=CC=CC2=CC3=CC=CC=C3C=C21 MWPLVEDNUUSJAV-UHFFFAOYSA-N 0.000 claims description 28
- YNPNZTXNASCQKK-UHFFFAOYSA-N phenanthrene Chemical compound C1=CC=C2C3=CC=CC=C3C=CC2=C1 YNPNZTXNASCQKK-UHFFFAOYSA-N 0.000 claims description 28
- 238000000855 fermentation Methods 0.000 claims description 20
- 230000004151 fermentation Effects 0.000 claims description 20
- 238000002835 absorbance Methods 0.000 claims description 4
- 239000011550 stock solution Substances 0.000 claims description 4
- 230000007613 environmental effect Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims 1
- 238000010790 dilution Methods 0.000 claims 1
- 238000005259 measurement Methods 0.000 claims 1
- 244000005700 microbiome Species 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 11
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- 238000002372 labelling Methods 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000003876 biosurfactant Substances 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- AFWTZXXDGQBIKW-UHFFFAOYSA-N C14 surfactin Natural products CCCCCCCCCCCC1CC(=O)NC(CCC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)O1 AFWTZXXDGQBIKW-UHFFFAOYSA-N 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 239000003129 oil well Substances 0.000 description 3
- NJGWOFRZMQRKHT-UHFFFAOYSA-N surfactin Natural products CC(C)CCCCCCCCCC1CC(=O)NC(CCC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)O1 NJGWOFRZMQRKHT-UHFFFAOYSA-N 0.000 description 3
- NJGWOFRZMQRKHT-WGVNQGGSSA-N surfactin C Chemical compound CC(C)CCCCCCCCC[C@@H]1CC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)O1 NJGWOFRZMQRKHT-WGVNQGGSSA-N 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 208000035404 Autolysis Diseases 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 206010057248 Cell death Diseases 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 230000028043 self proteolysis Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- ZCUQOPGIJRGJDA-UHFFFAOYSA-N 1-naphthalen-1-ylethane-1,2-diamine Chemical compound C1=CC=C2C(C(N)CN)=CC=CC2=C1 ZCUQOPGIJRGJDA-UHFFFAOYSA-N 0.000 description 1
- KAEDEGFCOPIKKM-UHFFFAOYSA-N 2-bromo-1-pyren-1-ylethanone Chemical compound C1=C2C(C(=O)CBr)=CC=C(C=C3)C2=C2C3=CC=CC2=C1 KAEDEGFCOPIKKM-UHFFFAOYSA-N 0.000 description 1
- 241000587112 Enterobacteriaceae sp. Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 229910001410 inorganic ion Inorganic materials 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 230000007269 microbial metabolism Effects 0.000 description 1
- 108091005601 modified peptides Proteins 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
Abstract
The invention relates to a method for quantifying lipopeptide in a complex system, which comprises the steps of mixing a complex system sample with polycyclic aromatic hydrocarbon, and detecting by using an ultraviolet spectrophotometer to determine the content of lipopeptide in the sample. Compared with the prior art, the method has the advantages of simple and convenient operation, short detection time, low cost, no need of large-scale instruments and equipment and the like.
Description
Technical Field
The invention belongs to the technical field of biosurfactant detection and evaluation, and particularly relates to quantitative detection of lipopeptide.
Background
Lipopeptide is a biosurfactant which is produced by microbial metabolism, has excellent performance, is most deeply researched so far and has great application potential. Among them, Surfactin (Surfactin) is the most representative lipopeptide, has not only good surface activity but also special biological activity, and has been used in the industries of medicine, cosmetics, food, oil extraction and the like.
At present, lipopeptide is obtained by wild bacteria fermentation, and fermentation substrates of lipopeptide comprise complex and various organic matters (such as yeast extract, peptone and the like) and also contain various inorganic ions. Meanwhile, in the later stage of fermentation, substances such as protein, nucleic acid and the like can be generated due to cell autolysis, and the system composition is very complex. In general, Lipopeptides are Produced by fermentation using wild bacteria in a low yield of about 0.5-1.5g/L (Young J, Yang S, Mu B. structural characterization of Lipid from Enterobacteriaceae sp. strain N18 derivatives of Lipid Science & Technology,2015,117 (890-898), Li Y M, Haddad N I A, Yang S Z, et al. variants of Lipid Science & Technology,2015,117 (890-11) modified peptides of Lipid Science N221 in fusion media compositions evaluation of Lipid Science, MS. strain Journal of culture of protein, strain S235, strain S18, strain S2, strain S221 in strain culture of protein, strain S12, strain S235, strain S12, strain S10, strain S10. 12. and strain S2. strain S.A. (Biostrain S.A.),44. strain S., 2008,150(3):305-325). For fermentation broth samples, the prior art generally adopts a method of drying and weighing after acid precipitation to quantify lipopeptides in the fermentation broth. Because organic nutrients which are not fully consumed and utilized and proteins, nucleic acids and the like derived from cell autolysis at the later stage of fermentation exist in the fermentation liquor, some of the substances can be precipitated together with the lipopeptide, so that the lipopeptide has errors in quantification. For environmental samples, such as oil well production fluids, the lipopeptide content is low, generally in the ppm range. Although, technical methods for labeling lipopeptides by ultraviolet or fluorescent groups to realize high-sensitivity detection have been established at present, such as detection by labeling amino acids with a BS-TFA reagent after hydrolysis of lipopeptides, HPLC detection by labeling lipopeptides with bromoacetylpyrene, fluorescent detection by labeling Surfactin with naphthylethylenediamine, direct detection by MALDI-TOF MS, and the like. Such methods require some manipulation or testing of lipopeptide purification, labeling reactions, HPLC or MS. Therefore, the method has the defects of complex operation, long detection time, high cost, large-scale instrument and equipment and the like.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a technical method for quickly detecting lipopeptide based on the characteristic that a biosurfactant has a solubilized anthracene, phenanthrene and other compounds with ultraviolet absorption groups, so that the defects in the prior art are overcome, and the simple, convenient and quick detection and quantification of the lipopeptide in a complex system are realized.
The purpose of the invention can be realized by the following technical scheme: a method for quantifying lipopeptide in a complex system is characterized in that a complex system sample is mixed with polycyclic aromatic hydrocarbon, and an ultraviolet spectrophotometer is used for detecting to determine the content of lipopeptide in the sample.
The complex system sample is fermentation broth stock solution or fermentation broth diluent or an environment sample when the lipopeptide is produced by microbial fermentation.
The polycyclic aromatic hydrocarbon is anthracene or phenanthrene.
The volume-mass ratio of the complex system sample to the polycyclic aromatic hydrocarbon is 1:1(mL/mg, v/w).
The complex system sample is mixed with polycyclic aromatic hydrocarbon and then placed at 30 ℃ for 5 hours.
When the polycyclic aromatic hydrocarbon is anthracene, a complex system sample and an anthracene mixed sample are used for measuring absorbance OD under 254nm by using an ultraviolet spectrophotometer254nmOD in terms of lipopeptide content (mg/L)254nmX 454.5.
When the polycyclic aromatic hydrocarbon is phenanthrene, measuring the absorbance OD of a complex system sample and a phenanthrene mixed sample at 254nm by using an ultraviolet spectrophotometer254nmOD in terms of lipopeptide content (mg/L)254nmX 238.1.
Compared with the prior art, the invention adopts the compound anthracene or phenanthrene with the ultraviolet absorption group to be mixed with fermentation liquor stock solution or fermentation liquor diluent or an environment sample when the lipopeptide is produced by microbial fermentation, and the anthracene or phenanthrene is solubilized into the sample by utilizing the characteristic that the lipopeptide biosurfactant can solubilize the anthracene or phenanthrene. The solubilization quantity of anthracene or phenanthrene is in direct proportion to the lipopeptide content in the sample, so that the lipopeptide in the sample can be easily quantified by measuring the quantity of anthracene or phenanthrene at 254 nm. The method has the advantages of simple operation, short detection time, low cost, no need of large-scale instruments and equipment, and the like.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Example 1
15mL of stock solution of the lipopeptide fermentation liquor is added with 15mg of anthracene crystal and placed for 5 hours at 35 ℃. Centrifuging at 5000rpm for 5min, using deionized water as control, and measuring OD with supernatant254nm0.9, lipopeptide content (mg/L) 0.9 × 454.5 409.05 mg/L.
Example 2
And (3) 15mL of lipopeptide fermentation broth diluent (1 part of fermentation broth diluted by 9 parts of deionized water), adding 15mg of phenanthrene crystals, and standing at 35 ℃ for 5 hours. Centrifuging at 5000rpm for 5min, using deionized water as control, and measuring OD with supernatant254nm0.5, lipopeptide content (mg/L) 0.5 × 238.1 119.05 mg/L.
Example 3
And (3) carrying out weak gel and microbial flooding combination on the North China oil field to improve the oil well produced liquid in the recovery ratio test area, centrifugally removing oil, removing sediment, taking 15mL of sample, adding 15mg of phenanthrene crystal, and standing at 35 ℃ for 5 h. Centrifuging at 5000rpm for 5min, using deionized water as control, and measuring OD with supernatant254nm0.1, lipopeptide content (mg/L) 0.1 × 238.1 mg/L23.81 mg/L.
Example 4
And centrifugally removing oil and sediment of 15mL of the produced liquid of the oil well in the enhanced recovery test area of the North China oil field by microbial flooding, adding 15mg of anthracene crystal, and standing at 35 ℃ for 5 hours. Centrifuging at 5000rpm for 5min, using deionized water as control, and measuring OD with supernatant254nm0.03 mg/L lipopeptide content (mg/L) 0.03 × 454.5 mg/L13.6 mg/L.
Claims (7)
1. A method for quantifying lipopeptide in a complex system is characterized in that a complex system sample is mixed with polycyclic aromatic hydrocarbon, and an ultraviolet spectrophotometer is used for detecting to determine the content of lipopeptide in the sample.
2. The method of claim 1, wherein the sample of the complex system is a fermentation broth stock solution or a dilution of a fermentation broth or an environmental sample during the production of the lipopeptide by fermentation with a microorganism.
3. The method of claim 1, wherein the polycyclic aromatic hydrocarbon is anthracene or phenanthrene.
4. The method of claim 1, wherein the volume-to-mass ratio of the sample to the polycyclic aromatic hydrocarbon is 1:1(mL/mg, v/w).
5. The method of claim 1, wherein the sample of the complex system is mixed with the polycyclic aromatic hydrocarbon and then kept at 30 ℃ for 5 hours.
6. The method of claim 1, wherein when the polycyclic aromatic hydrocarbon is anthracene, the absorbance OD of the mixed sample of the polycyclic aromatic hydrocarbon and the anthracene is measured at 254nm by an ultraviolet spectrophotometer254nmOD in terms of lipopeptide content (mg/L)254nmX 454.5.
7. The method of claim 1, wherein when the polycyclic aromatic hydrocarbon is phenanthrene, a complex system sample and a phenanthrene mixed sample are subjected to measurement of absorbance OD at 254nm by using an ultraviolet spectrophotometer254nmOD in terms of lipopeptide content (mg/L)254nmX 238.1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910883387.2A CN110672541A (en) | 2019-09-18 | 2019-09-18 | Method for quantifying lipopeptide in complex system |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910883387.2A CN110672541A (en) | 2019-09-18 | 2019-09-18 | Method for quantifying lipopeptide in complex system |
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| Publication Number | Publication Date |
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| CN110672541A true CN110672541A (en) | 2020-01-10 |
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| CN201910883387.2A Pending CN110672541A (en) | 2019-09-18 | 2019-09-18 | Method for quantifying lipopeptide in complex system |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102331407A (en) * | 2011-06-15 | 2012-01-25 | 天津膜天膜科技股份有限公司 | Method for detecting remaining trace tween 80 in water |
| US20150293110A1 (en) * | 2014-04-11 | 2015-10-15 | Surmodics Ivd, Inc. | Compositions and methods for in vitro diagnostic tests including zwitterionic solubilization reagent |
| CN107870168A (en) * | 2017-10-24 | 2018-04-03 | 苏州长光华医生物医学工程有限公司 | A kind of method and device for determining critical micelle concentration of surfactant |
-
2019
- 2019-09-18 CN CN201910883387.2A patent/CN110672541A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102331407A (en) * | 2011-06-15 | 2012-01-25 | 天津膜天膜科技股份有限公司 | Method for detecting remaining trace tween 80 in water |
| US20150293110A1 (en) * | 2014-04-11 | 2015-10-15 | Surmodics Ivd, Inc. | Compositions and methods for in vitro diagnostic tests including zwitterionic solubilization reagent |
| CN107870168A (en) * | 2017-10-24 | 2018-04-03 | 苏州长光华医生物医学工程有限公司 | A kind of method and device for determining critical micelle concentration of surfactant |
Non-Patent Citations (2)
| Title |
|---|
| SHUDONG LI.ET AL: ""Effect of rhamnolipid biosurfactant on solubilization of polycyclic aromatic hydrocarbons"", 《MARINE POLLUTION BULLETIN》 * |
| 侯晓虹: "《普通高等院校环境科学与工程类系列规划教材 环境污染物分析》", 31 January 2017, 中国建材工业出版社 * |
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Application publication date: 20200110 |
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