CN105572069A - Rapid detection method of cis-3-hydroxyL-proline in fermentation liquor - Google Patents
Rapid detection method of cis-3-hydroxyL-proline in fermentation liquor Download PDFInfo
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- CN105572069A CN105572069A CN201610035855.7A CN201610035855A CN105572069A CN 105572069 A CN105572069 A CN 105572069A CN 201610035855 A CN201610035855 A CN 201610035855A CN 105572069 A CN105572069 A CN 105572069A
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- proline
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- fermentation liquor
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- 238000000855 fermentation Methods 0.000 title claims abstract description 12
- 230000004151 fermentation Effects 0.000 title claims abstract description 12
- 238000001514 detection method Methods 0.000 title abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 19
- 239000000243 solution Substances 0.000 claims description 18
- BJBUEDPLEOHJGE-DMTCNVIQSA-N cis-3-hydroxy-L-proline Chemical compound O[C@@H]1CC[NH2+][C@@H]1C([O-])=O BJBUEDPLEOHJGE-DMTCNVIQSA-N 0.000 claims description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 claims description 5
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 claims description 5
- FZRCKLPSHGTOAU-UHFFFAOYSA-N 6-amino-1,4-dimethylcyclohexa-2,4-diene-1-carbaldehyde Chemical group CC1=CC(N)C(C)(C=O)C=C1 FZRCKLPSHGTOAU-UHFFFAOYSA-N 0.000 claims description 3
- 238000004737 colorimetric analysis Methods 0.000 claims description 2
- 230000002596 correlated effect Effects 0.000 claims description 2
- 238000002203 pretreatment Methods 0.000 claims description 2
- 239000000047 product Substances 0.000 claims description 2
- 239000010414 supernatant solution Substances 0.000 claims description 2
- 239000012916 chromogenic reagent Substances 0.000 claims 1
- VDQQXEISLMTGAB-UHFFFAOYSA-N chloramine T Chemical compound [Na+].CC1=CC=C(S(=O)(=O)[N-]Cl)C=C1 VDQQXEISLMTGAB-UHFFFAOYSA-N 0.000 abstract description 3
- 229960001479 tosylchloramide sodium Drugs 0.000 abstract description 3
- 230000035484 reaction time Effects 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000004811 liquid chromatography Methods 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000012153 distilled water Substances 0.000 description 7
- 239000007788 liquid Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 2
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 229960002591 hydroxyproline Drugs 0.000 description 2
- 238000009413 insulation Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 235000011149 sulphuric acid Nutrition 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- 229910016374 CuSO45H2O Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 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
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- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a rapid detection method of cis-3-hydroxyL-proline in fermentation liquor, and aims at providing the rapid detection method of cis-3-hydroxyL-proline, which is convenient to use, simple and fast, and high in flexibility. Compared with a common efficient liquid chromatography or chloramine-T method, the rapid detection method has the advantages that expensive instruments are not needed, operation is easy, the reaction time is short, the number of required samples is small and sensitivity is high. The work efficiency is greatly improved, and wide application and popularization value is achieved.
Description
Technical field
The invention belongs to spectrophotometry field, relate to a kind of detection method, be related specifically to the method for quick of cis-3-hydroxy L-proline in a kind of fermentation liquor.
Background technology
Cis-3-hydroxy L-proline mainly contains chemical synthesis and microbe transformation method two kinds of methods are produced.Wherein microbe transformation method has the advantage such as nontoxic and becomes the main method that production cis-3-hydroxy L-proline takes.In current detection fermentation liquor, the method for cis-3-hydroxy L-proline content mainly contains high performance liquid chromatography and chloramine-t method.Wherein, expensive equipment needed for high performance liquid chromatography, complicated operation, is not suitable for promoting.Chloramine-t method sensitivity is low, and the reaction time is longer, and can with other material generation chromogenic reactions in fermentation liquor.
Summary of the invention
For above-mentioned deficiency, the object of the present invention is to provide a kind of reagent avoiding using toxicity high, troublesome operation simplified, can detect the content of sample fast and effectively, this project detection method sampling amount is few, and detection time is not long, and technique is simple.
For solving the problems of the technologies described above, technical scheme provided by the invention is such:
In fermentation liquor, a method for quick for cis-3-hydroxy L-proline, comprises the steps: successively
1) to the product centrifugal treating that need detect;
2) get step 1) in supernatant solution, add 0.2mLCuSO45H2O, 0.5mLNaOH, 0.5mLH2O2,0.8mLH2SO4,2mL developer successively;
3) by step 2) in solution put into 70 DEG C of water-baths heating three minutes, ice-water bath one minute immediately after taking-up.
4) by step 3) in solution put into cuvette, adopt colourimetry L-hydroxyproline is wherein measured thus obtains correlated results.
Further, step 2) described in CuSO45H2O concentration: 0.05mol/L; NaOH concentration: 2.5mol/L; H2O2:0.5%; H2SO4 concentration: 4mol/L;
Further, described developer: take 5g paradime thylaminobenzaldehyde, uses 45mL deionized water, slowly adds 55mL n-propanol.
The invention has the beneficial effects as follows: provide a kind of reagent avoiding using toxicity high, troublesome operation simplified, can detect the content of sample fast and effectively, this project detection method sampling amount is few, and detection time is not long, and technique is simple.
Embodiment
Embodiment
1. the present embodiment introduces the compound method of solution and the reagent used in the present invention and detects the flow and method of operation.
1), preparation pre-treatment reagent: accurately draw 1mL fermentation liquor, with 9000rpm centrifugal two minutes.
2), CuSO4 solution is prepared: accurately take 1.24g cupric sulfate pentahydrate, with distilled water constant volume to 100mL, stirring and dissolving.
3), NaOH solution is prepared: accurately take 10 NaOH, with distilled water constant volume to 100mL, stirring and dissolving.
4), preparation H2O2 solution: accurately draw 2mL30%H2O2 solution, with distilled water constant volume to 120mL, stir.
5), preparation H2SO4 solution: accurately draw 39.2g concentrated sulfuric acid solution, slowly join in distilled water, with distilled water constant volume to 100mL, stir.
6), developer is prepared: accurately take 5g paradime thylaminobenzaldehyde, use 45mL distilled water, slowly add 55mL n-propanol.This reagent is oxidizable, needs airtight Cord blood.
7), standard solution preparation: the L-hydroxyproline adding distil water in the volumetric flask of 100mL getting 0.1g dissolves, and with distilled water constant volume to 100mL, is the mark liquid of 1000ppm.(this mark liquid should be placed on 4 DEG C of stored refrigerated)
8), detection method: get 0.2mL fermented liquid supernatant liquid and add 0.8mL water, add 0.2mLCuSO4 solution afterwards, then add 0.5mLNaOH solution successively, 0.5mL0.5%H2O2,0.8mLH2SO4 solution, 2mL developer shake up and move in 70 DEG C of water-baths by test tube rapidly afterwards, insulation 3min, again with cold water cooling, finally measure light absorption value with ultraviolet spectrophotometer at 560nm wavelength place.Absorbance is substituted into regression equation and obtains hydroxyproline content.
2. the present embodiment introduce the inventive method detect the typical curve of cis-3-hydroxy L-proline.
Detection method: be configured to 20mg/L, 40mg/L, 60mg/L, 80mg/L, 100mg/L solution respectively in test tube, add 0.2mLCuSO4 solution afterwards, then add 0.5mLNaOH solution successively, 0.5mL0.5%H2O2,0.8mLH2SO4 solution, 2mL developer shake up and move in 70 DEG C of water-baths by test tube rapidly afterwards, insulation 3min, again with cold water cooling, finally measure light absorption value with ultraviolet spectrophotometer at 560nm wavelength place.Absorbance is substituted into regression equation and obtains hydroxyproline content.
Testing result is as follows:
The typical curve formulated as shown in Figure 1.
What finally illustrate is, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (4)
1. the method for quick of cis-3-hydroxy L-proline in fermentation liquor, is characterized in that, comprise the steps: successively
1) pre-treatment reagent process is added to the product that need detect;
2) get step 1) in supernatant solution, add oxygenant and chromogenic reagent;
3) by step 2) in solution put into cuvette, adopt colourimetry L-hydroxyproline is wherein measured thus obtains correlated results.
2. the method for quick of cis-3-hydroxy L-proline in a kind of fermentation liquor according to claim 1, is characterized in that, step 1) sample needs centrifugal treating.
3. the method for quick of cis-3-hydroxy L-proline in a kind of fermentation liquor according to claim 1, it is characterized in that, described oxygenant is H2O2.
4. the method for quick of cis-3-hydroxy L-proline in a kind of fermentation liquor according to claim 1, it is characterized in that, described developer is paradime thylaminobenzaldehyde.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610035855.7A CN105572069A (en) | 2016-01-19 | 2016-01-19 | Rapid detection method of cis-3-hydroxyL-proline in fermentation liquor |
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| CN201610035855.7A CN105572069A (en) | 2016-01-19 | 2016-01-19 | Rapid detection method of cis-3-hydroxyL-proline in fermentation liquor |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030092153A1 (en) * | 1993-09-07 | 2003-05-15 | Akio Ozaki | Process for producing cis-3-hydroxy-L-proline |
| CN101852737A (en) * | 2010-03-10 | 2010-10-06 | 谱尼测试科技（北京）有限公司 | Method for determining tryptophan content in food through photometry |
| CN101871893A (en) * | 2009-04-21 | 2010-10-27 | 陈延霞 | Colorimetric method for determining free L-hydroxyproline (L-HYP) in milk powder |
| CN104894152A (en) * | 2015-05-26 | 2015-09-09 | 江南大学 | Method for producing cis-4-hydroxy-L-proline through recombinant escherichia coli |
| CN105203478A (en) * | 2015-09-15 | 2015-12-30 | 陕西省食品药品检验所 | Fast detection method of leather-hydrolyzed protein L-hydroxyproline |
-
2016
- 2016-01-19 CN CN201610035855.7A patent/CN105572069A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030092153A1 (en) * | 1993-09-07 | 2003-05-15 | Akio Ozaki | Process for producing cis-3-hydroxy-L-proline |
| CN101871893A (en) * | 2009-04-21 | 2010-10-27 | 陈延霞 | Colorimetric method for determining free L-hydroxyproline (L-HYP) in milk powder |
| CN101852737A (en) * | 2010-03-10 | 2010-10-06 | 谱尼测试科技（北京）有限公司 | Method for determining tryptophan content in food through photometry |
| CN104894152A (en) * | 2015-05-26 | 2015-09-09 | 江南大学 | Method for producing cis-4-hydroxy-L-proline through recombinant escherichia coli |
| CN105203478A (en) * | 2015-09-15 | 2015-12-30 | 陕西省食品药品检验所 | Fast detection method of leather-hydrolyzed protein L-hydroxyproline |
Non-Patent Citations (3)
| Title |
|---|
| 丁毅 等: "农产品质量安全快速检测技术-微波辅助水解比色法快速测定动物水解蛋白中羟脯氨酸的研究", 《现代科学仪器》 * |
| 曹建中 等: "《老年骨内科学》", 31 August 1996, 人民卫生出版社 * |
| 魏东 陈文慧: "羟脯氨酸含量的测定方法优化", 《现代中西医结合杂志》 * |
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