CN104276990A - Method for decoloring L-hydroxyproline fermentation broth by utilizing active carbon - Google Patents
Method for decoloring L-hydroxyproline fermentation broth by utilizing active carbon Download PDFInfo
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- CN104276990A CN104276990A CN201310286354.2A CN201310286354A CN104276990A CN 104276990 A CN104276990 A CN 104276990A CN 201310286354 A CN201310286354 A CN 201310286354A CN 104276990 A CN104276990 A CN 104276990A
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- fermented liquid
- gac
- oxyproline
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
- C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
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Abstract
The invention relates to a method for decoloring an L-hydroxyproline fermentation broth by utilizing active carbon. The method comprises pretreatment of active carbon, pretreatment of the fermentation broth, continuous decoloring of the L-hydroxyproline fermentation broth, regeneration of active carbon and other steps. According to the method, continuous decoloring is performed on the L-hydroxyproline fermentation broth by filling a chromatography column with powdered active carbon, and compared with a process of using powdered active carbon for decoloring in batches, active carbon can be repeatedly used for multiple times, column filling does not need repeatedly performing, regeneration can be directly performed after decoloring, the labor intensity is reduced, and the cost of purchasing active carbon is reduced. Additionally, the whole process is performed at room temperature, decoloring on a large scale is relatively easy to operate, high-temperature danger is avoided, energy is saved and cost is reduced. The method is mainly applied to removal of pigments in the L-hydroxyproline fermentation broth and is suitable for industrial application.
Description
Technical field
The present invention relates to a kind of method utilizing gac to decolour to L-oxyproline fermented liquid, be applicable to the decolorization that fermentation method prepares L-oxyproline.
Background technology
L-oxyproline (Hydroxyproline, Hyp) is chiral molecules, does not belong to 20 kinds of common amino acids, has 4 kinds of isomerss.This tests oxyproline used is trans-4-hydroxy-l-proline (trans-4-Hydroxy-L-Proline).The distribution of L-oxyproline is very wide, is the important composition composition of collagen, gelatin, plant cell wall protein matter and Secondary Metabolites of Microorganisms.L-oxyproline had played irreplaceable effect in food, medicine, makeup etc. in the last few years, can as the fodder additives of infant or baby food with some nonmammalians, also play effects such as regulating fat emulsification, antitumor, hypertension, also as additive, anti-oxidant, radiation-resistant effect can be had in addition in makeup.
Pigment in fermented liquid mainly contains three kinds of sources: raw material self with pigment, microbial metabolism generates in fermenting process pigment and fermented liquid in the pigment that generates of residual sugar and some nitrogenous source class substance reaction.Pigment can be divided into three types again: ionic, polarity and nonpolar.In whole fermenting process, fermented liquid is generally in acid state, and in sour environment, pigment mostly is acidic ionic.The adsorption of gac is mainly nonspecific, but the change of some envrionment conditions is conducive to the pigment in charcoal absorption fermented liquid, thus reaches the object removing pigment.Production mainly gelatin hydrolysate method and the fermentation method of current L-oxyproline, but which kind of produces the method for L-oxyproline all with the generation of pigment, and these pigments have a strong impact on carrying out smoothly of separation and Extraction work.
Not yet there is the relevant report about the decolouring of L-oxyproline fermented liquid at present.Gac is utilized to have the report that the L-oxyproline that hydrolysis method is produced decolours: (Shen Jinyu, Zhang Peng fly. and proline(Pro), oxyproline decoloration process are studied. the 9th national biochemical industry academic conference, 2000).The report of fermentative Production L-oxyproline has: utilize intestinal bacteria to produce the method (Unexamined Patent 3-266995) of trans-4-hydroxyl-L-oxyproline from 4-hydroxyl-2-oxoglutaric acid; The method (European patent EP 0547898A2) utilizing mould direct fermentation to produce; Streptomycete is utilized to transform the method (J.Biol.Chem of L-PROLINE, Journal of Biotogical Chemistry, 254,6684-6690,1979, Biochem.Biophys and Res, Biochemical and Biophysical Research Communication, 120,45-51,1984).
Summary of the invention
Current fermentative Production L-oxyproline produces with a large amount of pigment, have a strong impact on carrying out smoothly of later separation extraction work, and not yet have the article about the decolouring of L-oxyproline fermented liquid published at present, product must reach again certain transmittance and just meet production standard, and the problem to be solved in the present invention is to provide a kind of method utilizing gac to decolour to L-oxyproline fermented liquid.
The present invention, by changing character and the external environment thereof of fermented liquid, utilizes the decoloring ability of gac efficiently, was both removed as far as possible by the pigment in L-oxyproline fermented liquid, reduces again the absorption to L-oxyproline as far as possible.For the present invention that solves the problem technical scheme used is as follows:
Utilize the method that gac decolours to L-oxyproline fermented liquid, the method comprises the following steps:
(1) pre-treatment of fermented liquid: L-oxyproline fermented liquid is in the centrifugal 20min of 8000r/min high speed desktop refrigerated centrifuge, and removing thalline and solid substance, getting supernatant, to be placed in 4 DEG C of chromatography cabinets for subsequent use.
(2) pre-treatment of gac: powdered active carbon deionized water is carried out stirring and washing, repeatedly several times, leave standstill to supernatant without obvious impurity, then the 0.02mol/L hydrochloric acid that deionized water adds two volumes is discarded, leave standstill two hours, finally be washed till close to neutral with deionized water, dry for standby in baking oven.
(3) continuous stripping of L-oxyproline fermented liquid: the powdered active carbon taking 4.0g drying; load in chromatography column; with the salt pickling post that pH value is about 4; guarantee bubble-free in the middle of gac; then L-oxyproline fermented liquid is with the flow velocity upper prop of 1mL/min, decolouring at room temperature carry out, according in Chinese Pharmacopoeia to amino acid solution transmittance detect regulation; be with reference to selecting 430nm to measure wavelength for transmittance with deionized water, as outflow fermented liquid transmittance T
430lower than 97% time stop fermented liquid upper prop.
(4) regeneration of activated carbon: first pass into deionized water in chromatography column and wash away not by the fermented liquid decoloured, now elutant recoverable, then the sodium hydroxide solution being heated to 70 DEG C through thermostat water bath is passed into, flow velocity is 0.5mL/min, does with reference to transmittance T to effluent liquid with deionized water
430be greater than till 99.9%, finally with the gac after deionized water wash regeneration, be washed till close to neutral.
(5) gac after regeneration can repeating step (3) and (4)
In step (1), fermented liquid used is that the Escherichia coli fermentation 40h of genetic engineering modified mistake obtains, L-hydroxyproline content about 12g/L, and pH value is about 6.5, and the absorbancy being 430nm place at wavelength is 29.4.
In step (2), gac surface area per weight used can reach 500 ~ 1000m
2/ g.
In step (3), chromatography column used is the glass column of φ 25mm × 250mm, has removable filter paper bottom chromatography column, prevents gac with outflows such as fermented liquids.
In step (4), concentration of sodium hydroxide solution used is 6% (w/v).
Step (3) and (4) circulation time, the decolorizing effect of gac can die down gradually.
Beneficial effect:
1. choice of powder shaped activated carbon compares granular active carbon larger specific surface area, thus has stronger adsorpting pigment ability, uses less gac just can to decolour bulk fermentation liquid, reduces the number of times of repeatedly living again, and reduces labour intensity, saves labour cost.
2. select continuous stripping to compare traditional decolouring in batches more simple, improve the utilization ratio of gac, be applicable to industrial-scale decolouring.
3. select continuous stripping, can Direct Regeneration after decolouring completes, the chromatography column that need not more renew, decolorization operations is convenient more in batches, reduces labour intensity.
Embodiment
General explanation: fermented liquid involved by embodiment is obtain after the Escherichia coli fermentation 40h of genetic modification, L-hydroxyproline content about 12g/L, and pH value is about 6.5, and the absorbancy being 430nm place at wavelength is 29.4.
The measuring method of L-oxyproline: chloramine-t method.Concrete steps are as follows: 1. prepare L-hydroxyproline standard liquid: concentration is respectively 1mg/L, 2mg/L, 4mg/L, 5mg/L.Blank solution is deionized water.Treat test sample: fermented liquid centrifuging and taking supernatant, dilute certain multiple, in test tube, constant volume is to 2500 μ L.2. add 1mL chloramine-T, add 1mL developer again after 20min, add a cover mixing, be placed in 60 DEG C of water-baths and heat 20min, take out and be placed on standing 1min in cold water.3. A is surveyed with spectrophotometer at 560nm place
560, the L-Hydroxyproline concentration treating test sample is then calculated according to typical curve.L-oxyproline rate of loss is for L-oxyproline minimizing concentration in decolouring secondary fermentation liquid is divided by L-Hydroxyproline concentration in decolouring primary fermentation liquid.
Embodiment 1:
The concrete decoloring method of continuous stripping carries out according to the following steps.
(1) L-oxyproline fermented liquid is in the centrifugal 20min of 8000r/min high speed desktop refrigerated centrifuge, and removing thalline and solid substance, get supernatant and be placed in 4 DEG C of chromatography cabinets.
(2) powdered active carbon deionized water is carried out stirring and washing, repeatedly several times, leave standstill to supernatant without obvious impurity, then the 0.02mol/L hydrochloric acid that deionized water adds two volumes is discarded, leave standstill two hours, be finally washed till close to neutral with deionized water, dry in baking oven.
(3) powdered active carbon of 4.0g drying is taken; load in chromatography column; with the salt pickling post that pH value is about 4; guarantee bubble-free in the middle of gac; then L-oxyproline fermented liquid is with the flow velocity upper prop of 1mL/min; decolouring is at room temperature carried out, and is with reference to selecting 430nm to measure wavelength for transmittance with deionized water, as outflow fermented liquid transmittance T
430lower than 97% time stop fermented liquid upper prop.
(4) regeneration of activated carbon: first pass into deionized water in chromatography column and wash away not by the fermented liquid decoloured, now elutant recoverable, then passing into and being heated to 70 DEG C of concentration through thermostat water bath is the sodium hydroxide solution of 6%, flow velocity is 0.5mL/min, does with reference to transmittance T to effluent liquid with deionized water
430be greater than till 99.9%, finally with the gac after deionized water wash regeneration, be washed till close to neutral.
(5) gac after regeneration can repeating step (3) and (4).A rear decoloring effect of decolouring is as follows: process fermented liquid cumulative volume 205mL, transmittance 99.0%, L-oxyproline rate of loss 12.0% after the mixing of decolouring fermented liquid, regeneration of activated carbon rate 91.8%.
Embodiment 2:
The concrete decoloring method of continuous stripping carries out according to the following steps.
(1) L-oxyproline fermented liquid is in the centrifugal 20min of 8000r/min high speed desktop refrigerated centrifuge, and removing thalline and solid substance, get supernatant and be placed in 4 DEG C of chromatography cabinets.
(2) powdered active carbon deionized water is carried out stirring and washing, repeatedly several times, leave standstill to supernatant without obvious impurity, then the 0.02mol/L hydrochloric acid that deionized water adds two volumes is discarded, leave standstill two hours, be finally washed till close to neutral with deionized water, dry in baking oven.
(3) powdered active carbon of 6.0g drying is taken; load in chromatography column; with the salt pickling post that pH value is about 4; guarantee bubble-free in the middle of gac; then L-oxyproline fermented liquid is with the flow velocity upper prop of 1mL/min; decolouring is at room temperature carried out, and is with reference to selecting 430nm to measure wavelength for transmittance with deionized water, as outflow fermented liquid transmittance T
430lower than 97% time stop fermented liquid upper prop.
(4) regeneration of activated carbon: first pass into deionized water in chromatography column and wash away not by the fermented liquid decoloured, now elutant recoverable, then passing into and being heated to 70 DEG C of concentration through thermostat water bath is the sodium hydroxide solution of 6%, flow velocity is 0.5mL/min, does with reference to transmittance T to effluent liquid with deionized water
430be greater than till 99.9%, finally with the gac after deionized water wash regeneration, be washed till close to neutral.
(5) gac after regeneration can repeating step (3) and (4).A rear decoloring effect of decolouring is as follows: process fermented liquid cumulative volume 345mL, transmittance 99.1%, L-oxyproline rate of loss 11.5% after the mixing of decolouring fermented liquid, regeneration of activated carbon rate 92.3%.
Embodiment 3:
The concrete decoloring method of continuous stripping carries out according to the following steps.
(1) L-oxyproline fermented liquid is in the centrifugal 20min of 8000r/min high speed desktop refrigerated centrifuge, and removing thalline and solid substance, get supernatant and be placed in 4 DEG C of chromatography cabinets.
(2) powdered active carbon deionized water is carried out stirring and washing, repeatedly several times, leave standstill to supernatant without obvious impurity, then the 0.02mol/L hydrochloric acid that deionized water adds two volumes is discarded, leave standstill two hours, be finally washed till close to neutral with deionized water, dry in baking oven.
(3) powdered active carbon of 8.0g drying is taken; load in chromatography column; with the salt pickling post that pH value is about 4; guarantee bubble-free in the middle of gac; then L-oxyproline fermented liquid is with the flow velocity upper prop of 2mL/min; decolouring is at room temperature carried out, and is with reference to selecting 430nm to measure wavelength for transmittance with deionized water, as outflow fermented liquid transmittance T
430lower than 97% time stop fermented liquid upper prop.
(4) regeneration of activated carbon: first pass into deionized water in chromatography column and wash away not by the fermented liquid decoloured, now elutant recoverable, then passing into and being heated to 70 DEG C of concentration through thermostat water bath is the sodium hydroxide solution of 6%, flow velocity is 1mL/min, does with reference to transmittance T to effluent liquid with deionized water
430be greater than till 99.9%, finally with the gac after deionized water wash regeneration, be washed till close to neutral.
(5) gac after regeneration can repeating step (3) and (4).A rear decoloring effect of decolouring is as follows: process fermented liquid cumulative volume 400mL, transmittance 99.1%, L-oxyproline rate of loss 12% after the mixing of decolouring fermented liquid, regeneration of activated carbon rate 91.5%.
Claims (6)
1. utilize the method that gac decolours to L-oxyproline fermented liquid, it is characterized in that, the method comprises the following steps:
(1) pre-treatment of fermented liquid.
(2) pre-treatment of gac.
(3) continuous stripping of L-oxyproline fermented liquid: pretreated fermented liquid passes into the chromatography column that gac is housed, flow measurement fluid transmittance.
(4) regeneration of activated carbon: pass into regenerator in the backward chromatography column that decoloured, the absorbancy of flow measurement fluid.
(5) gac after regeneration can repeating step (3) and (4).
2. the gac as claimed in claim 1 method of decolour to L-oxyproline fermented liquid, it is characterized in that, in step (1), fermented liquid used is the Recombinant E. coli Fermentation Broth of product L-oxyproline.
3. the gac as claimed in claim 1 method of decolouring to L-oxyproline fermented liquid, it is characterized in that, in step (2), gac used is powdered active carbon, and surface area per weight is 500 ~ 1000m
2/ g.
4. the gac as claimed in claim 1 method of decolouring to L-oxyproline fermented liquid, it is characterized in that, in step (3), flow measurement fluid transmittance wavelength used is 430nm.
5. the gac as claimed in claim 1 method of decolouring to L-oxyproline fermented liquid, it is characterized in that, in step (4), regenerator used is for changing the reagent of environmental pH residing for gac.
6. the gac as claimed in claim 1 method of decolouring to L-oxyproline fermented liquid, it is characterized in that, in step (4), flow measurement fluid absorbancy wavelength used is 430nm.
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| CN201310286354.2A CN104276990B (en) | 2013-07-04 | 2013-07-04 | A kind of method that utilization activated carbon decolourizes to L hydroxyprolines zymotic fluid |
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| CN201310286354.2A CN104276990B (en) | 2013-07-04 | 2013-07-04 | A kind of method that utilization activated carbon decolourizes to L hydroxyprolines zymotic fluid |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105777603A (en) * | 2016-04-01 | 2016-07-20 | 天津市敬业精细化工有限公司 | Method for extracting L-hydroxyproline from L-hydroxyproline fermentation liquor |
| CN105837488A (en) * | 2016-03-31 | 2016-08-10 | 天津市敬业精细化工有限公司 | Hydroxyproline fermentation production technology |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03195494A (en) * | 1989-12-26 | 1991-08-27 | Toray Ind Inc | Production of l-proline by fermentation |
| JPH0754180A (en) * | 1993-08-06 | 1995-02-28 | Seiwa Kasei:Kk | Method for separation and refining of hydroxyproline from completely hydrolyzed liquid of collagen |
| CN101348453A (en) * | 2008-08-29 | 2009-01-21 | 广东肇庆星湖生物科技股份有限公司 | Method for purifying proline |
-
2013
- 2013-07-04 CN CN201310286354.2A patent/CN104276990B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03195494A (en) * | 1989-12-26 | 1991-08-27 | Toray Ind Inc | Production of l-proline by fermentation |
| JPH0754180A (en) * | 1993-08-06 | 1995-02-28 | Seiwa Kasei:Kk | Method for separation and refining of hydroxyproline from completely hydrolyzed liquid of collagen |
| CN101348453A (en) * | 2008-08-29 | 2009-01-21 | 广东肇庆星湖生物科技股份有限公司 | Method for purifying proline |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105837488A (en) * | 2016-03-31 | 2016-08-10 | 天津市敬业精细化工有限公司 | Hydroxyproline fermentation production technology |
| CN105837488B (en) * | 2016-03-31 | 2019-02-15 | 天津市敬业精细化工有限公司 | A kind of hydroxyproline fermentation manufacturing technique |
| CN105777603A (en) * | 2016-04-01 | 2016-07-20 | 天津市敬业精细化工有限公司 | Method for extracting L-hydroxyproline from L-hydroxyproline fermentation liquor |
| CN105777603B (en) * | 2016-04-01 | 2019-02-15 | 天津市敬业精细化工有限公司 | A method of extracting L- hydroxyproline from L- hydroxyproline fermentation liquid |
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