CN107365810A - The method for producing high-purity D alanine - Google Patents
The method for producing high-purity D alanine Download PDFInfo
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- CN107365810A CN107365810A CN201710722210.5A CN201710722210A CN107365810A CN 107365810 A CN107365810 A CN 107365810A CN 201710722210 A CN201710722210 A CN 201710722210A CN 107365810 A CN107365810 A CN 107365810A
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Abstract
The method for providing production high-purity D alanine, this method include：The step of microbial inoculant is fermented into the fermentation medium containing 1.0% 1.5%w/v DL alanine；When the pH of zymotic fluid is more than 7.0, and the mass ratio of L alanine and D alanine is less than 1:When 4, the aqueous solution of nitric acid containing DL alanine is added to zymotic fluid, with control ph between 6.5 7.0.Using low concentration DL alanine as substrate, and DL alanine substrates are added when adjusting zymotic fluid pH, shorten fermentation period, completed fermentation period at 36 42 hours, prepare high concentration D alanine.
Description
Technical field
The present invention relates generally to the preparing technical field of D-alanine, and especially, it is related to production high-purity D-alanine
Method.
Background technology
D-alanine is present in most of organic organizations as ALANINE optical isomer, is such as used as cell membrane peptide
The constituent of wall sugar or peptide antibiotic, is a kind of important chiral organic compound.
D-alanine is mainly used on medicine production and food additives at present.
On medicinal, D-alanine is the chiral raw material that pharmaceutical industry carries out chiral synthesis, is mainly used in chiral drug, medicine
The synthesis of intermediate.It is currently used primarily in production New-type wide-spectrum antibiotic, D- Propanolamines and polypeptide etc..D-alanine is also in itself
Produce the raw material of vitamin B6.D-alanine has primary amine groups, can competitively substitute secondary amine and be generated with nitrite
VANSlyke reacts, and nitrogen and organic acid is decomposed into, so as to inhibit carcinogen N-nitrosodimethylamine DMNA generation.Cause
This, D-alanine is clinically employed as a kind of new cancer gene therapy method.
In food additives industry, D-alanine is mainly used in the flavor effect of increase chemical seasoning, improves artificial
The sense of taste of sweetener, improve the tart flavour of organic acid.Because D-alanine has strong sweet taste in itself, sense of taste value is 4, is in amino acid
One kind of most sweet tea.It can synthesize dipeptide sweetener alitame (2.2.4.4-tea-methylthiet with L-Aspartic acid
anylamine).Alitame sweet taste quality better, good taste, without peculiar smell and offending taste, and only part A Li afterwards
Sugared ginseng and organism metabolism, its maximum quantity of heat production are only equivalent to the 0.02% of suitable sugariness sucrose, meet people at present to low-heat
The pursuit of food.Play an important roll on the functional food of diabetes patient.These applications provide huge city for the product
Field space.
The preparation method of present D-alanine is mainly microbe fermentation method and chemical synthesis.
Microbe fermentation method D-Cycloserine generation D-alanine can be made by quarter butt Saccharomyces casei.But product
Concentration is low, and production cycle length, equipment investment is big, there is side reaction, and separation is more complicated, pollution is big.And because the outer of alanine disappears
The activity of rotation, the optical purity of product D-alanine is than relatively low.
The chemical synthesis process of D-alanine has two kinds, and one kind is to directly obtain D-alanine using dissymmetric synthesis,
Another kind is to obtain the DL body of DL-Alanine by chemical synthesis, recycles various method for optical resolution, obtains the ammonia of D- third
Acid.Chemical synthesis uses plurality of raw materials and process route, and cost is relatively low, and production scale is big, is adapted to industrialized production.But institute
Obtained product is all that DL- mixes type alanine, it is necessary to carries out optical resolution, can just obtain D-alanine.Currently used for separation
For the method for DL-Alanine because step is more, cost is high, and industrialization is relatively difficult.And chemosynthesis reaction mechanism is complicated, technique mistake
Journey is grown；Homochiral reagent or noble metal complexes are needed to make catalyst, production cost is high.
Current external industrialized production D-alanine generally uses " amino acid acylase Split Method ", the amino that the method utilizes
Acid is expensive, and production cost is still higher, is only used for the production of Small Scale Industry metaplasia.
Physiological action due to D-alanine is constantly found that it is using more and more extensive, it is necessary to research and develop a kind of suitable
Close heavy industrialization, the D-alanine production technology of production cost can be significantly reduced.
The content of the invention
In order to solve the above problems, the present invention adds using low concentration DL-Alanine as substrate when adjusting zymotic fluid pH
Add DL-Alanine substrate, shorten fermentation period, fermentation period is completed in 36-42 hours, prepare high concentration D-alanine.At least
The present invention is completed based on this purpose.Specifically, the present invention includes herein below.
(1) by microbial inoculant into the fermentation medium containing 1.0%-1.5%w/v DL-Alanines, and suitable for
The microorganism is cultivated in the environment of microorganism growth；
(2) when the mass ratio of ALANINE and D-alanine is less than 1:4, and the pH of zymotic fluid be more than 7.0 when, addition contains
The aqueous solution of nitric acid of DL-Alanine is to zymotic fluid, to control the mass ratio of ALANINE and D-alanine in the zymotic fluid to exist
1:4 to 1:In the range of 2 and pH value is in 6.5-7.0；
(3) when the amount that the D-alanine of crystal form accounts for total D-alanine is less than 9%, end contains DL-Alanine
Aqueous solution of nitric acid addition.
In some embodiments, in described method, concentration of the DL-Alanine in aqueous solution of nitric acid is 40-
60%w/v.
In some embodiments, in described method, the total amount of adding of DL-Alanine is with originating DL- during fermentation
The mass ratio of alanine is 1:0.8 to 1:1.2.
In some embodiments, in described method, when cultivate the microorganism 6 it is small when after, start addition and contain DL-
The aqueous solution of nitric acid of alanine is to zymotic fluid.
In some embodiments, in described method, in addition to (4) add diatomite and activity into the zymotic fluid
Charcoal, it is concentrated by evaporation, filters to take clear liquid, and condensing crystallizing.
In some embodiments, in described method, the condensing crystallizing of the step (4) includes：
When the clear liquid is concentrated into the 10%-20% of the fermentating liquid volume, same volume is added into the clear liquid
Isopropanol, stir, centrifuge and dry to obtain D-alanine.
In some embodiments, in described method, the concentration of nitric acid is 5-15%w/v in the aqueous solution of nitric acid.
In some embodiments, in described method, the fermentation medium include 0.5%-1.0% corn steep liquors,
0.05%-0.1% sodium dihydrogen phosphates, 0.1%-0.2% magnesium sulfate and 10-120% biotins.
In some embodiments, in described method, concentration of the diatomite in the zymotic fluid is 1.5-2%
W/v, concentration of the activated carbon in the zymotic fluid are 2.5%-3.0%w/v.
In some embodiments, in described method, the microorganism is to cultivate 24-26 on 30 ° -32 ° of inclined-plane
Hour obtains.
Beneficial effect：
(1) using low concentration DL-Alanine as fermentation substrate in the present invention, side adjusts fermented liquid during the fermentation
A certain amount of DL-Alanine is added on pH value side into fermented liquid, controls the concentration of fermentation substrate during fermentation reaction, improves
Fermentation efficiency.
(2) the whole fermentation period that D-alanine is produced in the present invention is 36-42 hours, shortens the production cycle, improves life
Produce efficiency.
Embodiment
Now describe the various exemplary embodiment of the present invention in detail, the detailed description is not considered as the limit to the present invention
System, and it is understood as the more detailed description to certain aspects of the invention, characteristic and embodiment.
It should be understood that heretofore described term is only to describe special embodiment, being not intended to limit this hair
It is bright.In addition, for the number range in the present invention, it is thus understood that specifically disclose the scope upper and lower bound and they it
Between each median.Median and any other statement value in any statement value or stated ranges or in the scope
Each less scope between interior median is also included in the present invention.These small range of upper and lower bounds can be independent
Ground is included or excluded in the range of.
Unless otherwise stated, all technologies used herein and scientific terminology have the routine in field of the present invention
The identical meanings that technical staff is generally understood that.Although the present invention only describes preferable method and material, the present invention's
Implement or can also be used and similar or equivalent any method described herein and material in testing.The institute mentioned in this specification
There is document to be incorporated by reference into, to the disclosure and description method related to the document and/or material.It is incorporated to any
When document conflicts, it is defined by the content of this specification.
It is open term, i.e., on "comprising" used herein, " comprising ", " having ", " containing " etc.
Mean including but not limited to.Any or all combinations on "and/or" used herein, including the things.Unless
It is otherwise noted, otherwise % refers to quality percent by volume.For example, 9% refers to contain 9 grams of materials in 100 milliliters of volumes.
DL-Alanine of the present invention refers to the mixture containing D-alanine and ALANINE simultaneously.For mixing
The ratio of D-alanine and ALANINE is not particularly limited in thing.Preferably as in the mixture of initiation material have etc.
The D and ALANINE of amount.
Microorganism as used in the present invention is the bacterial strain for having high L- isomers degrading activity.It is preferred that the microorganism is
Refer to ALANINE of degrading, and the bacterial strain non-degradable to D-alanine.Microorganism of the present invention can be used in the prior art
Known any bacterial strain, it is possible to use any bacterial strain with function of the present invention obtained by known technology.As obtaining
Taking the known method of bacterial strain of the present invention includes：
The step of soil sample is gathered in the soil rich in organic matter, the soil sample are the soil apart from ground 5-15cm；It is excellent
Selection of land, the soil are the orchard rich in saccharomycete, vegetable garden or wild fruit growth district；Propagation training is carried out to bacterial strain in the soil sample
Foster step；The step of all bacterial strains in the soil sample carry out culture of isolated；The step of aimed strain is obtained from all bacterial strains
Suddenly.The aimed strain refers to the bacterial strain very high to ALANINE degradation capability.
In certain embodiments, the microorganism for can secrete amino acid oxidase, L- isomers racemase or its
He can degrade ALANINE enzyme bacterial strain.For example, the microorganism be Candida, brevibacterium lactofermentus DCSR-26,
Brevibacterium lactofermentus HR5-43, Bacillus Brevis ATCC 8185, Ala-D45, Pseudomaonas striata or
Pierce mould 9980 bacterial strain of the small Mildy Way of spore.
The activity of holding microorganism is needed in the present invention, the activity refers to that microorganism has propagation, breeding or growth
Vigor.Preferably, the microorganism can not be cracking state.The cracking of microorganism reduces reaction rate.
It is important that the pH value in fermentation process is controlled in the present invention.With the progress of fermentation process, the pH in zymotic fluid
Value can reduce.Inventor has found that the degraded of ALANINE dies down, and reaction efficiency reduces when pH value is less than 6.5.PH preferably
Value is less than 6.8.On the other hand, if pH value is higher than 7.0, can extend between seasonable.Preferably, the pH in fermentation process is controlled to exist
Between 6.5-7.0, between more preferably 6.6-6.8.
The present invention during the course of the reaction, preferably when the crystal form of D-alanine it is of the total volume less than 9% when terminate to add
Add, preferably terminate to add when crystal is accounted for below 8%.Such as terminate to add when 5%.In certain embodiments, in zymotic fluid
The D-alanine of dissolved state is preferably more than 10%, preferably more than 12%, more preferably more than 15%, most preferably 16%.
In the present invention, the pH value of fermented liquid in fermentation medium is adjusted using the aqueous solution of nitric acid containing DL-Alanine,
DL-Alanine is added into fermented liquid while the regulation of fermented liquid pH value is carried out using aqueous solution of nitric acid so that fermentation
Fermentation substrate concentration and the pH value of fermented liquid match in liquid, when controlling fermented liquid pH value, the control addition ammonia of DL- third
The speed and quality of acid, the cell density in culture medium is kept, improve D-alanine production efficiency.Wherein, the matter of DL-Alanine
It is 40-60% that amount, which accounts for aqueous solution of nitric acid and the percentage of DL-Alanine gross mass, it is preferable that the quality of DL-Alanine accounts for nitric acid
The aqueous solution and the percentage of DL-Alanine gross mass are 45-55%, for example, the quality of DL-Alanine account for aqueous solution of nitric acid and
The percentage of DL-Alanine gross mass is 45%, 50% or 55%.And quality volume of the DL-Alanine in aqueous solution of nitric acid
Concentration is 50-55g/L, for example, mass-volume concentration of the DL-Alanine in aqueous solution of nitric acid be 50g/L, 51g/L, 52g/L,
53g/L, 54g/L or 55g/L.
The mass ratio for needing to control ALANINE and D-alanine in the fermentation process of the present invention is 1:4 to 1:In the range of 2.
The mass ratio of ALANINE and D-alanine is less than 1:4, then reaction speed slow down.On the other hand, if above 1:2, then react
Speed equally declines.It should be noted that after last time increases the aqueous solution of nitric acid containing DL-Alanine, with reaction
Progress, now the mass ratio of ALANINE and D-alanine be smaller than 1:4, even 0.
The mass ratio of the total amount of adding of DL-Alanine and starting DL-Alanine is 1 during being fermented in the present invention:0.8 to 1:
1.2.It is preferred that 1:1.
Preferably, after fermented and cultured is started more than 6 hours, start to add the aqueous solution of nitric acid containing DL-Alanine
To zymotic fluid.If the time is too short, such as nitric acid of the addition containing DL-Alanine is water-soluble within the 5th hour after fermentation is started
Liquid, then it is unfavorable for controlling the mass ratio of ALANINE and D-alanine in appropriate scope.If start 10 after fermented and cultured
After more than hour, start to add the aqueous solution of nitric acid containing DL-Alanine to zymotic fluid, then ALANINE and D-alanine
Mass ratio can be too low, is unfavorable for the lifting of reaction speed.It is preferred that after fermentation is started between 6-10 hours, for example, 7 hours, 8
Hour, start within 9 hours to add the aqueous solution of nitric acid containing DL-Alanine to zymotic fluid.
The microculture condition of the present invention is preferably as follows described：
Microorganism fungus kind is cultivated 24 hours for 31 DEG C on inclined-plane, is linked into the culture medium disinfected, wherein culture medium
Composition for 15 grams of DL-Alanines, 10 grams of corn steep liquors, 120 microgram biotins, 0.8 gram of sodium dihydrogen phosphate, 1.2 grams of magnesium sulfate,
Culture volume constant volume flows to 1000 milliliters of shaken cultivation 61 hours on shaking table, fermentation processes concentration of substrate and pH value
Add 150 grams altogether of DL-Alanine, then the diatomite of 19 grams of addition, 20.8 grams of activated carbon in zymotic fluid, 86 DEG C of heating
43 minutes, filtering, clear liquid is taken, be concentrated in vacuo, fermentation clear liquid is concentrated to original 120 milliliters, adds 98% isopropanol 120
Milliliter, stirring make D-alanine crystallize 28 hours at ambient temperature, and centrifugation, drying obtain 55 grams of D-alanine finished product.
Embodiment
Embodiment 1
Microorganism fungus kind (Bacillus Brevis ATCC 8185), culture 25 is small on the inclined-plane that slope is 30.5 °
When, then by microbial inoculant into fermentation medium, the culture medium includes：1.5gDL- alanine, 0.8g corn steep liquors, 120
μ g biotins, 0.9g sodium dihydrogen phosphates and 0.1g magnesium sulfate, and culture volume constant volume is to 100 milliliters.
For shaken cultivation after 6 hours, the mass ratio for detecting ALANINE and D-alanine is 1 on shaking table:5, zymotic fluid
PH is 7.5, utilizes DL-Alanine concentration in fermented liquid in the aqueous solution of nitric acid control fermentation medium containing DL-Alanine
And pH value, the mass ratio of ALANINE and D-alanine is controlled in the zymotic fluid 1:4 to 1:In the range of 2 and pH value is in 6.5-
It is 7.0 interior；The concentration of wherein described aqueous solution of nitric acid DL-Alanine is 50%, when the D-alanine of crystal form accounts for total ammonia of D- third
When the amount of acid is 2%, terminate addition, continue reaction until ALANINE is 0.Added along with aqueous solution of nitric acid in culture medium
DL-Alanine quality amounts to 15g.
Diatomite 1.8g, activated carbon 2.5g are added into fermented liquid, is heated 32 minutes in 86 DEG C of temperature, filtering, is taken clear
Liquid, it is concentrated in vacuo, clear liquid is concentrated to 15 milliliters (15% of fermented liquid), 98% 15 milliliters of isopropanol is added, in room
Stirred under the conditions of temperature, when the amount that the D-alanine of crystal form accounts for total D-alanine is 9%, terminate fermentation, D-alanine knot
Brilliant 25 hours, centrifugation, drying obtain D-alanine finished product 5.3g.Whole fermentation process 36 hours.
Embodiment 2
Microorganism fungus kind (Bacillus Brevis ATCC 8185) is cultivated 24 hours on the inclined-plane that slope is 31 °,
Then by microbial inoculant into fermentation medium, the culture medium includes：15gDL- alanine, 10g corn steep liquors, 120 μ g dimensions
Raw plain H, 0.8g sodium dihydrogen phosphate and 1.2g magnesium sulfate, and culture volume constant volume is to 1000 milliliters.
Shaken cultivation 6.5 hours on shaking table, the mass ratio for detecting ALANINE and D-alanine is 1:4.5, zymotic fluid
PH when being 8, using DL-Alanine is dense in fermented liquid in the aqueous solution of nitric acid control fermentation medium containing DL-Alanine
Degree and pH value, the mass ratio of ALANINE and D-alanine is controlled in the zymotic fluid 1:4 to 1:Exist in the range of 2 with pH value
It is 6.5-7.0 interior；Wherein, the concentration of the aqueous solution of nitric acid DL-Alanine is 45%.When the D-alanine of crystal form accounts for total D-
When the amount of alanine is 5%, terminate addition, continue reaction until ALANINE is 0.Culture medium is added along with aqueous solution of nitric acid
In DL-Alanine quality amount to 150g.
Diatomite 19g, activated carbon 20.8g are added into fermented liquid, is heated 43 minutes in 86 DEG C of temperature, filtering, is taken clear
Liquid, it is concentrated in vacuo, clear liquid is concentrated to 120 milliliters (12% of fermented liquid), adds 98% 120 milliliters of isopropanol,
Stirred under room temperature condition, when the amount that the D-alanine of crystal form accounts for total D-alanine is 9%, terminate fermentation, D-alanine
Crystallization 28 hours, centrifugation, drying obtain D-alanine finished product 55g.Whole fermentation process 40 hours.
Embodiment 3
Microorganism fungus kind (Bacillus Brevis ATCC 8185) is cultivated 26 hours on the inclined-plane that slope is 31 °,
Then by microbial inoculant into fermentation medium, the culture medium includes：13gDL- alanine, 10g corn steep liquors, 150 μ g dimensions
Raw plain H, 0.68g sodium dihydrogen phosphate and 1.5g magnesium sulfate, and culture volume constant volume is to 1000 milliliters.
The mass ratio of shaken cultivation 7 hours on shaking table, ALANINE and D-alanine is 1:6, and the pH of zymotic fluid is
When 8.5, using in the aqueous solution of nitric acid control fermentation medium containing DL-Alanine in fermented liquid DL-Alanine concentration and
PH value, the mass ratio of ALANINE and D-alanine is controlled in the zymotic fluid 1:4 to 1:In the range of 2 and pH value is in 6.5-
It is 7.0 interior；Wherein, the concentration of the aqueous solution of nitric acid DL-Alanine is 55%.When the D-alanine of crystal form accounts for total ammonia of D- third
When the amount of acid is 8.1%, terminate addition, continue reaction until ALANINE is 0.Added along with aqueous solution of nitric acid in culture medium
DL-Alanine quality amount to 130g.
Diatomite 16g, activated carbon 2.5g are added into fermented liquid, is heated 40 minutes in 89 DEG C of temperature, filtering, is taken clear
Liquid, it is concentrated in vacuo, clear liquid is concentrated to 160 milliliters (16% of fermented liquid), adds 98% 160 milliliters of isopropanol,
Stirred under room temperature condition, D-alanine crystallizes 25 hours, when the amount that the D-alanine of crystal form accounts for total D-alanine is 9%
When, terminate fermentation, centrifugation, drying obtain D-alanine finished product 58g.Whole fermentation process 35 hours.
Comparative example 1
Microorganism fungus kind (Bacillus Brevis ATCC 8185) is cultivated 28 hours on the inclined-plane that slope is 30 °,
Then by microbial inoculant into 100 milliliters of fermentation mediums, the culture medium except the content of DL-Alanine be changed into 15% with
It is identical with the culture medium in embodiment 1 outside.
Shaken cultivation 48 hours on shaking table, controlled in fermentation medium ferment using aqueous solution of nitric acid during the fermentation
PH value in liquid is in 6.5-7.0.Continue reaction until ALANINE is 0.
Diatomite 1.8g, activated carbon 2.5g are added into fermented liquid, is heated 32 minutes in 86 DEG C of temperature, filtering, is taken clear
Liquid, it is concentrated in vacuo, clear liquid is concentrated to 15 milliliters (15% of fermented liquid), 98% 15 milliliters of isopropanol is added, in room
Stirred under the conditions of temperature, centrifugation, drying obtain D-alanine finished product.Whole fermentation process 65 hours.
Comparative example 2
Microorganism fungus kind (Bacillus Brevis ATCC 8185) is cultivated 24 hours on the inclined-plane that slope is 31 °,
Then by microbial inoculant into 1000 milliliters of fermentation mediums, the culture medium is changed into 15% except the content of DL-Alanine
It is identical with the culture medium in embodiment 2 in addition.
Shaken cultivation 5 hours on shaking table, it is 1 to detect the mass ratio of ALANINE and D-alanine in zymotic fluid:1.5
The pH6.8 of zymotic fluid, utilize the pH in fermented liquid in the aqueous solution of nitric acid control fermentation medium containing 50%DL- alanine
Value is in 6.5-6.8.When the amount that the D-alanine of crystal form accounts for total D-alanine is 12.2%, terminates addition, continue anti-
Should be 0 up to ALANINE.The DL-Alanine quality added along with aqueous solution of nitric acid in culture medium amounts to 25g.
Diatomite 1.8g, activated carbon 2.5g are added into fermented liquid, is heated 32 minutes in 86 DEG C of temperature, filtering, is taken clear
Liquid, it is concentrated in vacuo, clear liquid is concentrated to 15 milliliters (15% of fermented liquid), 98% 15 milliliters of isopropanol is added, in room
Stirred under the conditions of temperature, centrifugation, drying obtain D-alanine finished product.Whole fermentation process 70 hours.
Comparative example 3
Microorganism fungus kind (Bacillus Brevis ATCC 8185) is cultivated 24 hours on the inclined-plane that slope is 31 °,
Then by microbial inoculant into 1000 milliliters of fermentation mediums, the culture medium is changed into 13% except the content of DL-Alanine
It is identical with the culture medium in embodiment 3 in addition.
Shaken cultivation 12 hours on shaking table, it is 1 to detect the mass ratio of ALANINE and D-alanine in zymotic fluid:5, hair
The pH7.6 of zymotic fluid, utilize ALANINE and the ammonia of D- third in the aqueous solution of nitric acid control fermentation medium containing 50%DL- alanine
The mass ratio of acid is 1:4 to 1:In the range of 2 and pH value is in 6.5-7.0.When the D-alanine of crystal form accounts for total D-alanine
Amount be 2.3% when, terminate addition, continue reaction until ALANINE be 0.Added along with aqueous solution of nitric acid in culture medium
DL-Alanine quality amounts to 15g.
Diatomite 1.8g, activated carbon 2.5g are added into fermented liquid, is heated 32 minutes in 86 DEG C of temperature, filtering, is taken clear
Liquid, it is concentrated in vacuo, clear liquid is concentrated to 15 milliliters (15% of fermented liquid), 98% 15 milliliters of isopropanol is added, in room
Stirred under the conditions of temperature, centrifugation, drying obtain D-alanine finished product.Whole fermentation process 45 hours.
Comparative example 4
Microorganism fungus kind (Bacillus Brevis ATCC 8185) is cultivated 24 hours on the inclined-plane that slope is 31 °,
Then by microbial inoculant into 1000 milliliters of fermentation mediums, the culture medium is changed into 13% except the content of DL-Alanine
It is identical with the culture medium in embodiment 3 in addition.
Shaken cultivation 12 hours on shaking table, it is 1 to detect the mass ratio of ALANINE and D-alanine in zymotic fluid:5, hair
The pH7.6 of zymotic fluid, utilize ALANINE and the ammonia of D- third in the aqueous solution of nitric acid control fermentation medium containing 50%DL- alanine
The mass ratio of acid is 1:4 to 1:In the range of 2 and pH value is in 6.5-7.0.When the D-alanine of crystal form accounts for total D-alanine
Amount be 10% when, terminate addition, continue reaction until ALANINE be 0.Added along with aqueous solution of nitric acid in culture medium
DL-Alanine quality amounts to 118g.
Diatomite 1.8g, activated carbon 2.5g are added into fermented liquid, is heated 32 minutes in 86 DEG C of temperature, filtering, is taken clear
Liquid, it is concentrated in vacuo, clear liquid is concentrated to 15 milliliters (15% of fermented liquid), 98% 15 milliliters of isopropanol is added, in room
Stirred under the conditions of temperature, centrifugation, drying obtain D-alanine finished product.Whole fermentation process 69 hours.
In the case of without departing substantially from the scope or spirit of the invention, the embodiment of description of the invention can be done more
Kind is improved and change, and this will be apparent to those skilled in the art.Other realities obtained by the specification of the present invention
It is apparent obtain for technical personnel to apply mode.Present specification and embodiment are only exemplary.
Claims (10)
1. a kind of method for producing high-purity D-alanine, it comprises the following steps：
(1) by microbial inoculant into the fermentation medium containing 1.0%-1.5%w/v DL-Alanines, and suitable for micro- life
The microorganism is cultivated in the environment of thing growth；
(2) when the mass ratio of ALANINE and D-alanine is less than 1:4, and the pH of zymotic fluid be more than 7.0 when, addition contain DL-
The aqueous solution of nitric acid of alanine is to zymotic fluid, to control in the zymotic fluid mass ratio of ALANINE and D-alanine 1:4
To 1:In the range of 2 and pH value is in 6.5-7.0；
(3) when the amount that the D-alanine of crystal form accounts for total D-alanine is less than 9%, the nitre containing DL-Alanine is terminated
The addition of aqueous acid.
2. according to the method for claim 1, wherein concentration of the DL-Alanine in aqueous solution of nitric acid is 40-60%
w/v。
3. according to the method for claim 1, wherein the total amount of adding of DL-Alanine is with originating the ammonia of DL- third during fermentation
The mass ratio of acid is 1:0.8 to 1:1.2.
4. according to the method for claim 1, start addition wherein after when the culture microorganism 6 is small and contain the ammonia of DL- third
The aqueous solution of nitric acid of acid is to zymotic fluid.
5. according to the method for claim 1, wherein also adding diatomite and activity into the zymotic fluid including step (4)
Charcoal, it is concentrated by evaporation, filters to take clear liquid, and condensing crystallizing.
6. according to the method for claim 5, wherein the condensing crystallizing of the step (4) includes：
When the clear liquid is concentrated into the 10%-20% of the fermentating liquid volume, the isopropyl of same volume is added into the clear liquid
Alcohol, stir, centrifuge and dry to obtain D-alanine.
7. according to the method for claim 1, wherein the concentration of nitric acid is 5-15%w/v in the aqueous solution of nitric acid.
8. according to the method for claim 1, wherein the fermentation medium includes 0.5%-1.0% corn steep liquors, 0.05%-
0.1% sodium dihydrogen phosphate, 0.1%-0.2% magnesium sulfate and 10-120% biotins.
9. according to the method for claim 5, wherein concentration of the diatomite in the zymotic fluid is 1.5-2%w/v,
Concentration of the activated carbon in the zymotic fluid is 2.5%-3.0%w/v.
10. according to the method for claim 1, wherein the microorganism is that 24-26 hours are cultivated on 30 ° -32 ° of inclined-plane
Obtain.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1884565A (en) * | 2006-05-29 | 2006-12-27 | 安徽华恒生物工程有限公司 | Process for producing D-alanine using microbe |
| CN104140987A (en) * | 2013-05-07 | 2014-11-12 | 南京中医药大学 | A constant-pH feeding fermentation method for production of high-purity D-alanine |
| CN105624223A (en) * | 2014-10-29 | 2016-06-01 | 宜兴市前成生物有限公司 | Method for preparing DL-alanine and D-alanine |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1884565A (en) * | 2006-05-29 | 2006-12-27 | 安徽华恒生物工程有限公司 | Process for producing D-alanine using microbe |
| CN104140987A (en) * | 2013-05-07 | 2014-11-12 | 南京中医药大学 | A constant-pH feeding fermentation method for production of high-purity D-alanine |
| CN105624223A (en) * | 2014-10-29 | 2016-06-01 | 宜兴市前成生物有限公司 | Method for preparing DL-alanine and D-alanine |
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