CN105274179B - A kind of technique of extraction l-Isoleucine - Google Patents
A kind of technique of extraction l-Isoleucine Download PDFInfo
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- CN105274179B CN105274179B CN201510744298.1A CN201510744298A CN105274179B CN 105274179 B CN105274179 B CN 105274179B CN 201510744298 A CN201510744298 A CN 201510744298A CN 105274179 B CN105274179 B CN 105274179B
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Abstract
The invention discloses a kind of techniques of extraction L isoleucines comprising：1) mixed fermentation；2) it filters；3) concentration weight is molten；4) ion exchange.Present invention process fermentation efficiency is high, and extractive technique is easy, efficient, has a vast market foreground.
Description
Technical field
The invention belongs to biofermentation industry l-Isoleucine extraction process fields, specifically provide a kind of different bright ammonia of extraction L-
The technique of acid.
Background technology
L-Isoleucine, also known as L- isoleucines belong to neutral amino acid, branch are referred to as with Valine, L-Leu
Amino acid is mainly used for compound amino acid infusion, the infusion of three branched-chain amino acids, amino as one kind in amino acid starting material medicine
Sour oral solution etc. is important one of amino acid starting material medicine for treating hepatopathy, hepatic coma, the weak diseases such as weak, while
It is very wide to be widely used in fields, the market prospects such as food, sports health product and feed addictive.
The basic extracting method of l-Isoleucine has the precipitation method, full embrane method and ion-exchange.The precipitation method have it is easy to operate,
The advantages that product purity is high is extracted, its shortcoming is that precipitating reagent, which is a kind of benezene material, has carcinogenicity, is easily remained in the product,
And operating process is highly acid extraction, the problem of there is security risk, exist simultaneously waste water treatmentntrol difficult.Although full embrane method is useless
Water is few, but can not detach the miscellaneous amino acid being close with l-Isoleucine molecular weight, causes the product heteroacid extracted high,
Repetition crystallization can only be leaned on to prepare the l-Isoleucine of high-purity.Ion-exchange has at low cost, easy to operate, extraction effect
Well, the advantages that equipment is simple, but the organic wastewater that the method generates causes post-processing cost higher.Currently, single extraction side
Method has been unable to meet the requirement of l-Isoleucine production technology, needs to seek more cost-effective method to solve this technology
Problem.
It is by the cleaner liquid downward ph adjustment after ceramic membrane degerming to 2-4.5, with strong acid tree that ion exchange blooming, which filters extraction method,
For fat by amino acid adsorbed, waste liquid is expelled to sewage disposal, is finally eluted the amino acid of absorption with ammonium hydroxide.L-Isoleucine
It extracts and contains a large amount of thalline in mother liquor, the content of protein is up to 80% or more in thalline, and amino acid classes and proportioning also compare
It is more complete, and containing nutriments such as abundant vitamin, nucleic acid, polysaccharide, make animal feeding-stuff containing somatic protein full of nutrition, keep away
Exempt from utility to discharge in vain, result in waste of resources.
Invention content
The object of the present invention is to provide a kind of techniques of extraction l-Isoleucine, and this method fermentation efficiency is high, can be disposable
Heteroacid and other impurity in zymotic fluid are removed, production cost is reduced, reduces discharge of wastewater.
The present invention is achieved by the following technical solution：
A kind of technique of extraction l-Isoleucine comprising following steps：
1) mixed fermentation；2) it filters；3) concentration weight is molten；4) ion exchange.
Specifically, the technique includes the following steps：
1) mixed fermentation：By Corynebacterium glutamicum ATCC14309 seed liquors and brevibacterium lactofermentus ATCC13869 seeds
Liquid is mixed according to 2: 1 volume ratio, is obtained seed mixture liquid, is transferred in fermentation medium and cultivates according to 5% inoculum concentration, temperature
30 DEG C, pH value 7.2, incubation time 60 hours obtains l-Isoleucine zymotic fluid；
2) it filters：L-Isoleucine zymotic fluid is used into ceramic membrane filter, collects mycoprotein and filtrate；
3) concentration weight is molten：The filtrate that step 2) obtains is evaporated concentration, is evaporated to 1/5th of original volume, then
Add, obtained weight solution molten with the distilled water of concentrate same volume weight；
4) ion exchange：
1. regulating step 3) pH value of gained weight solution is 5, it then passes to strong acid ion exchange resin column, carries out ion friendship
It changes；The strong acid ion exchange resin column is that three ion exchange resin columns are cascaded by pipeline and valve, and every
All there are one rewinding valves for one ion exchange resin column bottom；
2. liquid enters from resin column top, lower part discharge；Instantly when ninhydrin colour developing is met in drain, lower drain is sealed in the
The lower drain of second column is sealed in third column by two columns when second column develops the color；
3. be further continued for when third column develops the color outer row 2.5 it is small when, the developing solution of discharge is glutamic acid, based on alanine, this
When can remove 40% of total heteroacid in zymotic fluid；
4. after 2.5 hours, stopping the charging of the first column, weighed again in stream tank before being washed till from the first column to third column string with pure water
New absorption is used；
5. at the end of with pure water rinsing, the ammonium hydroxide for being 2% with mass fraction, under the amino acid parsing that resin is adsorbed
Come, in resolving when liquid pH value reaches 6.5 under column, closes rewinding valve and open string column valve door, string washes next pillar, with this
Analogize and continue to elute when liquid pH reaches 6.5 under third pillar column, column of no longer going here and there starts the outer last person when pH value reaches 8.5
Liquid 3 hours stops elution；
6. the feed liquid eluted in ion-exchange column is concentrated, concentration l-Isoleucine mass concentration is 10%；
7. the activated carbon of the feed liquid added 3 ‰ after concentration is warming up to 70 DEG C, material liquid pH value is adjusted 5.5, is decolourized 35 points
Clock, then filtering remove activated carbon；
8. the feed liquid after decoloration is concentrated into the 1/6 of original volume, then decrease temperature crystalline, when temperature is down to 20 DEG C, centrifuge
High speed centrifugation obtains crude product, and moisture is 17%；
9. by decolourizing in crude product input bleacher, then concentrates, crystallizes, centrifuging, is dry, crushing obtains finished product.
Preferably, the Corynebacterium glutamicum ATCC14309 seed liquors and brevibacterium lactofermentus ATCC13869 seed liquors
Concentration be 1 × 108CFU/mL。
Preferably, the fermentation medium group is divided into：Glucose 12%, corn steep liquor 5%, beef extract 1%, epsom salt
0.2%, dipotassium hydrogen phosphate 0.2%, ferrous sulfate 0.001%, VB1 0.0001%, pH 7.0.
The advantageous effect that the present invention obtains：
The present invention uses hybrid bacterial strain fermentation technique, and yield must improve, be cooperateed with mutually between strain, mutually not antagonism；
The present invention is detached the big molecular impurities such as mycoprotein in mother liquor using membrane filtration system, collect fermentation thalli, by with
Upper condition efficiently easily detaches the mycoprotein in l-Isoleucine mother liquor, recycling of the membrane separation technique to mycoprotein
Rate is up to 98%, has the advantages of simple operation, is more suitable for large-scale industrial production.L-Isoleucine extraction is using the present invention
After production technology, avoid to improve the repeatedly crystallization and ion-exchange that the purity of l-Isoleucine carries out；Which shorten extractions
The time of high-purity l-Isoleucine, remarkable in economical benefits.Using the different bright ammonia of L- in membrane filtration-ion-exchange combined method extraction zymotic fluid
Acid, extract yield reduce loss, to reduce production cost up to 80% or more.This application discloses l-Isoleucine extractions
A series of entire protocols, optimize product structure specification production procedure again.Adsorption process generate waste water be diluted acid or
Inorganic salt solution, recycling greatly reduce consumption of the resin regeneration to inorganic acid or inorganic salts for being used when resin regeneration.And
Ion exchange process of the present invention generates 1/2 of wastewater flow rate not as good as traditional handicraft, reduces the environmental protection pressure of enterprise.The application is not necessarily to
The chemical reagent such as flocculant are added, avoid feed safety hidden danger existing for flocculant monomer, the mycoprotein taste of acquisition is pure
Just, smell is fragrant and sweet, is rich in a variety of fermentation amino acid.
Specific implementation mode
In order to make those skilled in the art better understand the technical solutions in the application, having below in conjunction with the application
Body embodiment more clearly and completely describes the present invention, it is clear that described embodiment is only the application one
Divide embodiment, instead of all the embodiments.Based on the embodiment in the application, those of ordinary skill in the art are not making
The every other embodiment obtained under the premise of creative work, should all belong to the scope of protection of the invention.
Embodiment 1
A kind of technique of extraction l-Isoleucine, includes the following steps：
1) preparation of mixed fermentation liquid：By Corynebacterium glutamicum ATCC14309 seed liquors and brevibacterium lactofermentus
ATCC13869 seed liquors are mixed according to 2: 1 volume ratio, obtain seed mixture liquid, Corynebacterium glutamicum ATCC14309 seed liquors
Concentration with brevibacterium lactofermentus ATCC13869 seed liquors is about 1 × 108CFU/mL is transferred to according to according to 5% inoculum concentration
It is cultivated in fermentation medium, 30 DEG C of temperature, pH value 7.2, incubation time 60 hours obtains l-Isoleucine zymotic fluid；Fermentation training
Base group is supported to be divided into：Glucose 12%, corn steep liquor 5%, beef extract 1%, epsom salt 0.2%, dipotassium hydrogen phosphate 0.2%, sulphur
Sour ferrous iron 0.001%, VB1 0.0001%, pH 7.0.
2) it uses ceramic membrane filter, membrane aperture 60-70nm to filter the l-Isoleucine zymotic fluid of fermentation ends, collects
Mycoprotein and filtrate；
3) by step 2) obtain filtrate be evaporated concentration, be evaporated to 1/5th of original volume, then add with it is dense
The distilled water weight of contracting liquid phase same volume is molten, and weight solution is made；
4) the heavy solution for obtaining step 3) extracts l-Isoleucine using ion-exchange：
1. it is 5 to adjust pH value, strong acid ion exchange resin column (C100 cation exchange resins) is then passed to, carries out ion
It exchanges, three columns in series ion exchange, the strong acid ion exchange resin column is that three strong acid ion exchange resin columns pass through pipeline
It is cascaded with valve, and there are one rewinding valves for each strong acid ion exchange resin column bottom；
2. cleaner liquid enters according to 2.0 speeds that flow velocity is resin volume from resin column top, lower part discharge；Instantly drain
When meeting ninhydrin colour developing, lower drain is sealed in into second column, the lower drain of the second column is sealed in into third when second column develops the color
A column；
3. be further continued for when third column develops the color outer row continuous 2.5 it is small when, the developing solution that is discharged at this time is glutamic acid, alanine
Based on, 40% or so of total heteroacid in zymotic fluid can be removed at this time；
4. after drained 2.5 hours, stopping the charging of the first column, before being washed till from the first column to third column string with pure water in stream tank
Absorption is used again again；
5. at the end of with pure water rinsing, by prepared 2% (mass fraction) ammonium hydroxide, flow velocity is according to the 1 of resin volume
Speed parses the amino acid that resin is adsorbed, and in resolving when liquid pH value reaches 6.5 under column, closes rewinding valve
Open string column valve door, string wash next pillar, and so on when under third pillar column liquid pH arrival 6.5 when continue to elute, no longer
String column starts outer last person's liquid 3 hours when pH value reaches 8.5, stops elution；With air from first pillar to third column
Son flows tank after pushing back the ammonium hydroxide in column；In this way three columns in series elution can effective heteroacid, which includes a large amount of lysine,
Other amino acid such as arginine；
6. the feed liquid eluted in ion-exchange column is concentrated, it is 10% that can concentrate l-Isoleucine mass concentration；
7. the activated carbon of the feed liquid added 3 ‰ after concentration is warming up to 70 DEG C, material liquid pH value is adjusted 5.5, is decolourized 35 points
Clock, then through microfroc filter by activated carbon and contaminant filter；
8. the concentrated crystallization of feed liquid after decoloration, volume is that condensate precursor accumulates to after concentrating 1/6, then decrease temperature crystalline,
When temperature is down to 20 DEG C, centrifuge high speed centrifugation obtains wet crude product, and moisture is 17%；
9. crude product puts into decoloration and then concentration in bleacher according to the concentration of 4% (mass fraction), crystallization, centrifuges, dry
Dry, crushing obtains refined finished product.
After testing, under identical fermentation condition, the present invention is fermented using hybrid bacterial strain, with single Corynebacterium glutamicum
ATCC14309 fermentations are compared, l-Isoleucine output increased 26%；
The l-Isoleucine that the present invention obtains is detected through HPLC, purity 99.0%, and white appearance is bright, miscellaneous amino acid
It is only according to quantity 1%, reaches the requirement of pharma grade product.
Listed above is only the best specific embodiment of the present invention.It is clear that the invention is not restricted to which above example, may be used also
With there are many deformations.All changes that those skilled in the art directly can export or associate from present disclosure
Shape is considered as protection scope of the present invention.
Claims (2)
1. a kind of technique of extraction l-Isoleucine comprising following steps：
1) mixed fermentation：Corynebacterium glutamicum ATCC14309 seed liquors and brevibacterium lactofermentus ATCC13869 seed liquors are pressed
According to 2: 1 volume ratio mixing, seed mixture liquid is obtained, is transferred in fermentation medium and cultivates according to 5% inoculum concentration, temperature 30
DEG C, pH value 7.2, incubation time 60 hours obtains l-Isoleucine zymotic fluid；
The fermentation medium group is divided into：Glucose 12%, corn steep liquor 5%, beef extract 1%, epsom salt 0.2%, phosphoric acid
Hydrogen dipotassium 0.2%, ferrous sulfate 0.001%, VB1 0.0001%, pH 7.0 are mass percent above；
2) it filters：L-Isoleucine zymotic fluid is used into ceramic membrane filter, collects mycoprotein and filtrate；
3) concentration weight is molten：The filtrate that step 2) obtains is evaporated concentration, 1/5th of original volume is evaporated to, then adds
It is molten with the distilled water weight of concentrate same volume, weight solution is made；
4) ion exchange：Weight solution obtained by step 3) removes impurity by ion exchange, obtains l-Isoleucine feed liquid, then dense
It contracts, l-Isoleucine mass concentration is 10% in concentrate；The activated carbon that 3 ‰ mass fractions are added in concentrate, then heats up
To 70 DEG C, material liquid pH value is adjusted 5.5, is decolourized 35 minutes, then filtering removal activated carbon；Feed liquid after decoloration is concentrated into substance
Long-pending 1/6, then decrease temperature crystalline, when temperature is down to 20 DEG C, centrifuge high speed centrifugation obtains crude product；Crude product is put into bleacher
Then interior decoloration is concentrated, is crystallized, centrifuging, is dry, crushing obtains finished product.
2. technique according to claim 1, which is characterized in that the Corynebacterium glutamicum ATCC14309 seed liquors and breast
The concentration of acid fermentation brevibacterium ATCC13869 seed liquors is 1 × 108CFU/mL。
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107916281A (en) * | 2017-07-10 | 2018-04-17 | 广西多得乐生物科技有限公司 | A kind of method that gamma aminobutyric acid is isolated and purified from streptococcus acidi lactici fermented solution |
| CN108285911B (en) * | 2017-12-06 | 2021-04-16 | 新疆阜丰生物科技有限公司 | Process for extracting L-isoleucine by fermentation |
| CN108285912B (en) * | 2017-12-06 | 2021-03-30 | 新疆阜丰生物科技有限公司 | Method for preparing and extracting pharmaceutical grade valine by fermentation |
| CN109628516B (en) * | 2018-12-12 | 2021-09-28 | 新疆阜丰生物科技有限公司 | Production and extraction process of L-isoleucine |
| CN110607330B (en) * | 2019-10-16 | 2023-03-10 | 新疆阜丰生物科技有限公司 | Production process of L-isoleucine |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3262861A (en) * | 1960-11-18 | 1966-07-26 | Kyowa Hakko Kogyo Kk | Method for producing l-isoleucine by fermentation process |
| GB1213087A (en) * | 1968-03-13 | 1970-11-18 | Kyowa Hakko Kogyo Kk | Production of isoleucine by fermentation |
| EP0167132A2 (en) * | 1984-06-29 | 1986-01-08 | Ajinomoto Co., Inc. | Process for producing l-isoleucine by fermentation |
| CN1800148A (en) * | 2005-12-12 | 2006-07-12 | 无锡晶海氨基酸有限公司 | Cleaning production process of extracting L-isoleucine from fermented liquor using ion-exchange |
| CN101423851A (en) * | 2008-11-07 | 2009-05-06 | 潍坊祥维斯化学品有限公司 | Method for fermentation preparation of L-aminoacid |
-
2015
- 2015-10-28 CN CN201510744298.1A patent/CN105274179B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3262861A (en) * | 1960-11-18 | 1966-07-26 | Kyowa Hakko Kogyo Kk | Method for producing l-isoleucine by fermentation process |
| GB1213087A (en) * | 1968-03-13 | 1970-11-18 | Kyowa Hakko Kogyo Kk | Production of isoleucine by fermentation |
| EP0167132A2 (en) * | 1984-06-29 | 1986-01-08 | Ajinomoto Co., Inc. | Process for producing l-isoleucine by fermentation |
| CN1800148A (en) * | 2005-12-12 | 2006-07-12 | 无锡晶海氨基酸有限公司 | Cleaning production process of extracting L-isoleucine from fermented liquor using ion-exchange |
| CN101423851A (en) * | 2008-11-07 | 2009-05-06 | 潍坊祥维斯化学品有限公司 | Method for fermentation preparation of L-aminoacid |
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