CN103667382A - Method for producing L-glutamine by fermentation of microorganisms - Google Patents
Method for producing L-glutamine by fermentation of microorganisms Download PDFInfo
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Abstract
The invention belongs to the technical field of extraction and purification of amino acid derivative fermentation liquor and particularly relates to a method for producing L-glutamine by fermentation of microorganisms. The method comprises the following steps: preparation of the fermentation liquor: slope cultivation, shaking bacterial species in a shake flask, first-stage seed cultivation, second-stage seed cultivation and continuous fermentation; extraction and purification of fermented products: fermentation liquor pretreatment, ion exchange, secondary crystallization, decoloration impurity removal, nanofiltration membrane decoloration impurity removal and concentration, concentrated crystallization and drying to obtain a finished product. The method disclosed by the invention has the benefits that the contents of organic and inorganic impurities and the pigments in the subsequent extraction and purification liquor of glutamine fermentation liquor are obviously reduced, the yield of the glutamine is increased, the quality of a product is improved, the production cost is reduced, and the labor intensity of workers is greatly alleviated; the emission of solid and liquid waste such as water, acid, alkaline and activated carbon is greatly decreased, and the pollution to the environment is relieved.
Description
Technical field
The invention belongs to extraction and the purification techniques field of amino acid derivative fermented liquid, be specifically related to a kind of method of microorganism fermentative production L-glutaminate.
Background technology
Glutamine (Glutamin) is also known as bran amido acid, is the abundantest non-essential amino acid of content in human body, and is uniquely a kind ofly can directly pass through the amino acid of cerebrovascular barrier (BBB).In human body, be stored in skeletal muscle or blood.When injured or ill, glutamine may obtain enough amounts by the food absorbing containing Gln.Organism generates glutamine by glutamine synthetase catalysis L-glutamic acid and ammonium salt reaction.
L-glutaminate, this product is made accessory substance in food-processing, blending enriching substance.And L glutamine is body building and vigorous and graceful fan's important nutritional supplement.Its (hereinafter referred to as glutamine) is total free aminoacids the abundantest in muscle, accounts for 60% of human body total free aminoacids total amount.Fasting plasma glutamine concentration is 500-750umol/L.Glutamine is not indispensable amino acid, and it can be synthesized by L-glutamic acid, a word used in person's names propylhomoserin, Isoleucine in human body.At disease, nutritional status, under the stress situation such as not good or high-intensity exercise, body increases the demand of glutamine, so that self synthetic can not satisfying the demand.
L-glutaminate is widely used in fields such as medicine, nutritive health-care, food, and demand is large, is referred to as one of most important kind in nonessential amino acid.L-glutamine is the important materials of Development of Novel medicine, clinical treatment and sports nutrition health care, and all body tissues all utilize L-glutamine synthetic cell slurry albumen and nucleoprotein.Aspect physiologically active, outside the Pass having with gastrointestinal tract disease, animal science and animal and veterinary industry, in recent years, again as one of human cytotoxic weapon, strengthening immune system and cause concern, be expected to become the novel material of trophotherapy.Because L-glutamine not only has important scientific research, actual production and using value, L-glutamine is in the effect of physiological function importance simultaneously, and present domestic conventional production methods still seriously polluted, the production capacity output value is lower, therefore, through State Council and deputy to the National People's Congress's approval, Production of L-Glutamine by Microbial Fermentation is listed national Eleventh Five-Year Plan emphasis tackling key problem science and technology item in.
Fermented liquid → ceramic membrane filter → flocculation, decolouring+ceramic membrane filter → condensing crystal → centrifugal → dry → product.
Summary of the invention
In order to solve above-mentioned technical problem, the present invention utilizes ceramic membrane, reverse osmosis, nanofiltration membrane and two Crystallization Procedure to carry out the method for purifying L-glutaminate; Its yield of product and the qualification rate that adopt the method to obtain improve greatly.
The method of microorganism fermentative production L-glutaminate of the present invention is to solve above technical problem by following technical scheme:
The preparation method of L-glutaminate, the method comprises following step:
(1) preparation of fermented liquid:
The brevibacterium flavum MQG121 bacterial strain of take is fermentation strain;
(1) slant culture: peace roasting pipe bacterial classification is accessed to aseptic slant medium 28-32 ℃ and cultivate 20-24 hour;
(2) shaking flask bacterial classification: cultured slant strains is accessed to 28-32 ℃ of aseptic shake-flask seed substratum and cultivate 16-20 hour;
(3) first order seed is cultivated: cultured shaking flask kind liquid is accessed to aseptic first order seed substratum, and under 1:0.5 ventilation condition, mixing speed 240rpm, cultivates 12-16 hour for 28-32 ℃;
(4) secondary seed is cultivated: cultured one-level kind liquid is accessed to aseptic secondary seed medium, and under 1:0.5 ventilation condition, mixing speed 220rpm, cultivates 2-6 hour for 28-32 ℃;
(5) cultured secondary seed is inoculated in the fermention medium of sterilizing, under 1:0.3 ventilation condition, mixing speed 140rpm, fermenting process regulates pH value to 7.0-7.2 with liquefied ammonia, through 0-12 hours yeast culture, then regulates pH value to 5.5-5.7,12-44 hours synthetic cultivations of product, process is controlled PH with liquefied ammonia, and sugared content 2.0-0% is controlled in stream sugaring, obtains the fermented liquid containing glutamine product;
Above-mentioned fermention medium is:
Glucose 16-20% corn steep liquor 1-6%
ZnSO
4 0.5-2 mg/ml （NH
4）
2SO
4 3-7%
Molasses 0.5-2% KH
2pO
42-5%,
K
2HPO
4 2-5% NaH
2PO
4 0.1-0.4%,
VITMAIN B1 1-4mg/L CuSO
40.8-2mg/ml
MgSO
4 0.05-0.08%, MnSO
4 8-12mg/ml
Above-mentioned brevibacterium flavum bacterial strain (
brevibacterium flavum) on November 12nd, 2013, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.8468;
(2) leavened prod extracts refining:
Ceramic membrane filter: the fermented liquid under normal temperature state is sent into ceramic membrane circulating filtration and obtain fermentation clear liquid, in working cycle, use recirculated water for the cooling of ceramic membrane circulation feed liquid, the temperature of circulating fermentation liquid is remained on below 70 ℃, and pottery filter concentration liquid adds reverse osmosis water to clean out residual product; By ceramic membrane filter, remove thalline and the macromole impurity in fermented liquid, make pottery cleaner liquid.
Primary crystallization: by the vacuum concentration under 50 ℃, 80rpm condition of the feed liquid after concentrated, concentrated solution product content is 60%, vacuum tightness is-0.098Mpa, with recirculated water, with the cooling rate of 10-15 ℃/h, be cooled to 15 ℃, growing the grain is 4 hours again, growing the grain mixing speed is 20rpm, obtains wet product crystallization through whizzer is centrifugal;
Crude product back dissolving: first reverse osmosis water is warming up to 70 ℃, mixing speed is 40rpm, by wet product dissolving crystallized, the amount that is 10% by product content drops into crude product, with reagent hydrochloric acid, is adjusted to pH3.5, and crystallization is dissolved completely, makes dissolving crude product liquid.
Ion-exchange: by content be 10%, pH is 3.5, temperature be 45 ℃ feed liquid with 1 times of resin volume/hour flow velocity by 732 cationic exchange coloums, feed liquid by cationic exchange coloum flows into 330 anion-exchange columns with identical speed, by ion exchange resin, remove the organic and inorganic impurity in feed liquid, make from handing over liquid; With H type 732 Zeo-karbs, regulate feed liquid PH to 6.0-6.5.
Nanofiltration: will enter nanofiltration equipment circulating filtration under the feed liquid normal temperature state after the removal of impurities of cation and anion exchange post; In working cycle, use recirculated water for nanofiltration circulation feed liquid cooling, the temperature of the feed liquid that makes to circulate remains on below 50 ℃, and nanofiltration concentrated solution adds reverse osmosis water to clean out residual product; By nanofiltration, remove the organic molecule impurity in feed liquid, make nanofiltration dialysis feed liquid.
Decolouring removal of impurities is processed: with gac to nanofiltration dialysis feed liquid the removal of impurities of decolouring process, feed liquid is warming up to 70 ℃, add 0.3% medicinal 767 gacs, be incubated 30 minutes, mixing speed is 60rpm, after transmitance reaches more than 95%, by the special strainer circulating filtration of sweet smell, remove the gac in feed liquid, make destainer.
Reverse osmosis concentration: the feed liquid after decolouring is cooled to 40 ℃, enters reverse osmosis concentration device circulation and be concentrated into content and reach 15%; In working cycle, use recirculated water to lower the temperature for circulation fluid, keep feed temperature≤50 ℃, make reverse osmosis concentrated liquid.With H type 732 Zeo-karbs, regulate feed liquid PH to 6.0-6.5.
Secondary crystal: reverse osmosis concentrated liquid is squeezed into single-action condensing crystal tank, keeping thickening temperature is≤50 ℃, vacuum tightness is-0.098Mpa, mixing speed keeps 60rpm, when concentrated solution product content is more than 60%, keep mixing speed 20rpm, by the cooling rate of 15-15 ℃ /h of recirculated water, be down to 15 ℃, growing the grain 4 hours, obtains wet product crystallization through rotating speed 1200rpm whizzer is centrifugal;
Dry: wet product crystalline product is dry through double cone dryer, and its condition is: vacuum tightness-0.098Mpa, use temperature is the hot water circulation of 80 ℃ temperature≤50 ℃ that keep dry, dry 6h, treat product temperature≤40 ℃ packing, pack environment condition is: 18 ℃-25 ℃ of temperature, humidity≤70%.
Preferably, in the preparation process of above-mentioned fermented liquid:
The brevibacterium flavum MQG121 bacterial strain of take is fermentation strain;
(1) slant culture: peace roasting pipe bacterial classification is accessed to aseptic slant medium 28-32 ℃ and cultivate 20-24 hour;
(2) shaking flask bacterial classification: cultured slant strains is accessed to 28-32 ℃ of aseptic shake-flask seed substratum and cultivate 16-20 hour;
(3) first order seed is cultivated: cultured shaking flask kind liquid is accessed to aseptic first order seed substratum, and under 1:0.5 ventilation condition, mixing speed 240rpm, cultivates 12-16 hour for 28-32 ℃;
(4) secondary seed is cultivated: cultured one-level kind liquid is accessed to aseptic secondary seed medium, and under 1:0.5 ventilation condition, mixing speed 220rpm, cultivates 2-6 hour for 28-32 ℃;
(5) cultured secondary seed is inoculated in the fermention medium of sterilizing, under 1:0.3 ventilation condition, mixing speed 140rpm, fermenting process regulates pH value to 7.0-7.2 with liquefied ammonia, through 0-12 hours yeast culture, then regulates pH value to 5.5-5.7,12-44 hours synthetic cultivations of product, process is controlled PH with liquefied ammonia, and sugared content 2.0-0% is controlled in stream sugaring, obtains the fermented liquid containing glutamine product;
Preferred, above-mentioned fermention medium is:
Glucose 18% corn steep liquor 1%
ZnSO
4 1.5 mg/ml （NH
4）
2SO
4 4%
Molasses 1.5% KH
2pO
42.5%,
K
2HPO
4 2.5% kH
2PO
4 0.1%,
VITMAIN B1 1mg/L CuSO
41mg/ml
MgSO
4 0.05%, MnSO
4 8mg/ml
Brevibacterium flavum bacterial strain of the present invention (
brevibacterium flavum) on November 12nd, 2013, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.8468; Preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode: 100101.
Beneficial effect of the present invention is:
(1) in the treating process of L-glutaminate, utilize ceramic membrane equipment to remove production bacterium and the high molecular weight protein in fermented liquid, replace traditional sheet frame sterilization technique, reduced floor space and labour intensity; Adopt ceramic membrane to carry out the degerming of glutamine ferment liquid, not only can reclaim albumen, due to the raising of filtered liquid quality, the consumption that can also significantly reduce ion exchange resin is (by resin 100 kg/ms original to the exchange capacity of product
3bring up to 150 kg/m
3), two Crystallization Procedure can significantly reduce thalline, organism and inorganics foreign matter content in glutamine subsequent extracted refined liquid, improve the yield of glutamine;
(3) adopt ultra-filtration membrane and sodium membrane filtration technology to remove organic pigment and the small organic molecule in feed liquid, organic impurity in feed liquid is reduced greatly, improved the transmittance of feed liquid, reduced the consumption of gac;
(4) adopt reverse osmosis to concentrate pre-treatment, reduced the concentrated destruction to product of high temperature, reduced the use of steam, reduced energy consumption, reduced sewage discharge, guarantee to enter the quality of ion exchange column feed liquid, strengthened the adsorptive power of ion exchange column to product, reduced the consumption of resin; Ion-exchange is resolved liquid glutamine content and is brought up to 8% by traditional 5.5%, from handing over liquid purity to be greatly enhanced, has improved quality product, has reduced production cost, has greatly alleviated workman's labour intensity.
(5) at product enriching stage, adopt general in the world membrane concentration separating technology to replace traditional concentrating under reduced pressure technique, reduced the destruction to product, reduced again production cost.And adopted sodium filter membrane production technique, and improved the quality of product, also reduced the resin demand of fermentation method traditional technology in the past, greatly reduced the useless quantity dischargeds of solid-liquid such as water, acid, alkali, gac, reduced the pollution to environment.
By above-mentioned treatment process, finished product total recovery for fermented liquid can reach more than 85% (with respect to the clean L-glutaminate cubage of removing thalline), finished product first-time qualification rate reaches 100%, and traditional technology can only reach 50% total recovery, and first-time qualification rate only has 98%.
Accompanying drawing explanation
Fig. 1 is process flow sheet of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further described, so that those skilled in the art more understands the present invention, but with this, does not limit the present invention.
Extraction production technology
The method of microorganism fermentative production L-glutaminate, the method comprises following step:
(1) preparation of fermented liquid:
The brevibacterium flavum MQG121 bacterial strain of take is fermentation strain;
(1) slant culture: peace roasting pipe bacterial classification is accessed to aseptic slant medium 28-32 ℃ and cultivate 20-24 hour;
(2) shaking flask bacterial classification: cultured slant strains is accessed to 28-32 ℃ of aseptic shake-flask seed substratum and cultivate 16-20 hour;
(3) first order seed is cultivated: cultured shaking flask kind liquid is accessed to aseptic first order seed substratum, and under 1:0.5 ventilation condition, mixing speed 240rpm, cultivates 12-16 hour for 28-32 ℃;
(4) secondary seed is cultivated: cultured one-level kind liquid is accessed to aseptic secondary seed medium, and under 1:0.5 ventilation condition, mixing speed 220rpm, cultivates 2-6 hour for 28-32 ℃;
(5) cultured secondary seed is inoculated in the fermention medium of sterilizing, under 1:0.3 ventilation condition, mixing speed 140rpm, fermenting process regulates pH value to 7.0-7.2 with liquefied ammonia, through 0-12 hours yeast culture, then regulates pH value to 5.5-5.7,12-44 hours synthetic cultivations of product, process is controlled PH with liquefied ammonia, and sugared content 2.0-0% is controlled in stream sugaring, obtains the fermented liquid containing glutamine product;
Above-mentioned fermention medium is:
Glucose 18% corn steep liquor 1%
ZnSO
4 1.5 mg/ml （NH
4）
2SO
4 4%
Molasses 1.5% KH
2pO
42.5%,
K
2HPO
4 2.5% kH
2PO
4 0.1%,
VITMAIN B1 1mg/L CuSO
41mg/ml
MgSO
4 0.05%, MnSO
4 8mg/ml
Above-mentioned brevibacterium flavum bacterial strain (
brevibacterium flavum) on November 12nd, 2013, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.8468;
(2) leavened prod extracts refining:
Ceramic membrane filter: the fermented liquid under normal temperature state is sent into ceramic membrane circulating filtration and obtain fermentation clear liquid, in working cycle, use recirculated water for the cooling of ceramic membrane circulation feed liquid, the temperature of circulating fermentation liquid is remained on below 70 ℃, and pottery filter concentration liquid adds reverse osmosis water to clean out residual product; By ceramic membrane filter, remove thalline and the macromole impurity in fermented liquid, make pottery cleaner liquid.
Primary crystallization: by the feed liquid after concentrated at 50 ℃,, vacuum concentration under 80rpm condition, concentrated solution product content is 60%, vacuum tightness is-0.098Mpa, with recirculated water, with the cooling rate of 10-15 ℃/h, be cooled to 15 ℃, growing the grain is 4 hours again, and growing the grain mixing speed is 20rpm, obtains wet product crystallization through whizzer is centrifugal;
Crude product back dissolving: first reverse osmosis water is warming up to 70 ℃, mixing speed is 40rpm, by wet product dissolving crystallized, the amount that is 10% by product content drops into crude product, with reagent hydrochloric acid, is adjusted to pH3.5, and crystallization is dissolved completely; Make dissolving crude product liquid.
Ion-exchange: by content be 10%, pH is 3.5, temperature be 45 ℃ feed liquid with 1 times of resin volume/hour flow velocity by 732 cationic exchange coloums, feed liquid by cationic exchange coloum flows into 330 anion-exchange columns with identical speed, by ion exchange resin, remove the organic and inorganic impurity in feed liquid, make from handing over liquid;
Nanofiltration: will enter nanofiltration equipment circulating filtration under the feed liquid normal temperature state after the removal of impurities of cation and anion exchange post; In working cycle, use recirculated water for nanofiltration circulation feed liquid cooling, the temperature of the feed liquid that makes to circulate remains on below 50 ℃, and nanofiltration concentrated solution adds reverse osmosis water to clean out residual product; By nanofiltration, remove the organic molecule impurity in feed liquid, make nanofiltration dialysis feed liquid.
Decolouring removal of impurities is processed: with gac to nanofiltration dialysis feed liquid the removal of impurities of decolouring process, feed liquid is warming up to 70 ℃, add 0.3% medicinal 767 gacs, be incubated 30 minutes, mixing speed is 60rpm, after transmitance reaches more than 95%, by the special strainer circulating filtration of sweet smell, remove the gac in feed liquid, make destainer.
Reverse osmosis concentration: the feed liquid after decolouring is cooled to 40 ℃, enters reverse osmosis concentration device circulation and be concentrated into content and reach 15%; In working cycle, use recirculated water to lower the temperature for circulation fluid, keep feed temperature≤50 ℃, make reverse osmosis concentrated liquid.With H type 732 Zeo-karbs, regulate feed liquid PH to 6.0-6.5.
Secondary crystal: reverse osmosis concentrated liquid is squeezed into single-action condensing crystal tank, keeping thickening temperature is≤50 ℃, vacuum tightness is-0.098Mpa, mixing speed keeps 60rpm, when concentrated solution product content is more than 60%, keep mixing speed 20rpm, by the cooling rate of 15-15 ℃ /h of recirculated water, be down to 15 ℃, growing the grain 4 hours, obtains wet product crystallization through whizzer is centrifugal;
Dry: wet product crystalline product is dry through double cone dryer, and its condition is: vacuum tightness-0.098Mpa, use temperature is the hot water circulation of 80 ℃ temperature≤50 ℃ that keep dry, dry 6h, treat product temperature≤40 ℃ packing, pack environment condition is: 18 ℃-25 ℃ of temperature, humidity≤70%.
Wet product crystalline product is dry through bipyramid, and its condition is: vacuum tightness-0.098Mpa, and 80 ℃ of temperature, packing, condition is: 18 ℃-25 ℃ of temperature, humidity≤60%, transmittance >=82%, DE value≤2.5%, obtains finished product.
(2) L-glutaminate is at 50m
3fermentor tank extracts in producing, refining result statistics
production statistics is as follows
(3) inspection after construction result: pilot product is through check, and indices meets L-glutaminate FCC V standard, and main performance index refers to following table:
From above data, the present invention is with respect to traditional method, and floor space is little, has saved floor space; The yield of product brings up to 85% from 50%, and activated carbon dosage has reduced 50%, has saved greatly production cost, and disposable qualification rate brings up to 100% from 95%; Purity brings up to 99.6% from 98.5%.From above, to recently, the present invention has significant progress with respect to traditional method.
The present invention extracts workshop section and adopts ceramic membrane device processes, and the production bacterium that utilizes ceramic membrane equipment to remove in fermented liquid replaces traditional sheet frame sterilization technique, and impurity in feed liquid is reduced greatly; But also reduced sewage discharge, guaranteed to enter the quality of ion exchange column feed liquid, strengthened the adsorptive power of ion exchange column, reduced the consumption of resin and gac, ion-exchange is crossed flow liquid glutamine content and is reached 8%, extracts, refines total recovery and reach more than 85%, has improved quality product, reduce production cost, greatly alleviated workman's labour intensity.At product enriching stage, adopt general in the world membrane concentration separating technology to replace traditional concentrating under reduced pressure technique, reduced the destruction to product, saved again steam consumption, also reduced coal quantity of boiler, the resource consumptions such as water, electricity, coal reduce greatly, be conducive to save energy and reduce the cost, production cost is low.And adopted sodium filter membrane concentration technology, and improved the quality of product, also reduced the resin demand of fermentation method tradition extraction concentration technique in the past, greatly reduced the quantity discharged of the waste water such as water, acid, alkali, reduced the pollution to environment.
Embodiment 2
A method for microorganism fermentative production L-glutaminate, the method comprises following step:
(1) preparation of fermented liquid:
The brevibacterium flavum MQG121 bacterial strain of take is fermentation strain;
Slant culture: will pacify 28-32 ℃ of roasting pipe bacterial classification access slant medium and cultivate 20-24 hour;
Shaking flask bacterial classification: 28-32 ℃ of cultured slant strains access shake-flask seed substratum cultivated to 16-20 hour;
First order seed is cultivated: 28-32 ℃ of the first order seed substratum of cultured shaking flask kind liquid access sterilization cultivated to 12-16 hour;
Secondary seed is cultivated: cultivate 2-6 hour for secondary seed medium 28-32 ℃ that cultured one-level kind liquid access was sterilized; Cultured secondary seed is inoculated in the fermention medium of sterilizing, passes into sterile wind, regulate pH value to 6.2-6.8, through cultivation in 40-48 hour, obtain the fermented liquid containing glutamine product;
Above-mentioned fermention medium is:
Semi-lactosi 13%, beef peptone 2.0%
Murphy juice 8.6 %, bean sprout juice 6%,
Zulkovsky starch 4% agar 20g/L
Soy peptone 4.5% (NH
4) SO
46.8%,
NaCl 0.6 %， NaH
2PO
4 0.3%,
MgSO
40.06%, MnSO
4 10mg/L，
CuSO
41.5mg/L vitamins B
11.5mg/L;
Above-mentioned brevibacterium flavum bacterial strain (
brevibacterium flavum) on November 12nd, 2013, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.8468;
(2) leavened prod extracts refining:
Ceramic membrane filter: the fermented liquid under normal temperature state is sent into ceramic membrane filter, and adopting recirculated water in treating processes is the cooling of circulation feed liquid, and the temperature of fermented liquid is remained on below 70 ℃, and concentrated solution adds reverse osmosis water to clean out residual product;
Reverse osmosis concentration: to the feed liquid reverse osmosis concentration under normal temperature, making material liquid pH is 7.0, and in feed liquid, product content is 10%, making product content in concentrated rear feed liquid is 15%;
Primary crystallization: by the feed liquid primary crystallization after concentrated, in primary crystallization, thickening temperature is 50 ℃, and concentrated solution product content is 60%, vacuum tightness is-0.098Mpa, is down to normal temperature growing the grain 4 hours, mixing speed is 20rpm, obtains wet product crystallization through whizzer is centrifugal;
Crude product back dissolving: by wet product dissolving crystallized, the amount that is 10% by product content drops into crude product reverse osmosis water solvent, adjusts pH3.5, is heated to 50 ℃, under the rotating speed of 30rpm under agitation condition until dissolving completely;
Ion-exchange: by content be 10%, pH is 3.5, temperature be 45 ℃ feed liquid with 1 times of resin volume/hour flow velocity by 732 cationic exchange coloums, the feed liquid by cationic exchange coloum flows into 330 anion-exchange columns with identical speed, must be from handing over liquid;
Nanofiltration: will enter nano-filtration step under the feed liquid normal temperature state after the removal of impurities of cation and anion exchange post;
Decolouring removal of impurities is processed: with nanofiltration to feed liquid the removal of impurities of decolouring process, by from handing over liquid to remove the organic-inorganic pigment in feed liquid through nanofiltration equipment circulating filtration, after concentrated solution adds reverse osmosis water cleaning product residual, make feed liquid transmittance reach 85%;
Membrane concentration is separated: the feed liquid after decolouring is cooled to 40 ℃, enters reverse osmosis concentration device and be concentrated into content and reach 15%;
Secondary crystal: in secondary crystal step, keeping thickening temperature is 50 ℃, and concentrated solution product content is more than 60%, vacuum tightness is-0.098Mpa, is down to normal temperature growing the grain 4 hours, mixing speed is 20rpm.Obtain wet product crystallization through whizzer is centrifugal;
Wet product crystalline product is dry through bipyramid, and its condition is: vacuum tightness-0.098Mpa, and 80 ℃ of temperature, packing, condition is: 18 ℃-25 ℃ of temperature, humidity≤60%, transmittance >=82%, DE value≤2.5%, obtains finished product.
Claims (3)
1. the method for microorganism fermentative production L-glutaminate, is characterized in that, the method comprises following step:
(1) preparation of fermented liquid:
The brevibacterium flavum MQG121 bacterial strain of take is fermentation strain;
(1) slant culture: peace roasting pipe bacterial classification is accessed to aseptic slant medium 28-32 ℃ and cultivate 20-24 hour;
(2) shaking flask bacterial classification: cultured slant strains is accessed to 28-32 ℃ of aseptic shake-flask seed substratum and cultivate 16-20 hour;
(3) first order seed is cultivated: cultured shaking flask kind liquid is accessed to aseptic first order seed substratum, and under 1:0.5 ventilation condition, mixing speed 240rpm, cultivates 12-16 hour for 28-32 ℃;
(4) secondary seed is cultivated: cultured one-level kind liquid is accessed to aseptic secondary seed medium, and under 1:0.5 ventilation condition, mixing speed 220rpm, cultivates 2-6 hour for 28-32 ℃;
(5) cultured secondary seed is inoculated in the fermention medium of sterilizing, under 1:0.3 ventilation condition, mixing speed 140rpm, fermenting process regulates pH value to 7.0-7.2 with liquefied ammonia, through 0-12 hours yeast culture, then regulates pH value to 5.5-5.7,12-44 hours synthetic cultivations of product, process is controlled PH with liquefied ammonia, and sugared content 2.0-0% is controlled in stream sugaring, obtains the fermented liquid containing glutamine product;
Above-mentioned fermention medium is:
Glucose 16-20% corn steep liquor 1-6%
ZnSO
4 0.5-2 mg/ml （NH
4）
2SO
4 3-7%
Molasses 0.5-2% KH
2pO
42-5%,
K
2HPO
4 2-5% NaH
2PO
4 0.1-0.4%,
VITMAIN B1 1-4mg/L CuSO
40.8-2mg/ml
MgSO
4 0.05-0.08%, MnSO
4 8-12mg/ml
Above-mentioned brevibacterium flavum bacterial strain (
brevibacterium flavum) on November 12nd, 2013, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.8468;
(2) leavened prod extracts refining:
Ceramic membrane filter: the fermented liquid under normal temperature state is sent into ceramic membrane circulating filtration and obtain fermentation clear liquid, in working cycle, use recirculated water for the cooling of ceramic membrane circulation feed liquid, the temperature of circulating fermentation liquid is remained on below 70 ℃, and pottery filter concentration liquid adds reverse osmosis water to clean out residual product; By ceramic membrane filter, remove thalline and the macromole impurity in fermented liquid, make pottery cleaner liquid;
Primary crystallization: by the vacuum concentration under 50 ℃, 80rpm condition of the feed liquid after concentrated, concentrated solution product content is 60%, vacuum tightness is-0.098Mpa, with recirculated water, with the cooling rate of 10-15 ℃/h, be cooled to 15 ℃, growing the grain is 4 hours again, growing the grain mixing speed is 20rpm, obtains wet product crystallization through the whizzer of 1200rpm rotating speed is centrifugal;
Crude product back dissolving: first reverse osmosis water is warming up to 70 ℃, mixing speed is 40rpm, by wet product dissolving crystallized, the amount that is 10% by product content drops into crude product, with reagent hydrochloric acid, is adjusted to pH3.5, and crystallization is dissolved completely; Make dissolving crude product liquid;
Ion-exchange: by content be 10%, pH is 3.5, temperature be 45 ℃ feed liquid with 1 times of resin volume/hour flow velocity by 732 cationic exchange coloums, feed liquid by cationic exchange coloum flows into 330 anion-exchange columns with identical speed, by ion exchange resin, remove the organic and inorganic impurity in feed liquid, make from handing over liquid;
Nanofiltration: will enter nanofiltration equipment circulating filtration under the feed liquid normal temperature state after the removal of impurities of cation and anion exchange post; In working cycle, use recirculated water for nanofiltration circulation feed liquid cooling, the temperature of the feed liquid that makes to circulate remains on below 50 ℃, and nanofiltration concentrated solution adds reverse osmosis water to clean out residual product; By nanofiltration, remove the organic molecule impurity in feed liquid, make nanofiltration dialysis feed liquid;
Decolouring removal of impurities is processed: with gac to nanofiltration dialysis feed liquid the removal of impurities of decolouring process, feed liquid is warming up to 70 ℃, add 0.3% medicinal 767 gacs, be incubated 30 minutes, mixing speed is 60rpm, after transmitance reaches more than 95%, by the special strainer circulating filtration of sweet smell, remove the gac in feed liquid, make destainer;
Reverse osmosis concentration: the feed liquid after decolouring is cooled to 40 ℃, enters reverse osmosis concentration device circulation and be concentrated into content and reach 15%; In working cycle, use recirculated water to lower the temperature for circulation fluid, keep feed temperature≤50 ℃, make reverse osmosis concentrated liquid;
With H type 732 Zeo-karbs, regulate feed liquid PH to 6.0-6.5;
Secondary crystal: reverse osmosis concentrated liquid is squeezed into single-action condensing crystal tank, keeping thickening temperature is≤50 ℃, vacuum tightness is-0.098Mpa, mixing speed keeps 60rpm, when concentrated solution product content is more than 60%, keep mixing speed 20rpm, by the cooling rate of 15-15 ℃ /h of recirculated water, be down to 15 ℃, growing the grain 4 hours, obtains wet product crystallization through whizzer is centrifugal; With H type 732 Zeo-karbs, regulate feed liquid PH to 6.0-6.5;
Dry: wet product crystalline product is dry through double cone dryer, and its condition is: vacuum tightness-0.098Mpa, use temperature is the hot water circulation of 80 ℃ temperature≤50 ℃ that keep dry, dry 6h, treat product temperature≤40 ℃ packing, pack environment condition is: 18 ℃-25 ℃ of temperature, humidity≤70%.
2. the method for a kind of microorganism fermentative production L-glutaminate as claimed in claim 1, is characterized in that, in the preparation process of described fermented liquid:
The brevibacterium flavum MQG121 bacterial strain of take is fermentation strain;
(1) slant culture: peace roasting pipe bacterial classification is accessed to aseptic slant medium 28-32 ℃ and cultivate 20-24 hour;
(2) shaking flask bacterial classification: cultured slant strains is accessed to 28-32 ℃ of aseptic shake-flask seed substratum and cultivate 16-20 hour;
(3) first order seed is cultivated: cultured shaking flask kind liquid is accessed to aseptic first order seed substratum, and under 1:0.5 ventilation condition, mixing speed 240rpm, cultivates 12-16 hour for 28-32 ℃;
(4) secondary seed is cultivated: cultured one-level kind liquid is accessed to aseptic secondary seed medium, and under 1:0.5 ventilation condition, mixing speed 220rpm, cultivates 2-6 hour for 28-32 ℃;
(5) cultured secondary seed is inoculated in the fermention medium of sterilizing, under 1:0.3 ventilation condition, mixing speed 140rpm, fermenting process regulates pH value to 7.0-7.2 with liquefied ammonia, through 0-12 hours yeast culture, then regulates pH value to 5.5-5.7,12-44 hours synthetic cultivations of product, process is controlled PH with liquefied ammonia, and sugared content 2.0-0% is controlled in stream sugaring, obtains the fermented liquid containing glutamine product.
3. the method for a kind of microorganism fermentative production L-glutaminate as claimed in claim 1, is characterized in that, described fermention medium is:
Glucose 18% corn steep liquor 1%
ZnSO
4 1.5 mg/ml （NH
4）
2SO
4 4%
Molasses 1.5% KH
2pO
42.5%,
K
2HPO
4 2.5% kH
2PO
4 0.1%,
VITMAIN B1 1mg/L CuSO
41mg/ml
MgSO
4 0.05%, MnSO
4 8mg/ml 。
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| CN104830941A (en) * | 2015-04-29 | 2015-08-12 | 宁夏诚志万胜生物工程有限公司 | Microbial conversion method utilizing mixed bacteria to efficiently synthesize L-theanine |
| CN108285913A (en) * | 2017-12-09 | 2018-07-17 | 新疆阜丰生物科技有限公司 | A kind of technique preparing extraction L-Glutamine |
| CN108997159A (en) * | 2018-07-17 | 2018-12-14 | 廊坊梅花生物技术开发有限公司 | A kind of preparation method of L-Glutamine |
| CN110452938A (en) * | 2019-08-29 | 2019-11-15 | 江西省食品发酵研究所 | A kind of fermentation method for producing of high-yield glutamine |
| CN112301071A (en) * | 2020-11-04 | 2021-02-02 | 赤峰蒙广生物科技有限公司 | Method for producing adenine by fermentation method |
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