CN103695490A - High-purity arginine production process - Google Patents
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Abstract
The invention discloses a high-purity arginine production process which comprises the following steps: 1 preparation of a fermentation liquor comprising slant culture, preparing a shake flask strain, primary seed culture, secondary seed culture, and continuing fermentation; and 2 extracting and refining of a fermentation product comprising fermentation liquor pretreatment, secondary crystallization, decolorization and impurity removal treatment, membrane concentration and separation, drying, and packaging, to obtain a finished product. The high-purity arginine production process has the beneficial effects that the contents of the strain and organic and inorganic impurities in the subsequent extracting refined liquid of the arginine are remarkably reduced, the yield of the arginine is increased, the product quality is improved, the production cost is lowered, the labor intensity of a worker is greatly alleviated, the emission of solid and liquid wastes like water, acid, alkaline, and activated carbon are greatly reduced, and the environmental pollution is reduced.
Description
Technical field
The present invention relates to amino acid zymotic fluid high efficiency extraction purification techniques field, particularly relate to a kind of high purity extractive propylhomoserin production technique.
Background technology
Arginine 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 amino acid.L-arginine is the important mesostate of organism ornithine cycle, and all body tissues all utilize L-arginine synthetic cell slurry albumen and nucleoprotein.Aspect physiologically active, outside the Pass having with hormone inductions such as tethelin, Regular Insulin, hyperglycemic-glycogenolytic factors, in recent years, as vascular relaxing factor, cause concern again, be expected to become the novel material of trophotherapy.Because L-arginine not only has important scientific research, actual production and using value, L-arginine 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, fermentative Production arginine is listed national Eleventh Five-Year Plan emphasis tackling key problem science and technology item in.
At present domestic main dependence proteolysis method is produced arginine, and during this method operational cost, yield is low, technology stability is bad and environmental pollution is serious, is unsuitable for scale operation.Abroad particularly European Union member countries extremely emphasize non-animal-origin (non-animaL), make hair-hydrolyzation arginine product be subject to the restriction that develops on a large scale very much.Therefore, set up China's fermentative Production arginine industry very urgent, also have good development prospect.
Arginine extracts and to mainly contain two kinds of production technique: i.e. proteolysis extraction process; Bio-fermentation process.In bio-fermentation process, in fermented liquid, contain a large amount of organic and inorganic impurities, traditional rear extraction process technique does not have guarantee to quality product, and time-consuming.Prior art has adopted Plate Filtration while extracting, and the time is long, and yield is low, and loss is large.
Summary of the invention
Object of the present invention is exactly to provide a kind of high purity extractive propylhomoserin production technique for the defect of above-mentioned existence.By preparation method of the present invention, finished product total recovery for fermented liquid can reach more than 83% (to be calculated with respect to the clean arginine content of removing thalline), finished product first-time qualification rate reaches 99.9%, and traditional technology can only reach 50% total recovery, and first-time qualification rate only has 97%.
A kind of high purity extractive propylhomoserin production technology scheme of the present invention is comprise the preparation of fermented liquid and extract purification step, wherein the preparation of fermented liquid employing brevibacterium flavum MQA121.
MQA121 bacterial strain is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and postcode: 100101, its deposit number is: CGMCC No.8392, the Latin title of bacterial classification is
brevibacterium fLavum, the microorganism (strain) of ginseng certificate: MQA121, preservation date is on October 25th, 2013.
Brevibacterium flavum MQA121 strain characteristics of the present invention is: acid producing ability is strong, glucose acid invert ratio is high, stable performance.Can be used as bacterial classification and produce arginine.
The preparation of fermented liquid comprises following step:
(1) slant culture: by brevibacterium flavum MQA121 access slant medium, cultivate for 30 ℃ and obtain slant strains for 20 hours;
(2) shaking flask bacterial classification: 30 ℃ of cultured slant strains access shake-flask seed substratum are cultivated and obtained shake-flask seed liquid for 22 hours;
(3) first order seed is cultivated: 30 ℃ of the first order seed substratum of cultured shake-flask seed liquid access sterilization are cultivated 18 hours to obtain to one-level kind liquid;
(4) secondary seed is cultivated: 30 ℃ of cultivations of secondary seed medium that cultured primary seed solution access was sterilized obtain secondary seed solution for 4-6 hour;
(5) cultured secondary seed solution is inoculated in the fermention medium of sterilizing, fermenting process is added 2% glucose by weight percentage and 5.0% corn steep liquor, 0.8% (NH
4)
2sO
4, 0.2%KH
2pO
4, 0.3%K
2hPO
43H
2o, passes into sterile wind, adopts ammoniacal liquor to control pH value 7.1, through cultivation in about 68-72 hour, obtains the fermented liquid containing arginine product.
Slant medium contains by weight percentage: glucose 1.8%, (NH
4)
2sO
40.5%, KH
2pO
40.2%, K
2hPO
43H
2o0.2%, MgSO
47H
2o0.05%, MnSO
4h
2o0.002%, VITAMIN 50 g/L, agar powder 2.0%, pH6.9 ~ 7.3,121 ℃ of sterilizings 20 minutes.
Shake-flask seed substratum, first order seed substratum, secondary seed medium and fermention medium contain by weight percentage: initial sugar concentration 10%, urea 5%, yeast extract paste 0.3%, corn steep liquor CSL 0.5%, (NH
4)
2sO
45%, MgSO
40.05%, KH
2pO
40.15%, K
2hPO
40.05%, VITAMIN 50 μ g/L.
In step (5), ammoniacal liquor volumetric concentration is 1:1.
Leavened prod extracts refining comprising the following steps: (1) fermentation liquor pretreatment; (2) flocculation, decolouring, concentrated; (3) secondary crystal; (4) finished product.
Be specially:
(1) fermentation liquor pretreatment utilizes ceramic membrane equipment to remove production bacterium and the high molecular weight protein in fermented liquid.Ceramic membrane aperture 100-800nm, operation pressure reduction 0.15-0.32MPa, crossflow velocity 3m/s, intends stabilized flux 80-125L/m
2.h.
Fermented liquid directly passes through ceramic membrane circulating filtration, and it is the cooling of circulation feed liquid that process adopts recirculated water, and cycle stock liquid temp is controlled at below 60 ℃, and concentrated solution adds reverse osmosis water to clean out residual product.Filtration time is 13 hours, and cleaner liquid content is 60g/L, filtrate clarification, transparent, and pre-treatment yield is 80%.After ceramic membrane, clear liquid bacteria containing amount < 0.2%.
(2) flocculation, decolouring, pre-concentration by the cleaner liquid obtaining in step (1) with 1 times of volume of resin/hour flow velocity flow through acidulous cation resin adsorption production, with the 2.5N ammoniacal liquor of 2 times of resin volumes, with the flow velocity wash-out of 1 times of resin volume, obtain elutriant; Elutriant is the nano-filtration membrane equipment circulation decolouring of 1000 molecular weight by latus rectum, process is used circulating water cooling controlled circulation feed temperature lower than 55 ℃, with reverse osmosis water cleaning concentrate to product content lower than 0.2%, it is more than 90% obtaining printing opacity, the nanofiltration concentrated solution that content is 6%; Nanofiltration clear liquid is warming up to 70 ℃, under 80rpm whipped state, add 1% activated carbon decolorizing, be incubated 30 minutes, when clear liquid transmittance reaches, more than 95% by decarburizing machine, remove gac and obtain the clear liquid that decolours, decolouring clear liquid is through reverse-osmosis circulating, with circulating water cooling controlled circulation feed temperature, lower than 50 ℃, after reaching 15%, concentrated solution content obtains reverse osmosis concentrated liquid; Reverse osmosis concentrated liquid take 1 times of resin volume/hour flow velocity flow through and obtain resin anion(R.A) that content is 13% transmittance 98% exchange liquid through weak anion resin.
(3) concentrated, crystallization sucks condensing crystal tank by anionresin liquid, at-0.08Mpa, feed temperature, be controlled at and under the condition that 50 ℃, mixing speed are 80rpm, anionresin liquid be concentrated into feed liquid content and reach 80%, reduce rotating speed to 50rpm, with-5 ℃ of frozen water, feed liquid is cooled to 5 ℃ of growing the grains 8 hours.
(4) finished product is rested brilliant crystal solution through centrifugation, and the alcohol with 75% cleans crystallization and obtains wet product, and wet product is dried and obtains dry product through bipyramid, packs to obtain finished product under aseptic condition.
The invention has the advantages that, this extraction process adopts ceramic membrane to replace traditional Plate Filtration, has improved filtrate quality, has reduced floor space, has improved product yield, has reduced labour intensity.
Adopt membrane separation technique to replace traditional ion-exchange isolation technique, reduced resin demand, improved feed liquid quality and seen through, reduced activated carbon dosage, improved quality product.
Adopt membrane concentration technology to replace traditional concentrating under reduced pressure technology, reduced the destruction to product, reduced energy consumption, improved quality product.
Finally, by preparation method of the present invention, finished product total recovery for fermented liquid can reach more than 83% (to be calculated with respect to the clean arginine content of removing thalline), and finished product first-time qualification rate reaches 99.9%, and traditional technology can only reach 50% total recovery, first-time qualification rate only has 97%.
embodiment:
In order to understand better the present invention, with specific examples, describe technical scheme of the present invention in detail below, but the present invention is not limited thereto.
Embodiment 1
The preparation of fermented liquid comprises following step:
(1) slant culture: by brevibacterium flavum MQA121 access slant medium (glucose 1.8%, (NH
4)
2sO
40.5%, KH
2pO
40.2%, K
2hPO
43H
2o0.2%, MgSO
47H
2o0.05%, MnSO
4h
2o0.002%, VITAMIN 50 g/L, agar powder 2.0%, pH6.9 ~ 7.3,121 ℃ of sterilizings 20 minutes) 30 ℃ cultivate and within 20 hours, obtain slant strains;
(2) shaking flask bacterial classification: 30 ℃ of cultured slant strains access shake-flask seed substratum are cultivated and obtained shake-flask seed liquid for 22 hours;
(3) first order seed is cultivated: 30 ℃ of the first order seed substratum of cultured shake-flask seed liquid access sterilization are cultivated 18 hours to obtain to one-level kind liquid;
(4) secondary seed is cultivated: 30 ℃ of cultivations of secondary seed medium that cultured primary seed solution access was sterilized obtain secondary seed solution for 4-6 hour;
(5) cultured secondary seed solution is inoculated in the fermention medium of sterilizing, fermenting process is added 2% glucose and nutritive substance (corn steep liquor 5.0%, (NH
4)
2sO
40.8%, KH
2pO
40.2%, K
2hPO
43H
2o0.3%), pass into sterile wind, adopt ammoniacal liquor (1:1 volume) to control pH value 7.1, through cultivation in about 68-72 hour, obtain the fermented liquid containing arginine product.
Shake-flask seed substratum, first order seed substratum, secondary seed medium and fermentative medium formula are: initial sugar concentration 10%, urea 5%, yeast extract paste 0.3%, corn steep liquor CSL 0.5%, (NH
4)
2sO
45%, MgSO
40.05%, KH
2pO
40.15%, K
2hPO
40.05%, the plain 50 μ g/L of micro-life.
Leavened prod extracts refining comprising the following steps: (1) fermentation liquor pretreatment; (2) flocculation, decolouring, concentrated; (3) secondary crystal; (4) finished product.
Be specially:
(1) fermentation liquor pretreatment utilizes ceramic membrane equipment to remove production bacterium and the high molecular weight protein in fermented liquid.Ceramic membrane aperture 100-800nm, operation pressure reduction 0.15-0.32MPa, crossflow velocity 3m/s, intends stabilized flux 80-125L/m
2.h.
Fermented liquid directly passes through ceramic membrane circulating filtration, and it is the cooling of circulation feed liquid that process adopts recirculated water, and cycle stock liquid temp is controlled at below 60 ℃, and concentrated solution adds reverse osmosis water to clean out residual product.
(2) flocculation, decolouring, pre-concentration by the cleaner liquid obtaining in step (1) with 1 times of volume of resin/hour flow velocity flow through acidulous cation resin D116(Shanghai Jing Kai resin company limited) adsorption production, with the 2.5N ammoniacal liquor of 2 times of resin volumes, with the flow velocity wash-out of 1 times of resin volume, obtain elutriant; Elutriant is the nano-filtration membrane equipment circulation decolouring of 1000 molecular weight by latus rectum, process is used circulating water cooling controlled circulation feed temperature lower than 55 ℃, with reverse osmosis water cleaning concentrate to product content lower than 0.2%, it is more than 90% obtaining printing opacity, the nanofiltration concentrated solution that content is 6%; Nanofiltration clear liquid is warming up to 70 ℃, under 80rpm whipped state, add 1% activated carbon decolorizing, be incubated 30 minutes, when clear liquid transmittance reaches, more than 95% by decarburizing machine, remove gac and obtain the clear liquid that decolours, decolouring clear liquid is through reverse-osmosis circulating, with circulating water cooling controlled circulation feed temperature, lower than 50 ℃, after reaching 15%, concentrated solution content obtains reverse osmosis concentrated liquid; Reverse osmosis concentrated liquid take 1 times of resin volume/hour flow velocity flow through through weak anion resin D374(Shanghai Huazhen Science and Technology Co., Ltd.) obtain resin anion(R.A) that content is 13% transmittance 98% exchange liquid.
(3) concentrated, crystallization sucks condensing crystal tank by anionresin liquid, at-0.08Mpa, feed temperature, be controlled at and under the condition that 50 ℃, mixing speed are 80rpm, anionresin liquid be concentrated into feed liquid content and reach 80%, reduce rotating speed to 50rpm, with-5 ℃ of frozen water, feed liquid is cooled to 5 ℃ of growing the grains 8 hours.
(4) it is with the full-automatic discharging whizzer of 2000rpm rotating speed, crystallization is separated with mother liquor that finished product is rested brilliant crystal solution, alcohol with 75% cleans crystallization and obtains wet product, wet product is dried at vacuum tightness-0.08Mpa through bipyramid, under temperature 70 C condition, dry and within 1 hour, obtain the dry product of water content below 0.5%, dry product is 20 ℃-25 ℃ of temperature at environment, humidity≤60%, packs to obtain finished product under aseptic condition.
This extraction process adopts ceramic membrane to replace traditional Plate Filtration, has improved filtrate quality, has reduced floor space, has improved product yield, has reduced labour intensity.
Adopt membrane separation technique to replace traditional ion-exchange isolation technique, reduced resin demand, improved feed liquid quality and seen through, reduced activated carbon dosage, improved quality product.
Adopt membrane concentration technology to replace traditional concentrating under reduced pressure technology, reduced the destruction to product, reduced energy consumption, improved quality product.
Finally, by preparation method of the present invention, finished product total recovery for fermented liquid can reach more than 83% (to be calculated with respect to the clean arginine content of removing thalline), and finished product first-time qualification rate reaches 99.9%, and traditional technology can only reach 50% total recovery, first-time qualification rate only has 97%.
Ceramic membrane has bacteria-eliminating efficacy well to arginine fermented liquid, and bacteria-eliminating efficacy is as shown in table 1:
Table 1
As can be seen from Table 2 originally, the new technology adopting through the present invention is for arginic extraction, and first product qualification rate and extract yield are higher than prior art level.
Table 2
Adopt above-mentioned processing means, finished product total recovery for fermented liquid can reach more than 85% (to be calculated with respect to the clean arginine content 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%.Pilot product is through the third party inspection of Accessories during Binzhou Administration of Quality and Technology Supervision, and indices meets State Standard of the People's Republic of China, food safety national standard, and foodstuff additive, L-arginine GB 28306-2012 standards, main performance index refers to table 3:
Table 3
。
Claims (8)
1. a high purity extractive propylhomoserin production technique, is characterized in that, comprises the preparation of fermented liquid and extracts purification step;
The preparation of fermented liquid comprises following step:
(1) slant culture: by brevibacterium flavum MQA121 access slant medium, cultivate for 30 ℃ and obtain slant strains for 20 hours;
(2) shaking flask bacterial classification: 30 ℃ of cultured slant strains access shake-flask seed substratum are cultivated and obtained shake-flask seed liquid for 22 hours;
(3) first order seed is cultivated: 30 ℃ of the first order seed substratum of cultured shake-flask seed liquid access sterilization are cultivated 18 hours to obtain to one-level kind liquid;
(4) secondary seed is cultivated: 30 ℃ of cultivations of secondary seed medium that cultured primary seed solution access was sterilized obtain secondary seed solution for 4-6 hour;
(5) cultured secondary seed solution is inoculated in the fermention medium of sterilizing, fermenting process is added 2% glucose by weight percentage and 5.0% corn steep liquor, 0.8% (NH
4)
2sO
4, 0.2%KH
2pO
4, 0.3%K
2hPO
43H
2o, passes into sterile wind, adopts ammoniacal liquor to control pH value 7.1, through cultivation in about 68-72 hour, obtains the fermented liquid containing arginine product.
2. a kind of high purity extractive propylhomoserin production technique according to claim 1, is characterized in that, slant medium contains by weight percentage: glucose 1.8%, (NH
4)
2sO
40.5%, KH
2pO
40.2%, K
2hPO
43H
2o0.2%, MgSO
47H
2o0.05%, MnSO
4h
2o0.002%, VITAMIN 50 g/L, agar powder 2.0%, pH6.9 ~ 7.3,121 ℃ of sterilizings 20 minutes.
3. a kind of high purity extractive propylhomoserin production technique according to claim 1, it is characterized in that, shake-flask seed substratum, first order seed substratum, secondary seed medium and fermention medium contain by weight percentage: initial sugar concentration 10%, urea 5%, yeast extract paste 0.3%, corn steep liquor CSL 0.5%, (NH
4)
2sO
45%, MgSO
40.05%, KH
2pO
40.15%, K
2hPO
40.05%, VITAMIN 50 μ g/L.
4. a kind of high purity extractive propylhomoserin production technique according to claim 1, is characterized in that, in step (5), ammoniacal liquor volumetric concentration is 1:1.
5. a kind of high purity extractive propylhomoserin production technique according to claim 1, is characterized in that, leavened prod extracts refining comprising the following steps: 1. fermentation liquor pretreatment; 2. flocculate, decolour, concentrated; 3. secondary crystal; 4. finished product.
6. a kind of high purity extractive propylhomoserin production technique according to claim 5, is characterized in that, leavened prod extracts refining being specially:
1. fermentation liquor pretreatment utilizes ceramic membrane equipment to remove production bacterium and the high molecular weight protein in fermented liquid, and fermented liquid directly passes through ceramic membrane circulating filtration, and it is the cooling of circulation feed liquid that process adopts recirculated water;
2. flocculate, decolour, pre-concentration by the cleaner liquid obtaining in step (1) with 1 times of volume of resin/hour flow velocity flow through acidulous cation resin adsorption production, with the 2.5N ammoniacal liquor of 2 times of resin volumes, with the flow velocity wash-out of 1 times of resin volume, obtain elutriant; Elutriant is the nano-filtration membrane equipment circulation decolouring of 1000 molecular weight by latus rectum, process is used circulating water cooling controlled circulation feed temperature lower than 55 ℃, with reverse osmosis water cleaning concentrate to product content lower than 0.2%, it is more than 90% obtaining printing opacity, the nanofiltration concentrated solution that content is 6%; Nanofiltration clear liquid is warming up to 70 ℃, under 80rpm whipped state, add 1% activated carbon decolorizing, be incubated 30 minutes, when clear liquid transmittance reaches, more than 95% by decarburizing machine, remove gac and obtain the clear liquid that decolours, decolouring clear liquid is through reverse-osmosis circulating, with circulating water cooling controlled circulation feed temperature, lower than 50 ℃, after reaching 15%, concentrated solution content obtains reverse osmosis concentrated liquid; Reverse osmosis concentrated liquid take 1 times of resin volume/hour flow velocity flow through and obtain resin anion(R.A) that content is 13% transmittance 98% exchange liquid through weak anion resin;
3. concentrated, crystallization sucks condensing crystal tank by anionresin liquid, anionresin liquid is concentrated into feed liquid content and reaches 80%, reduces rotating speed to 50rpm, with-5 ℃ of frozen water, feed liquid is cooled to 5 ℃ of growing the grains 8 hours;
4. finished product is rested brilliant crystal solution through centrifugation, and the alcohol with 75% cleans crystallization and obtains wet product, and wet product is dried and obtains dry product through bipyramid, packs to obtain finished product under aseptic condition.
7. a kind of high purity extractive propylhomoserin production technique according to claim 6, is characterized in that, step is middle ceramic membrane aperture 100-800nm 1., operation pressure reduction 0.15-0.32MPa, and crossflow velocity 3m/s, intends stabilized flux 80-125L/m
2.h.
8. a kind of high purity extractive propylhomoserin production technique according to claim 6, is characterized in that, step 3. in the condition of condensing crystal for-0.08Mpa, feed temperature are controlled at 50 ℃, mixing speed, be 80rpm.
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Cited By (4)
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| CN107232393A (en) * | 2017-07-31 | 2017-10-10 | 武汉轻工大学 | A kind of preparation method of cottonseed meal feed |
| CN107759495A (en) * | 2017-11-10 | 2018-03-06 | 山东丰银饲料科技有限公司 | One kind extraction arginic techniques of L |
| CN108409609A (en) * | 2018-01-31 | 2018-08-17 | 山东民强生物科技股份有限公司 | Arginine electrodialysis extraction process |
| CN112813115A (en) * | 2020-11-11 | 2021-05-18 | 新疆阜丰生物科技有限公司 | Production process of high-purity L-arginine |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107232393A (en) * | 2017-07-31 | 2017-10-10 | 武汉轻工大学 | A kind of preparation method of cottonseed meal feed |
| CN107759495A (en) * | 2017-11-10 | 2018-03-06 | 山东丰银饲料科技有限公司 | One kind extraction arginic techniques of L |
| CN108409609A (en) * | 2018-01-31 | 2018-08-17 | 山东民强生物科技股份有限公司 | Arginine electrodialysis extraction process |
| CN112813115A (en) * | 2020-11-11 | 2021-05-18 | 新疆阜丰生物科技有限公司 | Production process of high-purity L-arginine |
| CN112813115B (en) * | 2020-11-11 | 2023-10-03 | 新疆阜丰生物科技有限公司 | Production process of high-purity L-arginine |
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