CN102051391B - Fermentation method for producing glutamine - Google Patents
Fermentation method for producing glutamine Download PDFInfo
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- CN102051391B CN102051391B CN 200910153202 CN200910153202A CN102051391B CN 102051391 B CN102051391 B CN 102051391B CN 200910153202 CN200910153202 CN 200910153202 CN 200910153202 A CN200910153202 A CN 200910153202A CN 102051391 B CN102051391 B CN 102051391B
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Abstract
The invention relates to a fermentation method for producing glutamine, and belongs to the technical field of fermentation. The glutamine can be produced by the fermentation method instead of a chemical synthesis method, so the production cost is low, the efficiency is high, and the pollution on environment can be relieved effectively. The fermentation method is a model of conversion of laboratory study into industrial production, the yield of the glutamine is almost the same as that of the glutamine in laboratories, namely the yield of the glutamine in the laboratories is 46.6g/L, and the yield of the glutamine obtained by the industrial production is 45g/L; and the fermentation method reaches a domestically advanced level.
Description
Technical field
The present invention relates to a kind of fermentation process of producing glutamine, belong to fermentation technical field.
Background technology
Glutamine is a kind of neutral multifunctional amino acid, becomes gradually the study hotspot of the subjects such as trophology, physiology, immunology because of its unique and complex physiological function.Glutamine has the body of keeping acid base equilibrium, regulate many physiological actions such as body's immunity, be the active drug for the treatment of intestinal ulcer, chronic gastritis, it is not only supplied raw materials for the metabolism of body matter and energy, and provides nitrogen for the biosynthesizing of nucleic acid, glycoprotein etc.Production about glutamine, because its special physico-chemical property, the method production difficulty synthetic with chemical industry is very large, and comparatively desirable method is that the precursor-L-glutamic acid by glutamine is raw material at present, the synthetic glutamine take the Raney alumel as the catalyzer chemical industry; But still there are the many problems of reactions steps, and need separating for several times, yield low (20%～40%), unstable; The conditional request such as PH, temperature is harsh, and the glutamine that is prone to multiple strong side reaction-generation again decomposes or forms Pyrrolidonecarboxylic acid.In addition, the separation of end product remains in larger technical difficulty, and cost is higher, also has the problems such as environmental pollution.Some domestic research institutions begin that in the nineties fermentative Production glutamine has been carried out some to be explored, and has obtained the higher bacterial strain of some output, all also is laboratory stage but produce glutamine.Glutamine all is import at present, and the yield of domestic production is very low.
Summary of the invention
To the objective of the invention is in order addressing the above problem, a kind of fermentation process of producing glutamine to be provided.
Above-mentioned technical purpose of the present invention is achieved by the following technical programs: produce the fermentation process of glutamine, in turn include the following steps:
(1) chooses weight ratio and be respectively 15%, 1.5%, 0.01%, 0.05%, 10%, 5% glucose, corn oar, potassium primary phosphate, zinc sulfate, ammonium sulfate, CaCO
3, rest part is water, then above-mentioned substance is stirred and make solution;
(2) after mentioned solution is made, add again manganous sulfate 120mg/L, copper sulfate 10mg/L, ferric sulfate 1mg/L, VITMAIN B1 2mg/L, vitamin H H 2mg/L, again stir, make the substrate of stable homogeneous;
(3) in the substrate that step (2) makes, inoculate brevibacterium flavum, and the control temperature is at 30-32 ℃;
(4) after PH being controlled at 7.0～7.2,24 hours in the 1st～24 hour, PH is controlled at 6.5, and fermentation 44-48 hour.
Brevibacterium flavum can have been bought on market, and its morphological characteristic, cultural characters and physiological property are as follows: quarter butt is to bar-shaped, and atrichia does not move, and does not form gemma, and colony edge is neat, and the surface is glossy, moistening, smooth, bacterium colony is translucent; Suitable culture temperature: 30 ℃, suitable storage temperature :-70 ℃.
During being to produce, the difficult point of fermentative Production glutamine do not know which kind should need to place what batching, particularly inorganic salt in fermented liquid need to put, and respectively puts the control of how many amounts and condition.The inventor has drawn above-mentioned prescription through long test and summary, and the output of glutamine can be risen to 45g/L by strict condition control, reaches advanced international level.
As preferably, after the fermentation time spent arrives 44 hours, when being separated, replenishes the glutamine that generates in the substrate glucose, corn oar, potassium primary phosphate, zinc sulfate, ammonium sulfate, CaCO
3, manganous sulfate, copper sulfate, ferric sulfate, VITMAIN B1, vitamin H H, to keep the stable of each substances content in the fermented liquid.
Scheme after preferred can realize continuous production, one of its difficulty is how glutamine is in time separated, and transforms to L-glutamic acid to avoid glutamine, and the inventor is through long-term practice, 44～48 minute separation glutamine of discovery after fermentation can obtain preferably yield; Two of difficulty is how to guarantee that brevibacterium flavum maintains stationary phase, prevents that it from changing to decline phase.The inventor finds after fermentation is carried out 24 hours PH control to be turned down slightly through long-term practice, and namely being controlled at 6.5 o'clock can effectively prolong stationary phase.
In sum, the present invention has following beneficial effect:
1, the alternative chemical industry synthetic method of the inventive method is produced glutamine, and so not only production cost is low, and efficient is high, and can effectively alleviate the pollution to environment;
2, the inventive method is that laboratory study is converted into industrial typical case, and the yield in its glutamine yield and laboratory is more or less the same, and the laboratory is 46.6g/L, and industrial production is 45g/L; Reach advanced international level.
Description of drawings
Fig. 1 is glutamine ferment conditional curve figure.
Embodiment
Below in conjunction with accompanying drawing the present invention is described in further detail.
This specific embodiment only is explanation of the invention; it is not limitation of the present invention; those skilled in the art can make the modification that does not have creative contribution to present embodiment as required after reading this specification sheets, but as long as all are subject to the protection of patent law in claim scope of the present invention.
Produce the fermentation process of glutamine, in turn include the following steps:
(1) chooses weight ratio and be respectively 15%, 1.5%, 0.01%, 0.05%, 10%, 5% glucose, corn oar, potassium primary phosphate, zinc sulfate, ammonium sulfate, CaCO
3, rest part is water, then above-mentioned substance is stirred and make solution;
(2) after mentioned solution is made, add again manganous sulfate 120mg/L, copper sulfate 10mg/L, ferric sulfate 1mg/L, VITMAIN B1 2mg/L, vitamin H H 2mg/L, again stir, make the substrate of stable homogeneous;
(3) in the substrate that step (2) makes, inoculate brevibacterium flavum, and the control temperature is at 30-32 ℃;
(4) after PH being controlled at 7.0～7.2,24 hours in the 1st～24 hour, PH is controlled at 6.5, and fermentation 44-48 hour.
After the fermentation time spent arrives 44 hours, when being separated, replenishes the glutamine that generates in the substrate glucose, corn oar, potassium primary phosphate, zinc sulfate, ammonium sulfate, CaCO
3, manganous sulfate, copper sulfate, ferric sulfate, VITMAIN B1, vitamin H H, to keep the stable of each substances content in the fermented liquid.
The pre-research of producing of glutamine industry
1 materials and methods
1.1 bacterial classification
Brevibacterium flavum (Brevibacterium flavum)
1.2 reagent and instrument
Corn steep liquor, glucose, other are domestic reagent (chemical pure)
1.2.1 instrument
721 type spectrophotometers, pH meter,
Fermentor tank: 1. the SF2116 of NBS company type automatic control tank is furnished with pH electrode and dissolved oxygen electrode;
2. back and forth without joint speed governing shaking table.
1.2.2 substratum
Fermention medium: glucose 15%, corn oar 1.5%, potassium primary phosphate 0.01%, zinc sulfate 0.05%, ammonium sulfate 10%, copper sulfate 10mg/L, manganous sulfate 120mg/L, CaCO3 5%, ferric sulfate 1mg/L; VITMAIN B1 2mg/L, vitamin H H 2mg/L; PH 6.5.
1.2.3 culture condition
The canned liquid measure 8L of 16L automatic control, 118 ℃ of sterilization 5min in kind amount 3%～5% access fermentor cultivation liquid, need control pH, air quantity, rotating speed, dissolved oxygen level by test after the cooling.Regularly sampling is rule at culture medium flat plate in the fermenting process, checks to have or not microbiological contamination, and measures pH, and glutamine output.
1.2.4 analytical procedure
1.2.4.1 glutamine is measured
Paper Chromatography: fermented liquid is through the centrifugal solid matter such as thalline of removing, and quantitative point sample on chromatographic paper is with the contrast of glutamine reference liquid.Make developping agent with propyl carbinol, Glacial acetic acid, water (4: 1: 1).Launch completely, dry up filter paper, develop the color with 0.5% triketohydrindene hydrate acetone soln.Elutriant is 0.1%CuSO
45H
2O: 75% ethanol (2: 38), survey the O.D value at 510nm behind the wash-out 30min, and compare glutamine content in the calculation sample with the standard glutamine.
1.2.4.2 the mensuration of fermented liquid residual sugar
Adopt the fixed sugared method of Fehling
1.2.4.3pH the mensuration of value
With acidometer or accurate pH test paper, fermenting process is measured with pH electrode.
2 experimental results and discussion
(1) pH is on the impact of glutamine ferment
Because thalline to the utilization of medium nutrient content and the formation of meta-bolites, constantly changes the pH of nutrient solution.(NH4) 2SO 4 in the substratum is utilized by thalline and sugar is utilized mesostates such as generating organic acid, and the pH value is descended; The formation of glutamine consumes a large amount of ammonia the pH value is descended.Therefore, need constantly to replenish nitrogenous source (ammonia) and regulate the pH value, until fermentation ends.The brevibacterium flavum that this research is adopted is under neutral and slight alkalinity condition, and fermentation produces accumulation L-glutamic acid; (pH 5.0～5.8) produce glutamine (seeing the following form) under acidic conditions.
Produce amine phase PH to the impact of glutamine
(2) dissolved oxygen is on the impact of glutamine
In glutamine ferment, we have observed variation and the impact of dissolved oxygen.Need a large amount of ATP by the synthetic glutamine of glucose biological, energy metabolism is very sensitive to anoxic.Dissolved oxygen is crossed when hanging down, and production capacity is inadequate, and metabolism turns to pyruvic acid biosynthesizing lactic acid, produces amine and is suppressed.When dissolved oxygen was too high, glucose then entered the complete oxidation system and carries out metabolism, was unfavorable for the further reductive amination of α-ketoglutaric acid, had weakened synthetic (the seeing the following form) of glutamine.
16L tank dissolved oxygen is on producing the impact of amine speed
(3) 16L automatic control tank fermentation control process and the results are shown in following table and Fig. 1
Continuous three batch fermentation results
Fig. 1 has shown, produces later on amine at 40 hours and maintains higher level.Show that this bacterial strain is the strain excellent of fermentative Production glutamine, have industrialized developing and be worth.
Claims (2)
1. produce the fermentation process of glutamine, in turn include the following steps:
(1) chooses weight ratio and be respectively 15%, 1.5%, 0.01%, 0.05%, 10%, 5% glucose, corn steep liquor, potassium primary phosphate, zinc sulfate, ammonium sulfate, CaCO
3, rest part is water, then above-mentioned substance is stirred and make solution;
(2) after mentioned solution is made, add again manganous sulfate 120mg/L, copper sulfate 10mg/L, ferric sulfate lmg/L, VITMAIN B1 2mg/L, vitamin H H 2mg/L, again stir, make the substrate of stable homogeneous;
(3) in the substrate that step (2) makes, inoculate brevibacterium flavum, and the control temperature is at 30～32 ℃;
(4) after pH being controlled at 7.0～7.2,24 hours in the 1st～24 hour, pH is controlled at 6.5, and fermented 44～48 hours.
2. the fermentation process of production glutamine according to claim 1, it is characterized in that: after the fermentation time spent arrives 44 hours, replenish glucose, corn steep liquor, potassium primary phosphate, zinc sulfate, ammonium sulfate, CaCO when the glutamine that generates in the substrate is separated
3, manganous sulfate, copper sulfate, ferric sulfate, the rope Bl that supports one's family, vitamin H H, to keep the stable of each substances content in the fermented liquid;
The morphological characteristic of described brevibacterium flavum, cultural characters and physiological property are as follows: quarter butt is to bar-shaped, and atrichia does not move, and does not form gemma, and colony edge is neat, and the surface is glossy, moistening, smooth, bacterium colony is translucent; Suitable culture temperature: 30 ℃, suitable storage temperature :-70 ℃.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103695492B (en) * | 2013-12-24 | 2016-04-06 | 山东民强生物科技股份有限公司 | A kind of method improving L-glutamine yield |
| CN103667382B (en) * | 2013-12-24 | 2015-09-02 | 山东民强生物科技股份有限公司 | A kind of fermentable produces the method for L-glutaminate |
| CN103695491B (en) * | 2013-12-24 | 2016-08-24 | 山东民强生物科技股份有限公司 | The process for purification of L-glutaminate |
| 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 |
| CN109371072A (en) * | 2018-10-18 | 2019-02-22 | 许传高 | A kind of technique for reducing glutamic acid fermentation microbiological contamination and improving fermentation efficiency |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1537945A (en) * | 2003-04-16 | 2004-10-20 | 江西诚志生物工程有限公司 | Fermentation method for preparing L-glutamine with high producing |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1537945A (en) * | 2003-04-16 | 2004-10-20 | 江西诚志生物工程有限公司 | Fermentation method for preparing L-glutamine with high producing |
Non-Patent Citations (2)
| Title |
|---|
| 岳峡等.黄色短杆菌ATCC 14067生物合成L-谷氨酰胺.《江西科学》.1988,第6卷(第2期),8-12. * |
| 王妙虎等.pH值对谷氨酰胺发酵的影响.《微生物学杂志》.1990,第10卷(第3期),52-54. * |
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