CN1539984A - Technique for producing ISN stable isotope labeling L-glutamic acid - Google Patents
Technique for producing ISN stable isotope labeling L-glutamic acid Download PDFInfo
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Abstract
A stable isotope marker process for preparing L-glutamic acid from 15N includes choosing fermenting bacterium, plant storage, plant activating, culturing seeds, preparing culture medium for fermenting, fermenting, and separation extracting.
Description
Technical field
The present invention relates to microbial fermentation and biological extraction process field, relate in particular to the production technique of stable isotope tagged compound.
Background technology
15The production of N stable isotope label L-L-glutamic acid can adopt organic synthesis method, microbial fermentation and proteolysis to separate preparation method etc.The employing synthesis method is fairly simple, but needs optical resolution, makes
15The N raw material availability is lower, and cost rises.Proteolysis is separated preparation method and is usually used in preparing aminoacids complex, and it is very difficult to separate all amino acid.And adopt direct fermentation production, a large amount of enrichments of target amino acid, separates fairly simple, but because the very difficult mark of organic nitrogen source in the fermentating formula, usually make L-L-glutamic acid-
15The abundance of N descends a lot, does not reach product requirement.At present, exist
15Also lose patent and bibliographical information in the production field of N stable isotope label L-L-glutamic acid.
Summary of the invention
Purpose of the present invention is exactly to provide a kind of causing solving for the defective that overcomes above-mentioned prior art existence
15The problem that the N abundance descends, and improve as far as possible
15The N utilization ratio
15The production technique of N stable isotope label L-L-glutamic acid.
Purpose of the present invention can be achieved through the following technical solutions:
15The production technique of N stable isotope label L-L-glutamic acid is characterized in that, this technology may further comprise the steps:
(1) selection of fermentation strain: be used for one of Corynebacterium glutamicum (Corynebacterium glutamicum) that L-L-glutamic acid produces, brevibacterium flavum (Brevibacterium flavum), brevibacterium lactofermentum (Brevibacterium lactofermentum), Beijing rod bacillus (Corynebacteriumpekinense), Corynebacterium crenatum (Corynebacterium);
(2) preservation inclined-plane preparation: above-mentioned fermentation strain is inoculated in the inclined-plane, and 29～37 ℃ of constant temperature culture 8～20 hours obtain the preservation inclined-plane, refrigerate stand-by;
(3) activated inclined plane preparation: the preservation inclined-plane that step (2) is obtained is inoculated in activated inclined plane, the same step of cultural method (2);
(4) seed culture: the activated inclined plane lawn that step (3) is obtained is inoculated in sterilized the shaking in the bottle of seed culture medium of being equipped with, and cultivates 8～20 hours at 29～37 ℃ of following shaking tables;
(5) preparation of fermention medium:
With the full-synthetic culture medium is minimum medium, adds a small amount of organic nitrogen source; Main prescription is as shown in the table:
Explanation
Carbon source glucose 6～14g/dL or sucrose 12～25g/dL
Inorganic nitrogen-sourced urea 0.2～1g/dL, ammoniacal liquor, liquefied ammonia or ammonium chloride,
Ammonium sulfate, ammonium nitrate ammonium salts are by carbon-nitrogen ratio 100: 15～30
Organic nitrogen source corn steep liquor, yeast extract paste, casein hydrolyzate or thalline water
Separate liquid 0～1mL/dL, amino nitrogen is dense in the organic nitrogen source of use
Degree 5～20g/dL
MgSO
4.7H
2O 0.02～0.08g/dL
MnSO
4.H
2O 2～20mg/L
FeSO
4.7H
2O 2～20mg/L
VH 100～600μg/L
VB1 50～1000μg/L
(6) zymotechnique
Step (4) cultured seed 2～10% is inoculated in sterilized dress by volume
Begin fermentation in steps in fermentation shake flask of (5) described fermention medium or the fermentor tank, shaker fermentation control condition is: 30～33 ℃ of fermentation initial temperatures, initial pH6.8～7.2, shaking speed 180～240r/min; Have a net increase of 0.2～0.6 (measuring when fermented liquid dilutes 20 times) in the OD value temperature is elevated to 37～39 ℃, improve rotating speed to 260～350r/min simultaneously; At the polysorbate60 that logarithmic growth added about 0～0.3mL/dL in early stage 8～16 hours, pH is in slight alkalinity for the fermentation Whole Process Control, adds
15N labeled urea, ammoniacal liquor or liquefied ammonia are regulated pH7.0～7.6, and stream adds 50% glucose control fermented liquid glucose concn, 2～5g/dL, fermentation time 18～60 hours; Fermentor tank control condition is: 30～33 ℃ of fermentation initial temperatures, and initial pH6.8～7.2, air flow 0.5～1.5VVM, tank pressure 0.01～0.05Mpa, dissolved oxygen 0.5～90% saturation ratio is by polyethers or silicone defoamer froth breaking; Have a net increase of 0.2～0.6 (measuring when fermented liquid dilutes 40 times) in the OD value temperature is elevated to 37～39 ℃, improve rotating speed simultaneously and guarantee that dissolved oxygen is in 30～50% saturation ratios; At the polysorbate60 of logarithmic growth adding in early stage 5～16 hours 0～0.3mL/dL, pH is in slight alkalinity for the fermentation Whole Process Control, adds
15N labeled urea, ammoniacal liquor or liquefied ammonia are regulated pH7.0～7.6, and stream adds 50% glucose control fermented liquid glucose concn, 2～5g/dL, fermentation time 18～60 hours;
(7) separation and Extraction
With the cultured fermented liquid of step (6), pass through fermentation liquor pretreatment, promptly add oxalic acid and remove metal ion, add flocculation agent such as polyacrylamide, add heat extraction albumen, the supernatant liquor that the bactofugation body obtains adopts branches such as isoelectric precipitation, ion exchange method to extract and obtains
15N stable isotope label L-glutamic acid solution is caught up with ammonia and is adopted activated carbon decolorizing, ethanol low temperature crystallization, vacuum-drying to obtain product by vacuum concentration.
Corynebacterium glutamicum is meant Corynebacterium glutamicum (Corynebacteriumglutamicum) ATCC13032 in the described step (1), one of AS1.805 and their mutant strain, brevibacterium flavum is meant brevibacterium flavum (Brevibacterium flavum) ATCC14067, the TC2568 temperature sensitive mutant, one of TSH5459 temperature sensitive mutant and their mutant strain, brevibacterium lactofermentum is meant brevibacterium lactofermentum (Brevibacterium lactofermentum) ATCC13869, one of AJ11360 and their mutant strain, Beijing rod bacillus is meant Beijing rod bacillus (Corynebacteriumpekinense) AS1.299,7338, S9114, one of WTH-1 and their mutant strain, Corynebacterium crenatum is meant Corynebacterium crenatum (Corynebacterium) AS1.542, one of B9 and their mutant strain.
In described step (5) fermention medium, organic nitrogen sources such as corn steep liquor, thalline hydrolyzed solution add minute quantity 0～1mL/dL, and amino nitrogen concentration 5～20g/dL in the organic nitrogen source of use is so that it is right
15The influence of N abundance is reduced to bottom line; Promote growth by direct interpolation raised growth factor vitamin H and VB1, reduce of the influence of the shortage of organic nitrogen source thalli growth.
The Control of Dissolved Oxygen is by adjusting mixing speed, air flow, tank pressure realization in the described step (6).
The control of permeability of cell membrane, cell transition improves temperature by have a net increase of at 0.2～0.6 o'clock in OD value, adds tensio-active agent such as polysorbate60 and the raising dissolved oxygen is realized in the described step (6).
The control of pH adds by stream in the described step (6)
15N labeled urea, ammoniacal liquor or liquefied ammonia are realized.
Add glucose concn 2～5g/dL in the 50g/dL glucose solution control fermented liquid by stream in the described step (6).
In the described step (7)
15The extraction process of N stable isotope label L-L-glutamic acid adopts special-purpose mass spectrograph of isotopic analysis or spectroscopic analysis analysis
15The N abundance.
If adopt conventional fermentation process,
15Though N stable isotope label L-L-glutamic acid product is higher relatively,
15The N abundance descends very big, often descends more than 3%, is difficult to reach the high abundance product requirement.Though adopt the full-synthetic culture medium fermentation right
15The influence of N abundance is little, and the acid amount is too low to make but produce
15The N raw material availability is not high.By changing fermentating formula and technology, the present invention obtains
15N stable isotope label L-glutamic acid yield ratio adopts the corresponding raising of full-synthetic culture medium fermentation and acid amount more than 10 ~ 50%, and
15N stable isotope label L-L-glutamic acid
15The N abundance descends and can reduce to below 1%, have in addition descend hardly, improved greatly
15The N raw material availability has reduced production cost.Simultaneously, guaranteeing product
15Under the N abundance condition, simplify fermentation and extraction process, because raw material is fully used and has been saved the raw material cost recovery greatly.The present invention can utilize low abundance and high abundance
15The N inorganic raw material satisfies different abundance product requirements.
The present invention utilizes the production of microorganism direct fermentation, and control organic nitrogen source addition directly adds production factor, by physical condition control permeability of cell membrane, and adopts low-glucose addition process improving acid production rate.Can make like this
15The N raw material is utilized effectively, and abundance descends to being unlikely influences the products production requirement.The present invention is meticulousr than traditional L-extraction technology of glutamic acid, and adopts special-purpose mass spectrograph of isotropic substance or analysis of spectral method
15The N abundance, purity check adopts ordinary method.
Embodiment
Embodiment 1
Use bacterial classification for being the temperature sensitive mutant TC2568 that the bacterium mutagenic and breeding obtains that sets out with brevibacterium flavum, the substratum of use comprises slant preservation substratum, slant activation substratum, shake-flask seed and fermention medium.The slant preservation substratum adopts identical with the slant activation substratum with conventional fermentation.Seed that uses and fermentation initial formulation are as shown in the table.
Seed formula, fermenting initial formulation
Glucose 3.0g/dL 6.0g/dL
Urea (abundance 5.31%) 0.5g/dL 0.5G/dL
Corn steep liquor 0.2mL/dL 0.05mL/dL
MgSO
4·7H
2O 0.04g/dL 0.06g/dL
MnSO
4·H
2O 10mg/L 200mg/L
FeSO
4·7H
2O 10mg/L 200mg/L
VH 100μg/L 200μg/L
VB1 150μg/L 350μg/L
It is 50g/dL that stream adds with glucose concn, and it is 20g/dL that stream adds with urea concentration, all separates autoclaving, and urea also divides and disappears in above-mentioned seed and the fermentation initial formulation.
Brevibacterium flavum TC2568 is inoculated in (250mL triangular flask loading amount 20mL) the above-mentioned seed culture medium from the inclined-plane, 30 ℃, 220rpm are in cultivating about 10 hours on the patrolling shaking table.By 5% inoculum size above-mentioned seed liquor is inoculated in (500mL loading amount 20mL in the fermention medium, connect 4 bottles altogether), under 33 ℃, 220rpm condition, cultivated about 10 hours, when PH drops to 6.4 left and right sides, begin stream and add 20g/dL urea control PH in slight alkalinity in the top fermentation of patrolling shaking table.Regulating leavening temperature when the OD value has a net increase of 0.4 left and right sides is 37 ℃, rotating speed 280rpm, ends up to fermentation.The remaining sugar concentration that the fermentation whole process adds 50g/dL glucose control fermented liquid by stream is about 3g/dL, and fermentation culture finished in 42 hours, and fermentation is average produces sour 64g/L.
With centrifugation (3000rpm, 10 minutes) after above-mentioned fermented liquid heating half an hour, resulting supernatant liquor is regulated PH to L-L-glutamic acid iso-electric point with hydrochloric acid, and is put in refrigerator isoelectric point crystallization (spending the night).The crystal that filtration is obtained transfers PH2.0 to go up Zeo-karb (H again after with water dissolution
+Type) absorption, washing back is with 1N ammoniacal liquor wash-out, with the L-L-glutamic acid that obtains-
15The single spot elutriant of N concentration, recrystallize and filter the crystal vacuum-drying that obtains obtain L-L-glutamic acid-
15N solid 4.37 grams.And go up Zeo-karb (H behind the mother liquor accent PH2.0 with isoelectric point crystallization
+Type) absorption, washing back 0.02N NH
4The Cl wash-out, with the L-L-glutamic acid that obtains-
15Upper prop again after the single spot elutriant of N concentrates, washing is with NH
4Change 0.5N ammoniacal liquor wash-out after Cl washes off again, elutriant repeat again to obtain as stated above L-L-glutamic acid-
15N solid 1.22 grams.Obtain 5.59 gram products altogether, obtain product abundance 5.29% through mass spectroscopy, abundance descends 0.377%.
If change corn steep liquor in the seed culture medium into 2.0mL/dL, corn steep liquor changes 1.0mL/dL in the fermention medium, can obtain 7.24 gram products, but abundance has only 4.74%, and abundance descends 10.73％。If do not add corn steep liquor in seed and the fermention medium, can obtain 3.21 gram products, abundance 5.31%, abundance descends hardly.Take all factors into consideration, effect of the present invention is remarkable.
Embodiment 2
Use bacterial classification for brevibacterium flavum being the temperature sensitive mutant TSH5459 that the bacterium mutagenic and breeding obtains that sets out.
15The N raw material changes 98.32% abundance urea into, and the substratum of use comprises slant preservation substratum, slant activation substratum, shake-flask seed and fermention medium.The slant preservation substratum adopts identical with the slant activation substratum with conventional fermentation.Seed that uses and fermentation initial formulation are as shown in the table.
Seed formula, fermenting initial formulation
Glucose 3.0g/dL 8.0g/dL
Urea (abundance 5.31%) 0.55g/dL 0.55g/dL
Corn steep liquor 0.6mL/dL 0.1mL/dL
MgSO
4·7H
2O 0.04g/dL 0.06g/dL
MnSO
4·H
2O 2mg/L 20mg/L
FeSO
4·7H
2O 2mg/L 20mg/L
VH 100μg/L 300μg/L
VB1 150μg/L 450μg/L
It is 50g/dL that stream adds with glucose concn, and it is 22g/dL that stream adds with urea concentration, all separates autoclaving, and urea also divides and disappears in above-mentioned seed and the fermentation initial formulation.
Brevibacterium flavum TSH5459 is inoculated in (250mL triangular flask loading amount 20mL) the above-mentioned seed culture medium from the inclined-plane, 31 ℃, 220rpm are in cultivating about 12 hours on the patrolling shaking table.By 5% inoculum size above-mentioned seed liquor is inoculated in (500mL loading amount 20mL in the fermention medium, connect 6 bottles altogether), cultivated about 10 hours in the top fermentation of patrolling shaking table under 33 ℃, 220rpm condition, begin stream and add 22g/dL urea when PH drops to 6.4 left and right sides, PH is in slight alkalinity in control.Regulating leavening temperature and be 37 ℃, rotating speed 280rpm when the OD value has a net increase of 0.5 left and right sides stops until fermentation.The fermentation whole process is 2～3g/dL by the remaining sugar concentration that stream adds 50g/dL glucose control fermented liquid, fermentation culture 40～46 hours.The sour 62g/L of the average product of fermentation.
The separating and extracting method that adopts obtains product 6.0 grams at last altogether with embodiment 1, obtains product abundance 97.73% through mass spectroscopy, product purity 99.44%, and abundance descends 0.6%, satisfies the products production requirement fully.
Claims (8)
1.
15The production technique of N stable isotope label L-L-glutamic acid is characterized in that, this technology may further comprise the steps:
(1) selection of fermentation strain: be used for one of Corynebacterium glutamicum (Corynebacterium glutamicum) that L-L-glutamic acid produces, brevibacterium flavum (Brevibacterium flavum), brevibacterium lactofermentum (Brevibacterium lactofermentum), Beijing rod bacillus (Corynebacteriumpekinense), Corynebacterium crenatum (Corynebacterium);
(2) preservation inclined-plane preparation: above-mentioned fermentation strain is inoculated in the inclined-plane, and 29～37 ℃ of constant temperature culture 8～20 hours obtain the preservation inclined-plane, refrigerate stand-by;
(3) activated inclined plane preparation: the preservation inclined-plane that step (2) is obtained is inoculated in activated inclined plane, the same step of cultural method (2);
(4) seed culture: the activated inclined plane lawn that step (3) is obtained is inoculated in sterilized the shaking in the bottle of seed culture medium of being equipped with, and cultivates 8～20 hours at 29～37 ℃ of following shaking tables;
(5) preparation of fermention medium:
With the full-synthetic culture medium is minimum medium, adds a small amount of organic nitrogen source; Main prescription is as shown in the table:
Explanation
Carbon source glucose 6～14g/dL or sucrose 12～25g/dL
Inorganic nitrogen-sourced urea 0.2～1g/dL, ammoniacal liquor, liquefied ammonia or ammonium chloride,
Ammonium sulfate, ammonium nitrate ammonium salts are by carbon-nitrogen ratio 100: 15～30
Organic nitrogen source corn steep liquor, yeast extract paste, casein hydrolyzate or thalline water
Separate liquid 0～1mL/dL, amino nitrogen is dense in the organic nitrogen source of use
Degree 5～20g/dL
MgSO
4.7H
2O 0.02～0.08g/dL
MnSO
4.H
2O 2～20mg/L
FeSO
4.7H
2O 2～20mg/L
VH 100～600μg/L
VB1 50～1000μg/L
(6) zymotechnique
Step (4) cultured seed 2～10% is inoculated in sterilized fermentation shake flask that the described fermention medium of step (5) is housed or the fermentor tank and begins fermentation by volume, shaker fermentation control condition is: 30～33 ℃ of fermentation initial temperatures, initial pH6.8～7.2, shaking speed 180～240r/min; When the OD value has a net increase of 0.2～0.6 (20 times of mensuration of fermented liquid dilution), temperature is elevated to 37～39 ℃, improves rotating speed to 260～350r/min simultaneously; At the polysorbate60 that logarithmic growth added about 0～0.3mL/dL in early stage 8～16 hours, pH is in slight alkalinity for the fermentation Whole Process Control, adds
15N labeled urea, ammoniacal liquor or liquefied ammonia are regulated pH7.0～7.6, and stream adds 50% glucose control fermented liquid glucose concn, 2～5g/dL, fermentation time 18～60 hours; Fermentor tank control condition is: 30～33 ℃ of fermentation initial temperatures, and initial pH6.8～7.2, air flow 0.5～1.5VVM, tank pressure 0.01～0.05Mpa, dissolved oxygen 0.5～90% saturation ratio is by polyethers or silicone defoamer froth breaking; When the OD value has a net increase of 0.2～0.6 (40 times of mensuration of fermented liquid dilution), temperature is elevated to 37～39 ℃, improves rotating speed simultaneously and guarantee that dissolved oxygen is in 30～50% saturation ratios; At the polysorbate60 of logarithmic growth adding in early stage 5～16 hours 0～0.3mL/dL, pH is in slight alkalinity for the fermentation Whole Process Control, adds
15N labeled urea, ammoniacal liquor or liquefied ammonia are regulated pH7.0～7.6, and stream adds 50% glucose control fermented liquid glucose concn, 2～5g/dL, fermentation time 18～60 hours;
(7) separation and Extraction
With the cultured fermented liquid of step (6), pass through fermentation liquor pretreatment, promptly add oxalic acid and remove metal ion, add flocculation agent such as polyacrylamide, add heat extraction albumen, the supernatant liquor that the bactofugation body obtains adopts branches such as isoelectric precipitation, ion exchange method to extract and obtains
15N stable isotope label L-glutamic acid solution is caught up with ammonia and is adopted activated carbon decolorizing, ethanol low temperature crystallization, vacuum-drying to obtain product by vacuum concentration.
2. according to claim 1
15The production technique of N stable isotope label L-L-glutamic acid, it is characterized in that, Corynebacterium glutamicum is meant Corynebacterium glutamicum (Corynebacteriumglutamicum) ATCC13032 in the described step (1), one of AS1.805 and their mutant strain, brevibacterium flavum is meant brevibacterium flavum (Brevibacterium flavum) ATCC14067, the TC2568 temperature sensitive mutant, one of TSH5459 temperature sensitive mutant and their mutant strain, brevibacterium lactofermentum is meant brevibacterium lactofermentum (Brevibacterium lactofermentum) ATCC13869, one of AJ11360 and their mutant strain, Beijing rod bacillus is meant Beijing rod bacillus (Corynebacteriumpekinense) AS1.299,7338, S9 114, one of WTH-1 and their mutant strain, Corynebacterium crenatum is meant Corynebacterium crenatum (Corynebacterium) AS1.542, one of B9 and their mutant strain.
3. according to claim 1
15The production technique of N stable isotope label L-L-glutamic acid, it is characterized in that in described step (5) fermention medium, organic nitrogen sources such as corn steep liquor, thalline hydrolyzed solution add minute quantity 0～1mL/dL, amino nitrogen concentration 5～20g/dL in the organic nitrogen source that uses is so that it is right
15The influence of N abundance is reduced to bottom line; Promote growth by direct interpolation raised growth factor vitamin H and VB1, reduce of the influence of the shortage of organic nitrogen source thalli growth.
4. according to claim 1
15The production technique of N stable isotope label L-L-glutamic acid is characterized in that, The Control of Dissolved Oxygen is by adjusting mixing speed, air flow, tank pressure realization in the described step (6).
5. according to claim 1
15The production technique of N stable isotope label L-L-glutamic acid, it is characterized in that the control of permeability of cell membrane, cell transition improves temperature by have a net increase of at 0.2～0.6 o'clock in OD value, adds tensio-active agent such as polysorbate60 and the raising dissolved oxygen is realized in the described step (6).
6. according to claim 1
15The production technique of N stable isotope label L-L-glutamic acid is characterized in that, the control of pH adds by stream in the described step (6)
15N labeled urea, ammoniacal liquor or liquefied ammonia are realized.
7. according to claim 1
15The production technique of N stable isotope label L-L-glutamic acid is characterized in that, adds glucose concn 2～5g/dL in the 50g/dL glucose solution control fermented liquid by stream in the described step (6).
8. according to claim 1
15The production technique of N stable isotope label L-L-glutamic acid is characterized in that, in the described step (7)
15The extraction process of N stable isotope label L-L-glutamic acid adopts special-purpose mass spectrograph of isotopic analysis or spectroscopic analysis analysis
15The N abundance.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101811992A (en) * | 2010-04-02 | 2010-08-25 | 上海化工研究院 | Preparation method for semicarbazide hydrochloride labeled by stable isotope 13C and 15N |
| CN101824444A (en) * | 2010-04-09 | 2010-09-08 | 上海化工研究院 | Method for preparing <13>C-labelled glucose |
| CN102399732A (en) * | 2011-12-13 | 2012-04-04 | 梅花生物科技集团股份有限公司 | Beijing corynebacterium and application thereof |
| CN101824445B (en) * | 2009-03-06 | 2013-01-23 | 中国科学院生态环境研究中心 | Method for preparing 13C stable isotope labeled cellulose |
| CN105296464A (en) * | 2015-11-25 | 2016-02-03 | 曾志刚 | Method for screening 25hydroxyvitamin D3 high-yielding strain and screening polysorbate-80 content of fermentation medium |
| CN107099563A (en) * | 2017-06-02 | 2017-08-29 | 卢松 | It is a kind of the method that power technology prepares monosodium glutamate such as to utilize |
| CN112501222A (en) * | 2020-12-15 | 2021-03-16 | 梁山菱花生物科技有限公司 | Fermentation method of glutamic acid |
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2003
- 2003-10-24 CN CN 200310108164 patent/CN1260365C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101824445B (en) * | 2009-03-06 | 2013-01-23 | 中国科学院生态环境研究中心 | Method for preparing 13C stable isotope labeled cellulose |
| CN101811992A (en) * | 2010-04-02 | 2010-08-25 | 上海化工研究院 | Preparation method for semicarbazide hydrochloride labeled by stable isotope 13C and 15N |
| CN101811992B (en) * | 2010-04-02 | 2012-11-14 | 上海化工研究院 | Preparation method for semicarbazide hydrochloride labeled by stable isotope 13C and 15N |
| CN101824444A (en) * | 2010-04-09 | 2010-09-08 | 上海化工研究院 | Method for preparing <13>C-labelled glucose |
| CN101824444B (en) * | 2010-04-09 | 2012-07-18 | 上海化工研究院 | Method for preparing <13>C-labelled glucose |
| CN102399732A (en) * | 2011-12-13 | 2012-04-04 | 梅花生物科技集团股份有限公司 | Beijing corynebacterium and application thereof |
| CN105296464A (en) * | 2015-11-25 | 2016-02-03 | 曾志刚 | Method for screening 25hydroxyvitamin D3 high-yielding strain and screening polysorbate-80 content of fermentation medium |
| CN107099563A (en) * | 2017-06-02 | 2017-08-29 | 卢松 | It is a kind of the method that power technology prepares monosodium glutamate such as to utilize |
| CN112501222A (en) * | 2020-12-15 | 2021-03-16 | 梁山菱花生物科技有限公司 | Fermentation method of glutamic acid |
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| CN1260365C (en) | 2006-06-21 |
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