CN118345128A - Method for improving fermentation yield of L-amino acid - Google Patents
Method for improving fermentation yield of L-amino acid Download PDFInfo
- Publication number
- CN118345128A CN118345128A CN202310071484.8A CN202310071484A CN118345128A CN 118345128 A CN118345128 A CN 118345128A CN 202310071484 A CN202310071484 A CN 202310071484A CN 118345128 A CN118345128 A CN 118345128A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- corynebacterium
- amino acid
- sodium benzoate
- medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000855 fermentation Methods 0.000 title claims abstract description 86
- 230000004151 fermentation Effects 0.000 title claims abstract description 85
- 238000000034 method Methods 0.000 title claims abstract description 26
- 150000008575 L-amino acids Chemical class 0.000 title claims abstract description 21
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims abstract description 49
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 claims abstract description 30
- 235000010234 sodium benzoate Nutrition 0.000 claims abstract description 30
- 239000004299 sodium benzoate Substances 0.000 claims abstract description 30
- 239000004473 Threonine Substances 0.000 claims abstract description 24
- 229960002898 threonine Drugs 0.000 claims abstract description 24
- 239000002609 medium Substances 0.000 claims description 25
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 20
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 17
- 239000008103 glucose Substances 0.000 claims description 17
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 13
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 13
- 229960003237 betaine Drugs 0.000 claims description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 12
- 229910052799 carbon Inorganic materials 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 241000588724 Escherichia coli Species 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 11
- 235000019764 Soybean Meal Nutrition 0.000 claims description 9
- 240000008042 Zea mays Species 0.000 claims description 9
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 9
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 235000005822 corn Nutrition 0.000 claims description 9
- 238000011218 seed culture Methods 0.000 claims description 9
- 239000004455 soybean meal Substances 0.000 claims description 9
- 239000007836 KH2PO4 Substances 0.000 claims description 8
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 8
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 8
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 8
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 8
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 8
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 8
- 239000001301 oxygen Substances 0.000 claims description 8
- 229910052760 oxygen Inorganic materials 0.000 claims description 8
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 8
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 7
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 7
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 239000000413 hydrolysate Substances 0.000 claims description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 5
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 5
- 229910052564 epsomite Inorganic materials 0.000 claims description 5
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 5
- 239000011686 zinc sulphate Substances 0.000 claims description 5
- 241000186216 Corynebacterium Species 0.000 claims description 4
- 241000186145 Corynebacterium ammoniagenes Species 0.000 claims description 4
- 241000424760 Corynebacterium crenatum Species 0.000 claims description 4
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 4
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 4
- 229960002989 glutamic acid Drugs 0.000 claims description 4
- 229960000310 isoleucine Drugs 0.000 claims description 4
- 235000019766 L-Lysine Nutrition 0.000 claims description 3
- 229930182844 L-isoleucine Natural products 0.000 claims description 3
- 239000004472 Lysine Substances 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 229960003646 lysine Drugs 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 3
- 241001644925 Corynebacterium efficiens Species 0.000 claims description 2
- 241000337023 Corynebacterium thermoaminogenes Species 0.000 claims description 2
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 241000894007 species Species 0.000 claims description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 abstract description 15
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 abstract description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 10
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 5
- 239000001384 succinic acid Substances 0.000 abstract description 5
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 239000006227 byproduct Substances 0.000 abstract description 2
- 210000000170 cell membrane Anatomy 0.000 abstract description 2
- 239000007795 chemical reaction product Substances 0.000 abstract description 2
- 238000005265 energy consumption Methods 0.000 abstract description 2
- 230000035699 permeability Effects 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 239000000047 product Substances 0.000 abstract description 2
- 230000037358 bacterial metabolism Effects 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 description 12
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 8
- 235000011187 glycerol Nutrition 0.000 description 5
- 229940107700 pyruvic acid Drugs 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 108010078791 Carrier Proteins Proteins 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 125000005842 heteroatom Chemical group 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 101150117659 rhtA gene Proteins 0.000 description 2
- 101150033014 rhtB gene Proteins 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229960002449 glycine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 101150094644 rhtC gene Proteins 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a method for improving the fermentation yield of L-amino acid. In the process of producing L-amino acid by microbial fermentation, bacterial metabolism is changed by adding a certain amount of sodium benzoate into a fermentation medium, byproducts (such as citric acid and succinic acid) are reduced, and more nitrogen sources are converted into end products; changing permeability of cell membrane, reducing energy consumption, and increasing yield of main product (L-amino acid such as L-threonine).
Description
Technical Field
The invention relates to the field of fermentation engineering, in particular to a method for improving the fermentation yield of L-amino acid.
Background
At present, the L-amino acid is produced by adopting a microbial fermentation method, which mainly takes corn starch hydrolysis sugar or molasses as a carbon source and takes corn steep liquor, soybean meal hydrolysis liquid, yeast extract, peptone, ammonium salt, nitrate, urea, ammonia water, liquid ammonia and the like as nitrogen sources. The process optimization comprises dissolved oxygen, control of carbon source and nitrogen source, betaine dosage and other vitamins, etc.
At present, the transport proteins related to L-threonine in escherichia coli mainly comprise TdcC, rhtA, rhtB, rhtC and YecC, and besides TdcC, the transport proteins are export proteins. Among the export proteins, rhtA, rhtB and RhtC are currently most widely studied and used, wherein the specificity of RhtC is the highest for transporting threonine and energy is required.
Bacterial growth and production compete for the carbon source, and bacterial growth in large quantities consumes the carbon source for production, resulting in a decrease in the carbon source flow to the amino acids. In addition, during the fermentation process, along with the production of L-threonine, there is also a certain yield of hetero acids, such as glutamic acid, glycine, isoleucine and the like, and other organic acids, such as non-metabolized pyruvic acid, succinic acid and the like, wherein the related respiratory metabolism of the hetero acids is not easy to control.
Sodium benzoate is generally used as an additive in foods to play a role in preserving and preserving, and is also used for preparing a bacterial suspension so as to be convenient for long-term storage and transportation. Sodium benzoate has not been reported for use in increasing L-amino acid fermentation levels.
Disclosure of Invention
The invention aims to provide a method for improving the fermentation yield of L-amino acid.
To achieve the object of the present invention, in a first aspect, the present invention provides the use of sodium benzoate for increasing the fermentation yield of L-amino acids, preferably L-threonine, L-glutamic acid, L-lysine or L-isoleucine and the like.
In the present invention, the species used for fermentation include, but are not limited to, escherichia coli (ESCHERICHIA COLI), corynebacterium glutamicum (Corynebacterium glutamicum), corynebacterium validum (Corynebacterium efficiens), corynebacterium crenatum (Corynebacterium crenatum), corynebacterium ammoniagenes thermophilum (Corynebacterium thermoaminogenes) or Corynebacterium ammoniagenes (Corynebacterium aminogenes), preferably Escherichia coli MHZ-0216-5. Strain MHZ-0216-5 can be found in CN113846132A.
In a second aspect, the present invention provides a method for improving the fermentation yield of L-amino acids, wherein Escherichia coli is used as a fermentation strain, and a proper amount of sodium benzoate is added to a fermentation medium, so that the fermentation yield of L-amino acids is improved.
The method comprises the following steps: at the beginning of fermentation, 0.05-0.5g/L sodium benzoate is added to the fermentation medium.
The fermentation medium is as follows: corn steep liquor 5g/L, soybean meal hydrolysate 5g/L, glucose 40g/L,MgSO4·7H2O0.5g/L,KH2PO4 2g/L,FeSO4 20mg/L,MnSO4 20mg/L,ZnSO4 0.05mg/L,(NH4)2SO45g/L and betaine 1g/L.
The fermentation culture method comprises the following steps: inoculating the seed solution into a 50L fermentation tank containing 20L fermentation medium, introducing air at an air flow rate of 0.8vvm and a rotation speed of 300-600rpm, controlling pH7.0, and controlling the temperature at 37 ℃ and dissolved oxygen at 30%; the total fermentation time is 28-32h (preferably 30 h).
Feeding a carbon source when the residual sugar content in the fermentation liquid is 0.1g/L, controlling the glucose content in the fermentation liquid to be 0-5g/L, and simultaneously feeding 25% ammonia water to control the pH7.0 of the fermentation system; preferably, the carbon source is a 50% glucose solution.
The preparation method of the seed liquid comprises the following steps: the glycerol frozen strain is firstly coated on LB culture medium for activation culture overnight, a certain amount of bacterial colony is selected and inoculated in a 5L triangular flask filled with 1000mL of seed culture medium, the rotation speed of a shaking table is 120rpm, the temperature is 37 ℃, and the shaking culture is carried out for 8-12h until the strain concentration is OD 600 =10, so as to be used as seed liquid.
The seed culture medium is as follows: 15g/L corn steep liquor, 5g/L soybean meal hydrolysate, 20g/L,MgSO4·7H2O0.5g/L,KH2PO4 2g/L,FeSO4 10mg/L,MnSO4 10mg/L,(NH4)2SO4 5g/L g/L glucose and 0.5g/L betaine.
Or the method comprises the following steps: during fermentation for 12-20 h (preferably 16 h), 0.05-0.5g/L sodium benzoate is added into the fermentation medium.
Further, sodium benzoate is added to the fermentation medium (preferably for 16 h), the fermentation temperature is reduced to 34-36 ℃, dissolved oxygen is reduced to 15-25%, and glycerin and betaine are added to the final concentrations of 1-2g/L and 0.5g/L respectively, so as to further improve acid production.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
In the process of producing L-amino acid by microbial fermentation, a certain amount of sodium benzoate is added into a fermentation medium to change bacterial respiratory metabolism, reduce the production of byproducts (such as citric acid and succinic acid) and convert more nitrogen sources into end products; changing permeability of cell membrane, reducing energy consumption, and increasing yield of main product (L-amino acid such as L-threonine).
Detailed Description
The invention provides a method for improving the fermentation yield of L-amino acid (comprising optimization of culture medium and fermentation conditions), which adopts the following technical scheme:
1. Effect of sodium benzoate addition to fermentation Medium
(1) Preparing a seed culture medium and culturing seeds;
(2) Preparing a fermentation medium, adding sodium benzoate, and fermenting and culturing;
(3) Controlling fermentation conditions, adding 0.05-0.5g/L sodium benzoate into initial fermentation medium, and increasing L-amino acid (preferably L-threonine) yield.
2. Sodium benzoate is added during fermentation culture
Controlling fermentation conditions, and adding sodium benzoate after fermentation for 16 hours; the citric acid and the succinic acid are reduced, the content of pyruvic acid is increased, and the yield of L-threonine is improved.
3. And (3) fermenting for 16 hours, adding sodium benzoate, reducing the fermentation temperature to 34-36 ℃, reducing dissolved oxygen to control the dissolved oxygen to 15-25%, adding 1-2g/L glycerol as a carbon source, and simultaneously adding 0.5g/L betaine to further improve acid production.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
The strain used in the following examples was E.coli MHZ-0216-5, supplied by Langfang plum blossom biotechnology development Co., ltd. Strain MHZ-0216-5 can be found in CN113846132A.
Example 1 production method of L-threonine with sodium benzoate added to fermentation bed charge
The method comprises the following steps:
(1) L-threonine seed medium: 15g/L corn steep liquor, 5g/L soybean meal hydrolysate and 0.5g/L glucose 20g/L,MgSO4·7H2O 0.5g/L,KH2PO4 2g/L,FeSO4 10mg/L,MnSO4 10mg/L,(NH4)2SO4;5g/L, betaine.
(2) Seed culture: the strain adopts escherichia coli for production, the glycerol pipe frozen strain is firstly coated on an LB culture medium for activation culture overnight, a certain amount of bacterial colonies are selected and inoculated in a 5L triangular flask filled with 1000mL of seed culture medium, the bacterial colonies are put into a constant temperature reciprocating shaking table for shaking culture at the rotation speed of 120rpm and the temperature of 37 ℃ for 8-12 hours until the concentration of the strain in the seed liquid is OD 600 = 10, and then the culture is finished to be used as the seed liquid.
(3) L-threonine fermentation medium: corn steep liquor 5g/L, soybean meal hydrolysate 5g/L, glucose 40g/L,MgSO4·7H2O 0.5g/L,KH2PO4 2g/L,FeSO4 20mg/L,MnSO4 20mg/L,ZnSO4 0.05mg/L,(NH4)2SO4 5g/L, betaine 1g/L, and sodium benzoate 0.05 g/L, 0.1 g/L, 0.2 g/L, 0.3 g/L, 0.5g/L, respectively.
(4) Fermentation culture: the cultured seed liquid is inoculated into a 50L fermentation tank filled with 20L fermentation medium, the aeration rate is 0.8vvm, the rotating speed is 300-600rpm, the pH value is 7.0, the temperature is 37 ℃, and the dissolved oxygen is 30%.
Adding a carbon source into the fermentation liquor after the residual sugar content is 0.1g/L, controlling pH to 7.0 by taking ammonia water as a pH regulator, preparing 50% glucose solution as the added carbon source, controlling the glucose content in the fermentation liquor to be 0.1g/L, and taking 25% ammonia water as the added nitrogen source. The total fermentation time is 30 hours.
(5) Glucose content was determined using an SBA biosensor and L-threonine content was determined by HPLC. The results are shown in Table 1.
TABLE 1 fermentation results of sodium benzoate addition to initial fermentation Medium
It can be seen that small amounts of sodium benzoate have a positive effect on bacterial production and that increased amounts affect the growth of the bacteria themselves and thus the production of L-threonine.
Example 2 production of L-threonine by fed-batch sodium benzoate during fermentation
The method comprises the following steps:
(1) L-threonine seed medium: as in example 1.
(2) Seed culture: as in example 1.
(3) L-threonine fermentation medium: l-threonine fermentation medium: corn steep liquor 5g/L, soybean meal hydrolysate 5g/L, glucose 40g/L,MgSO4·7H2O 0.5g/L,KH2PO4 2g/L,FeSO4 20mg/L,MnSO4 20mg/L,ZnSO40.05mg/L,(NH4)2SO4 5g/L, betaine 1g/L.
(4) Fermentation culture: as in example 1.
Sodium benzoate at 0.2g/L was added at 12h, 16h, 20h of fermentation, respectively.
(5) Glucose content was determined using an SBA biosensor and L-threonine content was determined by HPLC. The results are shown in Table 2.
TABLE 2 post-fermentation results of sodium benzoate addition during fermentation
Sodium benzoate, g/L | 12h | 16h | 20h |
L-threonine, g/L | 111.5 | 114.8 | 109.1 |
Conversion, percent | 56.1 | 57.1 | 55.5 |
Succinic acid, g/L | 0.1 | 0.1 | 0.1 |
Citric acid, g/L | 0.1 | 0.1 | 0.1 |
Pyruvic acid, g/L | 0.2 | 0.3 | 0.3 |
OD600 | 36.1 | 37.6 | 38.4 |
Example 3 production method of L-threonine by fed-batch sodium benzoate during fermentation
The method comprises the following steps:
(1) L-threonine seed medium: as in example 2.
(2) Seed culture: as in example 2.
(3) L-threonine fermentation medium: l-threonine fermentation medium: corn steep liquor 5g/L, soybean meal hydrolysate 5g/L, glucose 40g/L,MgSO4·7H2O 0.5g/L,KH2PO4 2g/L,FeSO4 20mg/L,MnSO4 20mg/L,ZnSO40.05mg/L,(NH4)2SO4 5g/L, betaine 1g/L.
(4) Fermentation culture: as in example 2, 0.2g/L sodium benzoate was added during 16h of fermentation, and the temperature was lowered to 35℃and 0.5g/L betaine and 1.5g/L glycerol were fed in.
(5) Glucose content was determined using an SBA biosensor and L-threonine content was determined by HPLC at 118.5g/L. The conversion was 58.2%.
(6) Fermentation liquor OD 600 =36.5, succinic acid content 0.1g/L, citric acid content 0.1g/L and pyruvic acid content 0.3g/L.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. The application of sodium benzoate in improving the fermentation yield of L-amino acid;
preferably, the L-amino acid is L-threonine, L-glutamic acid, L-lysine or L-isoleucine.
2. Use according to claim 1, characterized in that the species used for fermentation is escherichia coli (ESCHERICHIA COLI), corynebacterium glutamicum (Corynebacterium glutamicum), corynebacterium validum (Corynebacterium efficiens), corynebacterium crenatum (Corynebacterium crenatum), corynebacterium ammoniagenes thermophilum (Corynebacterium thermoaminogenes) or corynebacterium ammoniagenes (Corynebacterium aminogenes).
3. A method for improving the fermentation yield of L-amino acid is characterized in that escherichia coli is used as a fermentation strain, and a proper amount of sodium benzoate is added into a fermentation culture medium, so that the fermentation yield of L-amino acid is improved;
preferably, the L-amino acid is L-threonine, L-glutamic acid, L-lysine or L-isoleucine.
4. A method according to claim 3, characterized in that the method comprises: at the beginning of fermentation, 0.05-0.5g/L sodium benzoate is added to the fermentation medium.
5. The method of claim 4, wherein the fermentation medium is: corn steep liquor 5g/L, soybean meal hydrolysate 5g/L, glucose 40g/L,MgSO4·7H2O 0.5g/L,KH2PO4 2g/L,FeSO4 20mg/L,MnSO4 20mg/L,ZnSO4 0.05mg/L,(NH4)2SO4 5g/L and betaine 1g/L.
6. A method according to claim 3, wherein the method of fermentation culture comprises: inoculating the seed solution into a 50L fermentation tank containing 20L fermentation medium, introducing air at an air flow rate of 0.8vvm and a rotation speed of 300-600rpm, controlling pH7.0, and controlling the temperature at 37 ℃ and dissolved oxygen at 30%; the total fermentation time is 28-32h;
Feeding a carbon source when the residual sugar content in the fermentation liquid is 0.1g/L, controlling the glucose content in the fermentation liquid to be 0-5g/L, and simultaneously feeding 25% ammonia water to control the pH7.0 of the fermentation system;
Preferably, the carbon source is a 50% glucose solution.
7. The method of claim 6, wherein the seed liquid preparation method comprises: the glycerol frozen strain is firstly coated on LB culture medium for activation culture overnight, a certain amount of bacterial colony is selected and inoculated in a 5L triangular flask filled with 1000mL seed culture medium, the rotation speed of a shaking table is 120rpm, the temperature is 37 ℃, and the shaking culture is carried out for 8-12h until the strain concentration is OD 600 =10, so as to be used as seed liquid;
The seed culture medium is as follows: 15g/L corn steep liquor, 5g/L soybean meal hydrolysate, 20g/L,MgSO4·7H2O0.5g/L,KH2PO4 2g/L,FeSO4 10mg/L,MnSO4 10mg/L,(NH4)2SO4 5g/L g/L glucose and 0.5g/L betaine.
8. A method according to claim 3, characterized in that the method comprises: and adding 0.05-0.5g/L sodium benzoate into the fermentation medium when fermenting for 12-20 h.
9. The method according to claim 8, wherein sodium benzoate is added to the fermentation medium while the fermentation temperature is lowered to 34-36 ℃ and dissolved oxygen is reduced to 15-25% and glycerol and betaine are added to a final concentration of 1-2g/L, 0.5g/L, respectively.
10. The method according to any one of claims 3 to 9, wherein the fermentation broth is escherichia coli MHZ-0216-5.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310071484.8A CN118345128A (en) | 2023-01-16 | 2023-01-16 | Method for improving fermentation yield of L-amino acid |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310071484.8A CN118345128A (en) | 2023-01-16 | 2023-01-16 | Method for improving fermentation yield of L-amino acid |
Publications (1)
Publication Number | Publication Date |
---|---|
CN118345128A true CN118345128A (en) | 2024-07-16 |
Family
ID=91820069
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310071484.8A Pending CN118345128A (en) | 2023-01-16 | 2023-01-16 | Method for improving fermentation yield of L-amino acid |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN118345128A (en) |
-
2023
- 2023-01-16 CN CN202310071484.8A patent/CN118345128A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP3550026B1 (en) | L-isoleucine-producing corynebacterium glutamicum fermentation medium and culture method | |
CN110396493B (en) | Culture medium composition and method for producing isoleucine | |
CN112626143B (en) | Fermentation method of L-lysine | |
CN112322673B (en) | Fermentation method of glutamic acid | |
CN112501221A (en) | Method for improving conversion rate of threonine and saccharic acid | |
CN109609566B (en) | Method for improving threonine yield | |
CN110885865B (en) | Method for producing alpha-glutamic acid by fermentation | |
CN118345128A (en) | Method for improving fermentation yield of L-amino acid | |
CN114045235B (en) | Method for producing single-cell protein and fermentable sugar by using methanotrophic bacteria | |
CN110878325B (en) | Optimized glutamic acid fermentation medium | |
CN112430633B (en) | Process for producing arginine by fermenting fed-batch culture solution | |
CN112251474B (en) | Method for improving fermentation yield and saccharic acid conversion rate of L-glutamic acid | |
CN113502308B (en) | Method for producing vitamin B12 by aerobic fermentation based on redox potential regulation | |
CN109609567B (en) | Green production method of L-tryptophan by using mycoprotein enzymolysis liquid to replace yeast powder | |
CN112195206A (en) | Amino acid fermentation process using liquid caustic soda to replace part of liquid ammonia | |
CN118345129A (en) | Method for improving fermentation yield of L-threonine | |
JP3074781B2 (en) | Production method of L-lysine by fermentation method | |
CN117987485A (en) | Method for producing L-threonine by fermentation, culture medium and application thereof | |
CN114875090B (en) | Method for producing lysine and application thereof | |
CN116606795A (en) | Method for improving amino acid fermentation acid production | |
CN112080533B (en) | Full-nutrition fed-batch fermentation control process for improving yield of L-isoleucine | |
CN112662609B (en) | Fermentation medium for increasing yield of beta-alanine and application method | |
CN116042753B (en) | Preparation method of corn steep liquor mycoprotein composite hydrolysate and preparation method of L-isoleucine | |
CN116656755A (en) | Culture medium sterilization method and application thereof in L-amino acid fermentation production | |
CN1210888A (en) | Process for producing saccharifying enzyme using waste molasses as main raw material |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |