CN114875089B - Method for improving fermentation efficiency of L-valine - Google Patents
Method for improving fermentation efficiency of L-valine Download PDFInfo
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- CN114875089B CN114875089B CN202210552375.3A CN202210552375A CN114875089B CN 114875089 B CN114875089 B CN 114875089B CN 202210552375 A CN202210552375 A CN 202210552375A CN 114875089 B CN114875089 B CN 114875089B
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- 238000000855 fermentation Methods 0.000 title claims abstract description 143
- 230000004151 fermentation Effects 0.000 title claims abstract description 143
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 title claims abstract description 110
- 229960004295 valine Drugs 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 35
- 235000013379 molasses Nutrition 0.000 claims abstract description 57
- 230000001580 bacterial effect Effects 0.000 claims abstract description 22
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims abstract description 16
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims abstract description 15
- 235000016068 Berberis vulgaris Nutrition 0.000 claims description 55
- 241000335053 Beta vulgaris Species 0.000 claims description 55
- 239000007788 liquid Substances 0.000 claims description 48
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 40
- 239000001963 growth medium Substances 0.000 claims description 40
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 38
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 36
- 239000008103 glucose Substances 0.000 claims description 28
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 22
- 229910052760 oxygen Inorganic materials 0.000 claims description 22
- 239000001301 oxygen Substances 0.000 claims description 22
- 240000008042 Zea mays Species 0.000 claims description 21
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 21
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 21
- 235000005822 corn Nutrition 0.000 claims description 21
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 20
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 20
- 239000000725 suspension Substances 0.000 claims description 20
- 238000011218 seed culture Methods 0.000 claims description 19
- 239000002609 medium Substances 0.000 claims description 18
- 239000000843 powder Substances 0.000 claims description 18
- 239000011780 sodium chloride Substances 0.000 claims description 18
- 239000007787 solid Substances 0.000 claims description 18
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 10
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 10
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 10
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 10
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 10
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 10
- 239000002504 physiological saline solution Substances 0.000 claims description 10
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 9
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 2
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 22
- 239000002253 acid Substances 0.000 abstract description 7
- 230000015572 biosynthetic process Effects 0.000 abstract description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 abstract description 4
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 abstract description 4
- 229960003237 betaine Drugs 0.000 abstract description 4
- 239000000463 material Substances 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 4
- 229940024606 amino acid Drugs 0.000 abstract description 3
- 150000001413 amino acids Chemical class 0.000 abstract description 3
- 229960002685 biotin Drugs 0.000 abstract description 3
- 235000020958 biotin Nutrition 0.000 abstract description 3
- 239000011616 biotin Substances 0.000 abstract description 3
- 230000009123 feedback regulation Effects 0.000 abstract description 3
- 230000002503 metabolic effect Effects 0.000 abstract description 3
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 230000007246 mechanism Effects 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 description 13
- 238000011081 inoculation Methods 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 8
- 239000001888 Peptone Substances 0.000 description 8
- 108010080698 Peptones Proteins 0.000 description 8
- 244000061458 Solanum melongena Species 0.000 description 8
- 235000002597 Solanum melongena Nutrition 0.000 description 8
- 239000008272 agar Substances 0.000 description 8
- 235000015278 beef Nutrition 0.000 description 8
- 229940041514 candida albicans extract Drugs 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 239000000284 extract Substances 0.000 description 8
- 235000019319 peptone Nutrition 0.000 description 8
- 229910001220 stainless steel Inorganic materials 0.000 description 8
- 239000010935 stainless steel Substances 0.000 description 8
- 239000012138 yeast extract Substances 0.000 description 8
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 7
- 235000011114 ammonium hydroxide Nutrition 0.000 description 7
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 6
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 6
- 239000004474 valine Substances 0.000 description 6
- 241001052560 Thallis Species 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 4
- 230000001276 controlling effect Effects 0.000 description 3
- 229940107700 pyruvic acid Drugs 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 235000020776 essential amino acid Nutrition 0.000 description 2
- 239000003797 essential amino acid Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 1
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical compound OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000000153 supplemental effect Effects 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/265—Micrococcus
- C12R2001/28—Micrococcus glutamicus ; Corynebacterium glutamicum
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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Abstract
A method for improving the fermentation efficiency of L-valine belongs to the technical field of amino acid fermentation. In order to solve the problems of long fermentation period, low acid yield and the like in the prior art of producing the L-valine by utilizing microbial fermentation, the invention provides a method for improving the fermentation efficiency of the L-valine by utilizing tryptophan by combining an L-valine biosynthesis way and a metabolic feedback regulation mechanism, and meanwhile, the bacterial activity is improved and the fermentation period is shortened by optimizing a sectional feeding process. The molasses which is a cheap material is fed in batches, so that the dosage of betaine and biotin is reduced, and the cost of production raw materials is lowered. The method for producing L-valine can be used for producing the L-valine with the yield of more than 90g/L and the conversion rate of 46 percent, and can be used for improving the fermentation efficiency of the L-valine.
Description
Technical Field
The invention belongs to the technical field of amino acid fermentation, and particularly relates to a method for improving fermentation efficiency of L-valine.
Background
L-valine is taken as one of essential amino acids, has multiple biological functions, has wide application in industries such as food, medicine, feed and the like, and is mainly obtained by a microbial fermentation method.
CN113025670a discloses a high-efficiency valine preparation method, which uses recombinant genetic engineering strain Sval031 as a production strain, and adopts control processes of properly optimizing culture medium components, adjusting dissolved oxygen, inoculum size and the like, and the fermentation is performed for 51h, wherein the valine yield is 74g/L, and the sugar-acid conversion rate is 54.8%.
CN105695526a discloses a fermentation process for improving acid production of L-valine by feeding materials step by step, by adjusting the formula of a culture medium and adopting a mode of feeding corn steep liquor process in a time-division mode and controlling dissolved oxygen step by step at the same time, biosynthesis of valine is promoted, feedback regulation in an amino acid biosynthesis pathway is avoided, generation of byproducts is reduced, the fermentation process produces L-valine for 55h in a fermentation period, acid production is 80g/L, and conversion rate is 32%.
Along with the increasing market demand of L-valine, the bottleneck problems of long fermentation period, high cost, low acid yield and the like still need to be faced when the large-scale production of L-valine is carried out. Therefore, there is a need to find a fermentation process with short fermentation period, low cost and high valine yield.
Disclosure of Invention
In order to solve the problems of long fermentation period, low acid yield and the like in the prior art of producing the L-valine by utilizing microbial fermentation, the invention provides a method for improving the fermentation efficiency of the L-valine by utilizing tryptophan by combining an L-valine biosynthesis way and a metabolic feedback regulation mechanism, and meanwhile, the bacterial activity is improved and the fermentation period is shortened by optimizing a sectional feeding process. The method specifically comprises the steps of inoculating seed liquid of corynebacterium glutamicum for producing L-valine into a fermentation medium containing tryptophan according to an inoculation amount of 10%, fermenting for 48 hours at a fermentation temperature of 32-33 ℃ and a pH value of 7.0-7.2 under the condition that dissolved oxygen is more than or equal to 30%, adding 80% of liquid glucose in the fermentation process to maintain the sugar concentration in a fermentation tank to be 10-20g/L, and adding nutritional small materials in batches in the early and middle stages of fermentation to obtain the L-valine fermentation liquid.
Further limiting, the seed liquid is obtained by inoculating corynebacterium glutamicum on a solid culture medium, culturing for 24-36h at 32 ℃ and pH of 6.0-7.0, eluting with physiological saline, and scattering to obtain bacterial suspension; inoculating the bacterial suspension into a seed culture medium, and culturing at 32 ℃ under the condition that dissolved oxygen is more than or equal to 30% to obtain seed liquid.
Further limited, the seed culture medium comprises 40-50g/L of corn steep liquor, 20g/L of glucose, 5-8g/L of yeast powder, 10g/L of sodium chloride, 2-3g/L of magnesium sulfate and the balance of water.
Further defined, the tryptophan content of the fermentation medium is 0.3-0.5g/L.
Further defined, the fermentation medium comprises tryptophan 0.3-0.5g/L, glucose 20-25g/L, potassium dihydrogen phosphate 5-7g/L, ammonium sulfate 5-6g/L, corn steep liquor 40-45g/L, magnesium sulfate 1-3g/L, VB 1 0.002-0.005g/L, VH 0.004-0.006g/L, and water in balance; adjusting pH to 6.0-7.0, sterilizing at 121deg.C for 20mim。
Further defined, the nutritional supplement is beet molasses.
Further defined, the ratio of the total amount of beet molasses make-up to the volume of initial fermentation medium is 25g: 1L-30 g:1L.
Further defined, the beet molasses is supplemented in the form of a solution, and the preparation method of the beet molasses solution is to calculate the total usage amount of the beet molasses, fix the volume of the beet molasses to 2000mL by distilled water, and sterilize for 15min at 118 ℃.
Further defined, the way of supplementing beet molasses is to supplement 1000mL of beet molasses solution at constant speed in the early and middle stages of fermentation respectively.
Further defined, the early period is 12 to 16 hours after the start of fermentation, and the mid period is 24 to 28 hours after the start of fermentation.
The invention has the beneficial effects that:
the synthesis path of L-valine is complex, pyruvic acid is mainly used as a precursor substance, and valine is finally formed through multi-step flow reaction. The invention combines the metabolic synthesis way of L-valine, adds a small amount of tryptophan into the fermentation medium, supplements the essential amino acid for the growth of thalli, reduces the conversion and utilization of phosphoenolpyruvate upstream of pyruvic acid to tryptophan, promotes the accumulation of a large amount of pyruvic acid, and further improves the acid yield of L-valine.
In order to solve the problem that early senescence occurs in the fermentation process of the thalli, the beet molasses is added in sections, the beet molasses has complex components, contains various nutrition growth factors such as betaine, biotin and the like, and is beneficial to the rapid synthesis of cell membranes of the thalli, the growth of the thalli, the stabilization of the activity of the thalli in the middle and later stages and further the shortening of the fermentation period after a proper amount of beet molasses is added. Meanwhile, betaine is added in the later fermentation period, so that the permeability of the membrane is increased, osmotic pressure is relieved, a methyl donor is provided, the transportation and secretion of products are facilitated, the feedback inhibition of byproducts is reduced, and the synthesis of L-valine is promoted. In addition, the molasses which is an inexpensive material is fed in batches, so that the dosage of betaine and biotin is reduced, and the cost of production raw materials is reduced.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to be limiting, but any modifications, equivalents, improvements, etc. within the spirit and principles of the present invention are intended to be included within the scope of the present invention.
The preservation number of the corynebacterium glutamicum producing L-valine used by the invention is CGMCC No.21700.
Example 1:
(1) Preparation of bacterial suspension: corynebacterium glutamicum producing L-valine is inoculated into 250mL eggplant bottle solid slant culture medium with liquid volume of 80mL, cultured for 30h at 32 ℃ and pH of 6.5, and eluted and scattered by 200mL physiological saline to obtain bacterial suspension. The solid culture medium consists of 5g/L glucose, 10g/L peptone, 10g/L beef extract, 2.5g/L sodium chloride, 5g/L yeast extract, 20g/L agar powder and the balance of water.
(2) Preparing seed liquid: inoculating 200mL of primary seed solution into a stainless steel fermentation tank filled with 3L of seed culture medium, and adjusting pressure, rotation speed and air volume at 32deg.C to maintain dissolved oxygen not less than 30% (wherein the pressure adjustment range is 0.04-0.1MPa, the rotation speed adjustment range is 200-600rpm, and the air volume adjustment range is 0.2-1 m) 3 Culturing under the condition of/h) until the seeds are mature, and obtaining seed liquid; the seed culture medium consists of 45g/L corn steep liquor, 20g/L glucose, 6g/L yeast powder, 10g/L sodium chloride, 2.5g/L magnesium sulfate and the balance of water.
(3) Fermentation: inoculating mature seed liquid with 10% of inoculation amount into fermentation medium composed of tryptophan 0.4g/L, glucose 22g/L, potassium dihydrogen phosphate 6g/L, ammonium sulfate 5.5g/L, corn steep liquor 42g/L, magnesium sulfate 2g/L, VB 1 0.004g/L, VH 0.005g/L, balance water, pH 6.5.
The fermentation control method comprises the following steps: the pH value is maintained at 7.0 and 32 ℃ by adopting 25% ammonia water automatic control, and the dissolved oxygen is maintained to be more than or equal to 30% by adjusting the pressure, the rotating speed and the air quantity, wherein the pressure adjustment range is 0.04-0.1MPa, the rotating speed adjustment range is 200-900 revolutions per minute, and the air quantity adjustment range is 0.2-1m 3 And/h. 1000mL of the fermentation liquid is respectively added at a constant speed in the early stage (12-16 h after the fermentation is started) and the middle stage (24-28 h after the fermentation is started)And (3) adding 80% of liquid glucose into the beet molasses solution subjected to high-temperature sterilization in the fermentation process to maintain the sugar concentration in the fermentation tank to be 10-20g/L until the fermentation is finished, so as to obtain the L-valine fermentation liquid. The ratio of the amount of beet molasses in the beet molasses solution to the initial fermentation volume feed liquid was 27g:1L beet molasses solution is prepared by calculating total dosage of beet molasses, metering volume of beet molasses to 2000mL with distilled water, and sterilizing at 118 deg.C for 15min. The total fermentation time of the method is 48 hours, the yield of L-valine is 90.5g/L through content detection and conversion rate calculation, and the conversion rate is 46.11%.
The conversion rate in the invention has the following calculation formula: (valine yield. Times.volume)/(fermentation bottom sugar + supplemental sugar). Times.100%.
Example 2:
(1) Preparation of bacterial suspension: the corynebacterium glutamicum producing L-valine is inoculated into a solid slant culture medium of a 250mL eggplant bottle with a liquid volume of 80mL, and is cultured for 24 hours at 32 ℃ and pH 6, and is eluted and scattered by 200mL of physiological saline, so as to obtain bacterial suspension. The solid culture medium consists of 5g/L glucose, 10g/L peptone, 10g/L beef extract, 2.5g/L sodium chloride, 5g/L yeast extract, 20g/L agar powder and the balance of water.
(2) Preparing seed liquid: inoculating 200mL of primary seed solution into a stainless steel fermentation tank filled with 3L of seed culture medium, and adjusting pressure, rotation speed and air volume at 32deg.C to maintain dissolved oxygen not less than 30% (wherein the pressure adjustment range is 0.04-0.1MPa, the rotation speed adjustment range is 200-600rpm, and the air volume adjustment range is 0.2-1 m) 3 Culturing under the condition of/h) until the seeds are mature, and obtaining seed liquid; the seed culture medium consists of 40g/L corn steep liquor, 20g/L glucose, 5g/L yeast powder, 10g/L sodium chloride, 2g/L magnesium sulfate and the balance of water.
(3) Fermentation: inoculating mature seed solution with 10% of inoculation amount into fermentation medium composed of tryptophan 0.3g/L, glucose 20g/L, potassium dihydrogen phosphate 5g/L, ammonium sulfate 5g/L, corn steep liquor 40g/L, magnesium sulfate 1g/L, VB 1 0.002g/L, VH 0.004g/L, balance water, pH 6.
The fermentation control method comprises the following steps: automatically controlling and maintaining pH at 7.0 and 32deg.C with 25% ammonia waterThe dissolved oxygen is maintained to be more than or equal to 30 percent by adjusting the pressure, the rotating speed and the air quantity, wherein the pressure adjusting range is 0.04-0.1MPa, the rotating speed adjusting range is 200-900 revolutions per minute, and the air quantity adjusting range is 0.2-1m 3 And/h. 1000mL of beet molasses solution sterilized at high temperature is respectively and constantly added in the early stage (12-16 h after the fermentation starts) and the middle stage (24-28 h after the fermentation starts), 80% of liquid glucose is added in the fermentation process, so as to maintain the sugar concentration in the fermentation tank at 10-20g/L until the fermentation is finished, and the L-valine fermentation liquid is obtained. The ratio of the amount of beet molasses in the beet molasses solution to the initial fermentation volume feed liquid was 25g:1L beet molasses solution is prepared by calculating total dosage of beet molasses, metering volume of beet molasses to 2000mL with distilled water, and sterilizing at 118 deg.C for 15min. The total fermentation time of the method is 48 hours, the yield of L-valine is 91.35g/L through content detection and conversion rate calculation, and the conversion rate is 46.64%.
Example 3:
(1) Preparation of bacterial suspension: corynebacterium glutamicum producing L-valine is inoculated into 250mL eggplant bottle solid slant culture medium with a liquid volume of 80mL, cultured for 36h at 32 ℃ and pH7, and eluted and scattered by 200mL physiological saline to obtain bacterial suspension. The solid culture medium consists of 5g/L glucose, 10g/L peptone, 10g/L beef extract, 2.5g/L sodium chloride, 5g/L yeast extract, 20g/L agar powder and the balance of water.
(2) Preparing seed liquid: inoculating 200mL of primary seed solution into a stainless steel fermentation tank filled with 3L of seed culture medium, and adjusting pressure, rotation speed and air volume at 32deg.C to maintain dissolved oxygen not less than 30% (wherein the pressure adjustment range is 0.04-0.1MPa, the rotation speed adjustment range is 200-600rpm, and the air volume adjustment range is 0.2-1 m) 3 Culturing under the condition of/h) until the seeds are mature, and obtaining seed liquid; the seed culture medium consists of corn steep liquor 50g/L, glucose 20g/L, yeast powder 8g/L, sodium chloride 10g/L, magnesium sulfate 3g/L, and water in balance.
(3) Fermentation: inoculating mature seed solution with 10% of inoculation amount into fermentation medium composed of tryptophan 0.5g/L, glucose 25g/L, potassium dihydrogen phosphate 7g/L, ammonium sulfate 6g/L, corn steep liquor 45g/L, magnesium sulfate 3g/L, VB 1 0.005g/L, VH 0.006g/L, balance water, pH7.
The fermentation control method comprises the following steps: the pH value is maintained at 7.2 and 33 ℃ by adopting 25% ammonia water automatic control, and the dissolved oxygen is maintained to be more than or equal to 30% by adjusting the pressure, the rotating speed and the air quantity, wherein the pressure adjustment range is 0.04-0.1MPa, the rotating speed adjustment range is 200-900 revolutions per minute, and the air quantity adjustment range is 0.2-1m 3 And/h. 1000mL of beet molasses solution sterilized at high temperature is respectively and constantly added in the early stage (12-16 h after the fermentation starts) and the middle stage (24-28 h after the fermentation starts), 80% of liquid glucose is added in the fermentation process, so as to maintain the sugar concentration in the fermentation tank at 10-20g/L until the fermentation is finished, and the L-valine fermentation liquid is obtained. The ratio of the amount of beet molasses in the beet molasses solution to the initial fermentation volume feed liquid was 30g:1L beet molasses solution is prepared by calculating total dosage of beet molasses, metering volume of beet molasses to 2000mL with distilled water, and sterilizing at 118 deg.C for 15min. The total fermentation time of the method is 48 hours, the yield of L-valine is 90.03g/L through content detection and conversion rate calculation, and the conversion rate is 46.32%.
Comparative example 1:
(1) Preparation of bacterial suspension: corynebacterium glutamicum producing L-valine is inoculated into 250mL eggplant bottle solid slant culture medium with liquid volume of 80mL, cultured for 30h at 32 ℃ and pH of 6.5, and eluted and scattered by 200mL physiological saline to obtain bacterial suspension. The solid culture medium consists of 5g/L glucose, 10g/L peptone, 10g/L beef extract, 2.5g/L sodium chloride, 5g/L yeast extract, 20g/L agar powder and the balance of water.
(2) Preparing seed liquid: inoculating 200mL of primary seed solution into a stainless steel fermentation tank filled with 3L of seed culture medium, and adjusting pressure, rotation speed and air volume at 32deg.C to maintain dissolved oxygen not less than 30% (wherein the pressure adjustment range is 0.04-0.1MPa, the rotation speed adjustment range is 200-600rpm, and the air volume adjustment range is 0.2-1 m) 3 Culturing under the condition of/h) until the seeds are mature, and obtaining seed liquid; the seed culture medium consists of 45g/L corn steep liquor, 20g/L glucose, 6g/L yeast powder, 10g/L sodium chloride, 2.5g/L magnesium sulfate and the balance of water.
(3) Fermentation: inoculating mature seed solution to hair according to 10% of inoculation amountIn the fermentation culture medium, the composition of the fermentation culture medium comprises 22g/L of glucose, 6g/L of monopotassium phosphate, 5.5g/L of ammonium sulfate, 42g/L of corn steep liquor, 2g/L of magnesium sulfate and VB 1 0.004g/L, VH 0.005g/L, balance water, pH 6.5.
The fermentation control method comprises the following steps: the pH value is maintained at 7.0 and 32 ℃ by adopting 25% ammonia water automatic control, and the dissolved oxygen is maintained to be more than or equal to 30% by adjusting the pressure, the rotating speed and the air quantity, wherein the pressure adjustment range is 0.04-0.1MPa, the rotating speed adjustment range is 200-900 revolutions per minute, and the air quantity adjustment range is 0.2-1m 3 And/h. And (3) adding 80% of liquid glucose in the fermentation process to maintain the sugar concentration in the fermentation tank to be 10-20g/L until the fermentation is finished, so as to obtain the L-valine fermentation liquid. The total fermentation time of the method is 53h, the L-valine is 70.5g/L through content detection and conversion rate calculation, and the conversion rate is 38.2%.
Comparative example 2:
(1) Preparation of bacterial suspension: corynebacterium glutamicum producing L-valine is inoculated into 250mL eggplant bottle solid slant culture medium with liquid volume of 80mL, cultured for 30h at 32 ℃ and pH of 6.5, and eluted and scattered by 200mL physiological saline to obtain bacterial suspension. The solid culture medium consists of 5g/L glucose, 10g/L peptone, 10g/L beef extract, 2.5g/L sodium chloride, 5g/L yeast extract, 20g/L agar powder and the balance of water.
(2) Preparing seed liquid: inoculating 200mL of primary seed solution into a stainless steel fermentation tank filled with 3L of seed culture medium, and adjusting pressure, rotation speed and air volume at 32deg.C to maintain dissolved oxygen not less than 30% (wherein the pressure adjustment range is 0.04-0.1MPa, the rotation speed adjustment range is 200-600rpm, and the air volume adjustment range is 0.2-1 m) 3 Culturing under the condition of/h) until the seeds are mature, and obtaining seed liquid; the seed culture medium consists of 45g/L corn steep liquor, 20g/L glucose, 6g/L yeast powder, 10g/L sodium chloride, 2.5g/L magnesium sulfate and the balance of water.
(3) Fermentation: inoculating mature seed liquid with 10% of inoculation amount into fermentation medium composed of tryptophan 0.4g/L, glucose 22g/L, potassium dihydrogen phosphate 6g/L, ammonium sulfate 5.5g/L, corn steep liquor 42g/L, magnesium sulfate 2g/L, VB 1 0.004g/L, VH 0.005g/L, balance water, pH 6.5.
The fermentation control method comprises the following steps: the pH value is maintained at 7.0 and 32 ℃ by adopting 25% ammonia water automatic control, and the dissolved oxygen is maintained to be more than or equal to 30% by adjusting the pressure, the rotating speed and the air quantity, wherein the pressure adjustment range is 0.04-0.1MPa, the rotating speed adjustment range is 200-900 revolutions per minute, and the air quantity adjustment range is 0.2-1m 3 And/h. And (3) adding 80% of liquid glucose in the fermentation process to maintain the sugar concentration in the fermentation tank to be 10-20g/L until the fermentation is finished, so as to obtain the L-valine fermentation liquid. The total fermentation time of the method is 53h, and the L-valine is 78.51g/L and the conversion rate is 39.30% through content detection and conversion rate calculation.
Comparative example 3:
(1) Preparation of bacterial suspension: corynebacterium glutamicum producing L-valine is inoculated into 250mL eggplant bottle solid slant culture medium with liquid volume of 80mL, cultured for 30h at 32 ℃ and pH of 6.5, and eluted and scattered by 200mL physiological saline to obtain bacterial suspension. The solid culture medium consists of 5g/L glucose, 10g/L peptone, 10g/L beef extract, 2.5g/L sodium chloride, 5g/L yeast extract, 20g/L agar powder and the balance of water.
(2) Preparing seed liquid: inoculating 200mL of primary seed solution into a stainless steel fermentation tank filled with 3L of seed culture medium, and adjusting pressure, rotation speed and air volume at 32deg.C to maintain dissolved oxygen not less than 30% (wherein the pressure adjustment range is 0.04-0.1MPa, the rotation speed adjustment range is 200-600rpm, and the air volume adjustment range is 0.2-1 m) 3 Culturing under the condition of/h) until the seeds are mature, and obtaining seed liquid; the seed culture medium consists of 45g/L corn steep liquor, 20g/L glucose, 6g/L yeast powder, 10g/L sodium chloride, 2.5g/L magnesium sulfate and the balance of water.
(3) Fermentation: inoculating mature seed liquid with 10% of inoculation amount into fermentation medium composed of tryptophan 0.4g/L, glucose 22g/L, potassium dihydrogen phosphate 6g/L, ammonium sulfate 5.5g/L, corn steep liquor 42g/L, magnesium sulfate 2g/L, VB 1 0.004g/L, VH 0.005g/L, balance water, pH 6.5.
The fermentation control method comprises the following steps: the pH value is maintained at 7.0 and 32 ℃ by adopting 25% ammonia water for automatic control, and the dissolved oxygen is maintained to be more than or equal to 30% by adjusting the pressure, the rotating speed and the air quantity, wherein the pressure adjusting range is 0.04-0.1MPa, and the rotating speed is changedThe adjusting range is 200-900 rpm, and the air quantity adjusting range is 0.2-1m 3 And/h. And (3) when the fermentation is carried out for 12-16 hours, adding beet molasses solution which is sterilized at high temperature at a constant speed, adding 80% liquid glucose in the fermentation process, and maintaining the sugar concentration in the fermentation tank to be 10-20g/L until the fermentation is finished, thus obtaining the L-valine fermentation liquid. The ratio of the amount of beet molasses in the beet molasses solution to the initial fermentation volume is 13.5g/L, and the preparation method of the beet molasses solution comprises calculating the total amount of beet molasses, metering the volume of beet molasses to 1000mL by distilled water, and sterilizing at 118 ℃ for 15min. The total fermentation time of the method is 50 hours, the L-valine is 86.39g/L through content detection and conversion rate calculation, and the conversion rate is 43.4%.
Comparative example 4:
(1) Preparation of bacterial suspension: corynebacterium glutamicum producing L-valine is inoculated into 250mL eggplant bottle solid slant culture medium with liquid volume of 80mL, cultured for 30h at 32 ℃ and pH of 6.5, and eluted and scattered by 200mL physiological saline to obtain bacterial suspension. The solid culture medium consists of 5g/L glucose, 10g/L peptone, 10g/L beef extract, 2.5g/L sodium chloride, 5g/L yeast extract, 20g/L agar powder and the balance of water.
(2) Preparing seed liquid: inoculating 200mL of primary seed solution into a stainless steel fermentation tank filled with 3L of seed culture medium, and adjusting pressure, rotation speed and air volume at 32deg.C to maintain dissolved oxygen not less than 30% (wherein the pressure adjustment range is 0.04-0.1MPa, the rotation speed adjustment range is 200-600rpm, and the air volume adjustment range is 0.2-1 m) 3 Culturing under the condition of/h) until the seeds are mature, and obtaining seed liquid; the seed culture medium consists of 45g/L corn steep liquor, 20g/L glucose, 6g/L yeast powder, 10g/L sodium chloride, 2.5g/L magnesium sulfate and the balance of water.
(3) Fermentation: inoculating mature seed liquid with 10% of inoculation amount into fermentation medium composed of tryptophan 0.4g/L, glucose 22g/L, potassium dihydrogen phosphate 6g/L, ammonium sulfate 5.5g/L, corn steep liquor 42g/L, magnesium sulfate 2g/L, VB 1 0.004g/L, VH 0.005g/L, balance water, pH 6.5.
The fermentation control method comprises the following steps: the pH value is automatically controlled and maintained at 7.0 and 32 ℃ by adopting 25% ammonia water, and the pressure is regulated,The rotating speed and the air quantity are used for maintaining the dissolved oxygen to be more than or equal to 30 percent, wherein the pressure adjustment range is 0.04-0.1MPa, the rotating speed adjustment range is 200-900 revolutions per minute, and the air quantity adjustment range is 0.2-1m 3 And/h. And (3) when fermentation is carried out for 24-48h, adding beet molasses solution which is sterilized at high temperature at a constant speed, adding 80% liquid glucose in the fermentation process, and maintaining the sugar concentration in the fermentation tank to be 10-20g/L until the fermentation is finished, thus obtaining the L-valine fermentation liquid. The ratio of the amount of beet molasses in the beet molasses solution to the initial fermentation volume is 13.5g/L, and the preparation method of the beet molasses solution comprises calculating the total amount of beet molasses, metering the volume of beet molasses to 1000mL by distilled water, and sterilizing at 118 ℃ for 15min. The total fermentation time of the method is 50h, the L-valine is 82.66g/L through content detection and conversion rate calculation, and the conversion rate is 41.5%.
Comparative example 5:
(1) Preparation of bacterial suspension: corynebacterium glutamicum producing L-valine is inoculated into 250mL eggplant bottle solid slant culture medium with liquid volume of 80mL, cultured for 30h at 32 ℃ and pH of 6.5, and eluted and scattered by 200mL physiological saline to obtain bacterial suspension. The solid culture medium consists of 5g/L glucose, 10g/L peptone, 10g/L beef extract, 2.5g/L sodium chloride, 5g/L yeast extract, 20g/L agar powder and the balance of water.
(2) Preparing seed liquid: inoculating 200mL of primary seed solution into a stainless steel fermentation tank filled with 3L of seed culture medium, and adjusting pressure, rotation speed and air volume at 32deg.C to maintain dissolved oxygen not less than 30% (wherein the pressure adjustment range is 0.04-0.1MPa, the rotation speed adjustment range is 200-600rpm, and the air volume adjustment range is 0.2-1 m) 3 Culturing under the condition of/h) until the seeds are mature, and obtaining seed liquid; the seed culture medium consists of 45g/L corn steep liquor, 20g/L glucose, 6g/L yeast powder, 10g/L sodium chloride, 2.5g/L magnesium sulfate and the balance of water.
(3) Fermentation: inoculating mature seed liquid with 10% of inoculation amount into fermentation medium composed of glucose 22g/L, potassium dihydrogen phosphate 6g/L, ammonium sulfate 5.5g/L, corn steep liquor 42g/L, magnesium sulfate 2g/L, VB 1 0.004g/L, VH 0.005g/L, balance water, pH 6.5.
The fermentation control method comprises the following steps: collectingAutomatically controlling and maintaining pH at 7.0 and 32deg.C with 25% ammonia water, and adjusting pressure, rotation speed and air volume to maintain dissolved oxygen at not less than 30%, wherein the pressure adjustment range is 0.04-0.1MPa, the rotation speed adjustment range is 200-900 rpm, and the air volume adjustment range is 0.2-1m 3 And/h. 1000mL of beet molasses solution sterilized at high temperature is respectively and constantly added in the early stage (12-16 h after the fermentation starts) and the middle stage (24-28 h after the fermentation starts), 80% of liquid glucose is added in the fermentation process, so as to maintain the sugar concentration in the fermentation tank at 10-20g/L until the fermentation is finished, and the L-valine fermentation liquid is obtained. The ratio of the amount of beet molasses in the beet molasses solution to the initial fermentation volume is 27g/L, and the preparation method of the beet molasses solution comprises calculating the total amount of beet molasses, and sterilizing the beet molasses with distilled water to 2000mL at 118 ℃ for 15min. The total fermentation time of the method is 48 hours, the yield of L-valine is 87.77g/L through content detection and conversion rate calculation, and the conversion rate is 44.01%.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. A method for improving the fermentation efficiency of L-valine is characterized in that seed liquid of corynebacterium glutamicum producing L-valine is inoculated into a fermentation medium containing tryptophan in an inoculum size of 10%, 48h is fermented at a fermentation temperature of 32-33 ℃ and a pH value of 7.0-7.2 under the condition that dissolved oxygen is more than or equal to 30%, 80% of liquid glucose is added in the fermentation process to maintain the sugar concentration in a fermentation tank to be 10-20g/L, and beet molasses is added in batches in the early and middle stages of fermentation to obtain L-valine fermentation liquid; the tryptophan content in the fermentation medium is 0.3-0.5 g/L; the ratio of the total amount of beet molasses added to the volume of the initial fermentation medium is 25g: 1L-30 g:1L.
2. The method according to claim 1, wherein the seed solution is obtained by inoculating Corynebacterium glutamicum on a solid medium, culturing at 32℃and pH 6.0-7.0 for 24-36h, eluting with physiological saline, and scattering to obtain a bacterial suspension; inoculating the bacterial suspension into a seed culture medium, and culturing at 32 ℃ under the condition that dissolved oxygen is more than or equal to 30% to obtain seed liquid.
3. The method of claim 2, wherein the seed medium comprises 40-50g/L corn steep liquor, 20-g/L glucose, 5-8-g/L yeast powder, 10-g/L sodium chloride, 2-3-g/L magnesium sulfate, and the balance water.
4. The method according to claim 1, wherein the fermentation medium has a composition of tryptophan 0.3-0.5g/L, glucose 20-25g/L, potassium dihydrogen phosphate 5-7g/L, ammonium sulfate 5-6g/L, corn steep liquor 40-45g/L, magnesium sulfate 1-3g/L, VB 1 0.002-0.005g/L, VH 0.004-0.006g/L, and water as the rest; the pH is adjusted to 6.0-7.0, and the mixture is sterilized at 121 ℃ for 20 mm.
5. The method of claim 1, wherein the beet molasses is supplemented in solution form and the beet molasses solution is prepared by calculating the total amount of beet molasses, metering the beet molasses to 2000ml with distilled water and sterilizing at 118 ℃ for 15min.
6. The method according to claim 5, wherein the beet molasses is fed in such a manner that 1000mL beet molasses solution is fed at a constant rate in the early and middle stages of fermentation, respectively.
7. The method of claim 6, wherein the early stage is 12h to 16h after the start of fermentation and the mid stage is 24h to 28h after the start of fermentation.
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| CN103215322A (en) * | 2013-04-12 | 2013-07-24 | 北京轻发生物技术中心 | Fermentation method for increasing yield of L-valine |
| CN105695526A (en) * | 2016-04-06 | 2016-06-22 | 河南巨龙生物工程股份有限公司 | Fermentation technique for enhancing L-valine acid productivity by multi-step material supplementation |
| CN106086099A (en) * | 2016-08-27 | 2016-11-09 | 河北圣雪大成制药有限责任公司 | L valine fermentation medium and the fermentation process with its production L valine |
| CN109652476A (en) * | 2019-02-25 | 2019-04-19 | 内蒙古拜克生物有限公司 | A kind of method of fermenting and producing Valine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103215322A (en) * | 2013-04-12 | 2013-07-24 | 北京轻发生物技术中心 | Fermentation method for increasing yield of L-valine |
| CN105695526A (en) * | 2016-04-06 | 2016-06-22 | 河南巨龙生物工程股份有限公司 | Fermentation technique for enhancing L-valine acid productivity by multi-step material supplementation |
| CN106086099A (en) * | 2016-08-27 | 2016-11-09 | 河北圣雪大成制药有限责任公司 | L valine fermentation medium and the fermentation process with its production L valine |
| CN109652476A (en) * | 2019-02-25 | 2019-04-19 | 内蒙古拜克生物有限公司 | A kind of method of fermenting and producing Valine |
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