CN116622785A - Method for reducing succinic acid byproduct of lactic acid fermentation - Google Patents
Method for reducing succinic acid byproduct of lactic acid fermentation Download PDFInfo
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- CN116622785A CN116622785A CN202210126828.6A CN202210126828A CN116622785A CN 116622785 A CN116622785 A CN 116622785A CN 202210126828 A CN202210126828 A CN 202210126828A CN 116622785 A CN116622785 A CN 116622785A
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 110
- 238000000855 fermentation Methods 0.000 title claims abstract description 68
- 230000004151 fermentation Effects 0.000 title claims abstract description 68
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 title claims abstract description 68
- 239000004310 lactic acid Substances 0.000 title claims abstract description 55
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 55
- 239000001384 succinic acid Substances 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 31
- 239000006227 byproduct Substances 0.000 title claims abstract description 5
- 239000003112 inhibitor Substances 0.000 claims abstract description 16
- 229960002523 mercuric chloride Drugs 0.000 claims abstract description 14
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims abstract description 14
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 claims abstract description 13
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 claims abstract description 11
- BEPAFCGSDWSTEL-UHFFFAOYSA-N dimethyl malonate Chemical compound COC(=O)CC(=O)OC BEPAFCGSDWSTEL-UHFFFAOYSA-N 0.000 claims abstract description 11
- IIJLLHGPEZBIIT-UHFFFAOYSA-N ethyl 3-nitropropanoate Chemical compound CCOC(=O)CC[N+]([O-])=O IIJLLHGPEZBIIT-UHFFFAOYSA-N 0.000 claims abstract description 10
- LTMRRSWNXVJMBA-UHFFFAOYSA-L 2,2-diethylpropanedioate Chemical compound CCC(CC)(C([O-])=O)C([O-])=O LTMRRSWNXVJMBA-UHFFFAOYSA-L 0.000 claims abstract description 6
- 108020003285 Isocitrate lyase Proteins 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 13
- 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 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000008103 glucose Substances 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 10
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 10
- 239000001301 oxygen Substances 0.000 claims description 10
- 229910052760 oxygen Inorganic materials 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 8
- 238000009423 ventilation Methods 0.000 claims description 8
- 241000588724 Escherichia coli Species 0.000 claims description 7
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 claims description 7
- 239000000920 calcium hydroxide Substances 0.000 claims description 7
- 229910001861 calcium hydroxide Inorganic materials 0.000 claims description 7
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 5
- 230000002053 acidogenic effect Effects 0.000 claims description 5
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 5
- 230000012010 growth Effects 0.000 claims description 5
- 241001052560 Thallis Species 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 4
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 claims description 3
- 239000000347 magnesium hydroxide Substances 0.000 claims description 3
- 229910001862 magnesium hydroxide Inorganic materials 0.000 claims description 3
- 230000003472 neutralizing effect Effects 0.000 claims description 3
- 241000228245 Aspergillus niger Species 0.000 claims description 2
- 241000193749 Bacillus coagulans Species 0.000 claims description 2
- 244000063299 Bacillus subtilis Species 0.000 claims description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 2
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 claims description 2
- 241000186673 Lactobacillus delbrueckii Species 0.000 claims description 2
- 241000186672 Lactobacillus delbrueckii subsp. bulgaricus Species 0.000 claims description 2
- 229940054340 bacillus coagulans Drugs 0.000 claims description 2
- 229940004208 lactobacillus bulgaricus Drugs 0.000 claims description 2
- 230000010261 cell growth Effects 0.000 claims 1
- 241000894007 species Species 0.000 claims 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 10
- 239000008367 deionised water Substances 0.000 description 8
- 229910021641 deionized water Inorganic materials 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 230000001276 controlling effect Effects 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
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- 230000015572 biosynthetic process Effects 0.000 description 5
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- 230000037361 pathway Effects 0.000 description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 5
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 5
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 4
- 108090000854 Oxidoreductases Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
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- 239000002054 inoculum Substances 0.000 description 4
- 238000009630 liquid culture Methods 0.000 description 4
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- WBLZUCOIBUDNBV-UHFFFAOYSA-N 3-nitropropanoic acid Chemical compound OC(=O)CC[N+]([O-])=O WBLZUCOIBUDNBV-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000003208 petroleum Substances 0.000 description 3
- 229920000747 poly(lactic acid) Polymers 0.000 description 3
- 239000004626 polylactic acid Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000012807 shake-flask culturing Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 101100242035 Bacillus subtilis (strain 168) pdhA gene Proteins 0.000 description 2
- 229930182843 D-Lactic acid Natural products 0.000 description 2
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 101100123255 Komagataeibacter xylinus aceC gene Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 101100134871 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) aceE gene Proteins 0.000 description 2
- 101150094017 aceA gene Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- SCJNCDSAIRBRIA-DOFZRALJSA-N arachidonyl-2'-chloroethylamide Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(=O)NCCCl SCJNCDSAIRBRIA-DOFZRALJSA-N 0.000 description 2
- 101150070136 axeA gene Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 229940022769 d- lactic acid Drugs 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- VXZQTXSCMRPKMH-UHFFFAOYSA-N diethyl 2-hydroxypropanedioate Chemical compound CCOC(=O)C(O)C(=O)OCC VXZQTXSCMRPKMH-UHFFFAOYSA-N 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- PTYVBEKOPJHZLJ-UHFFFAOYSA-N 2-nitropropanoic acid Chemical compound OC(=O)C(C)[N+]([O-])=O PTYVBEKOPJHZLJ-UHFFFAOYSA-N 0.000 description 1
- GHCVXTFBVDVFGE-UHFFFAOYSA-N 4-amino-6-chloro-1,3,5-triazin-2-ol Chemical compound NC1=NC(O)=NC(Cl)=N1 GHCVXTFBVDVFGE-UHFFFAOYSA-N 0.000 description 1
- KIUMMUBSPKGMOY-UHFFFAOYSA-L 5-[(3-carboxylato-4-nitrophenyl)disulfanyl]-2-nitrobenzoate Chemical compound C1=C([N+]([O-])=O)C(C(=O)[O-])=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C([O-])=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-L 0.000 description 1
- ODBLHEXUDAPZAU-ZAFYKAAXSA-N D-threo-isocitric acid Chemical compound OC(=O)[C@H](O)[C@@H](C(O)=O)CC(O)=O ODBLHEXUDAPZAU-ZAFYKAAXSA-N 0.000 description 1
- ODBLHEXUDAPZAU-FONMRSAGSA-N Isocitric acid Natural products OC(=O)[C@@H](O)[C@H](C(O)=O)CC(O)=O ODBLHEXUDAPZAU-FONMRSAGSA-N 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 102000011929 Succinate-CoA Ligases Human genes 0.000 description 1
- 108010075728 Succinate-CoA Ligases Proteins 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- GZCGUPFRVQAUEE-VANKVMQKSA-N aldehydo-L-glucose Chemical compound OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)C=O GZCGUPFRVQAUEE-VANKVMQKSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- ZIYVHBGGAOATLY-UHFFFAOYSA-N methylmalonic acid Chemical compound OC(=O)C(C)C(O)=O ZIYVHBGGAOATLY-UHFFFAOYSA-N 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- VNOYUJKHFWYWIR-ITIYDSSPSA-N succinyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)CCC(O)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 VNOYUJKHFWYWIR-ITIYDSSPSA-N 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- ODBLHEXUDAPZAU-UHFFFAOYSA-N threo-D-isocitric acid Natural products OC(=O)C(O)C(C(O)=O)CC(O)=O ODBLHEXUDAPZAU-UHFFFAOYSA-N 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
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- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Tropical Medicine & Parasitology (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for adding an isocitrate lyase specific inhibitor in a lactic acid fermentation process to block succinic acid generation as a fermentation byproduct. The specific inhibitor comprises one or more of methyl glycolate, dimethyl malonate, diethyl malonate, ethyl 3-nitropropionate, mercuric chloride or 5,5' -dithiobis (2-nitrobenzoic acid), and can effectively reduce the succinic acid content in lactic acid.
Description
Technical Field
The invention belongs to the technical field of biology, and mainly relates to a lactic acid fermentation production method
Background
Currently, petroleum-based chemicals are widely used as raw materials in the fields of polymeric materials, textiles, paints, organic solvents, and the like. However, in a large background of global warming and the growing shortage of petroleum resources, polylactic acid as a representative of biodegradable materials is considered as one of the main substitutes for petroleum-based plastics and the like. The D-lactic acid and the L-lactic acid with extremely high optical purity and extremely high chemical purity which are required in the processing of the polylactic acid are prepared by utilizing a large amount of cheap raw materials, are necessary for the development of the polylactic acid industry, and are new power for the development and transformation of the organic acid industry.
In order to obtain the lactic acid fermentation with high substrate conversion rate, high yield and high optical and chemical purity of target products, metabolic pathways are modified, and genetic engineering strains are used as lactic acid production bacteria.
There are three main synthesis routes of succinic acid, one of which is that fumaric acid is reduced to form succinic acid under the action of fumaric acid reductase; and secondly, succinyl-CoA is hydrolyzed to form succinic acid under the action of succinyl-CoA synthetase. And thirdly, the isocitric acid participates in the cyclic process of the half-aldehyde acid to form succinic acid (aceA). Under aerobic metabolism, the succinic acid formation process and the decomposition process are performed simultaneously, and escherichia coli hardly accumulates succinic acid. Under anaerobic metabolism, E.coli accumulates succinic acid under the action of fumaric acid reductase. The synthesis of lactic acid is often an anaerobic or low oxygen supply culture condition, and thus, it is necessary to block the synthesis of succinic acid by pathway modification. CN 104278003B, taking escherichia coli as an initial strain, knocking out the fumaric acid reductase gene frdABCD, and blocking the anaerobic generation path of succinic acid. CN 105705630A blocks the succinic acid anaerobic production pathway by knocking out the fumaric acid reductase gene frdA.
However, in practice, on the one hand, the genetic modification cost is high, the operation is complicated, on the other hand, although the anaerobic pathway of succinic acid is blocked, part of air still exists in the fermentation tank, or oxygen also exists in the fermentation liquor in the aerobic proliferation stage in the earlier stage of culture, although ventilation is stopped for anaerobic culture, the oxygen existing under the above conditions still enters cells, succinic acid is generated through the aerobic pathway, and accumulation of succinic acid is caused. If the aerobic succinic acid generation pathway is blocked by a genetic engineering means, bacteria cannot grow. But the generation of succinic acid not only can lead to the reduction of the conversion rate of lactic acid, but also can lead to the reduction of the yield in the process of separating and purifying lactic acid, and finally, the cost of high-chemical-purity lactic acid is increased.
Disclosure of Invention
Aiming at the problem that succinic acid generation cannot be completely inhibited in the lactic acid fermentation process, the invention blocks succinic acid generation by using an isocitrate lyase (aceA) specific inhibitor in the fermentation process.
In order to achieve the above effects, the present invention provides a method for reducing succinic acid, a byproduct of lactic acid fermentation, comprising the steps of:
(1) Culturing lactic acid producing bacteria seed liquid, and inoculating the lactic acid producing bacteria seed liquid into a fermentation tank containing a culture medium for bacterial growth; (2) When the concentration of the thalli reaches an OD600 of 10-50, adding an isocitrate lyase specific inhibitor, and then entering an acidogenic stage for lactic acid fermentation; the specific inhibitor is one or a combination of methyl glycolate, dimethyl malonate, diethyl malonate, ethyl 3-nitropropionate, mercuric chloride or 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB).
Preferably, the combination of inhibitors is a mixture of methyl glycolate, dimethyl malonate and mercuric chloride; or is a mixture of methyl glycolate, dimethyl malonate, diethyl malonate and ethyl 3-nitropropionate; a more preferred combination is a mixture of ethyl 3-nitropropionate, mercuric chloride, and 5,5' -dithiobis (2-nitrobenzoic acid).
The lactic acid producing strain in the step (1) is selected from one or more of escherichia coli recombinant bacteria, bacillus coagulans, bacillus subtilis, lactobacillus delbrueckii, lactobacillus bulgaricus, aspergillus niger and saccharomycetes.
The seed liquid in the step (1) is shake flask culture seed liquid and/or seed tank culture seed liquid, the culture medium of the shake flask culture seed liquid is LB culture medium, and shake flask culture conditions are as follows: LB culture medium is filled in the triangular flask, lactic acid producing strain glycerol tube is inoculated, and shake culture is carried out for 10-14h at 30-37 ℃ and 100-500 r/min.
The culture conditions of the seed tank culture seed solution are as follows: inoculating shake flask seeds into a seed tank of a liquid culture medium according to an initial 3-5% inoculum size, wherein the initial volume of the seed tank after inoculation is 25-60% of the working volume, controlling the temperature to be 30-37 ℃, maintaining the pH to be 6.8-7.5 by adding an alkaline neutralizing agent, controlling dissolved oxygen to be 20-60%, controlling ventilation to be 0.1-2.0vvm, and rotating at 200-1000r/min; when the concentration of the bacterial cells reaches an OD600 of 10-30, the bacterial cells are used as seed liquid to be inoculated into a fermentation tank.
The liquid medium is M9 medium, and the working volume of the seed tank is 40-60%, preferably 50-60%.
Controlling dissolved oxygen of the seed tank by 20-60%, preferably 20-40%, more preferably 20-30%;
the alkaline neutralizer is selected from one or more of calcium hydroxide, sodium hydroxide, magnesium hydroxide and ammonia water, preferably ammonia water.
The aeration gas is sterile air.
The temperature of the thallus growth stage in the step (1) is controlled to be 37-45 ℃, the pH is 6.8-7.5, the ventilation is 0.1-2.0vvm, the rotating speed is 200-1000r/min, and the dissolved oxygen is 10-30%;
the lactic acid fermentation process in the step (2) further comprises a process of maintaining the pH at 6.0-7.5 by adding an alkaline neutralizing agent.
The alkaline neutralizer is one or more of calcium hydroxide, sodium hydroxide, magnesium hydroxide and ammonia water, preferably calcium hydroxide.
The fermentation acidogenesis stage in step (2) further comprises a continuous fed-batch or fed-batch glucose process.
The culture conditions in the acidogenic stage in the step (2) are that the temperature is controlled to 37-50 ℃, preferably 37-45 ℃, more preferably 40-45 ℃ and the rotating speed is controlled to 100-500rpm, more preferably 100-300rpm.
The succinic acid-specific inhibitor is added at an OD600 of 15 to 45 in the step (2), more preferably 20 to 30.
The concentration of the specific inhibitor in the step (2) is 0.01-20mM, preferably 0.03-15mM.
Preferably, the combination of the plurality of inhibitors is a mixture of ethyl 3-nitropropionate, mercuric chloride and 5,5' -dithiobis (2-nitrobenzoic acid) with an action concentration of 0.01-0.05mM, preferably 0.03-0.05mM, and the composition ratio of the mixture is preferably (5-7): (2-3): (1-2).
The beneficial effects of the invention are as follows:
compared with the existing lactic acid production process, the invention adds the isocitrate lyase specific inhibitor in the lactic acid fermentation process, which not only does not affect the production of lactic acid, but also can effectively reduce the synthesis of succinic acid, and the inhibition rate can reach 60-100% at most.
Detailed Description
In order to further illustrate the beneficial effects of the present invention, the present invention will be described in detail with reference to the following examples, which are not to be construed as limiting the present invention.
The embodiment of the invention mainly comprises the following raw material information:
methyl glycolate was purchased from alfa under the designation a17870;
dimethyl malonate is available from CATO under the designation CCFD200172;
diethyl malonate was purchased from Sigma-Aldrich under the accession number 86320;
3-nitropropionic acid was purchased from Sigma-Aldrich under the designation N5636;
5,5' -dithiobis (2-nitrobenzoic acid) was purchased from Sigma-Aldrich under the designation D8310;
mercuric chloride was purchased from Shanghai test, cat No. 10013616;
the escherichia coli recombinant bacteria are purchased from China general microbiological culture Collection center, and the strain number is CGMCC 11059.
LB medium: 10g of peptone, 5g of yeast powder and 10g of NaCl, regulating the pH to 7.4 by using a 1M sodium hydroxide solution, fixing the volume to 1L by using deionized water, and sterilizing for 20min at 121 ℃;
m9 medium: 1M MgSO is firstly configured 4 ，MgSO 4 ·7H 2 Dissolving O2.46 g in 10ml deionized water, and sterilizing under high pressure for later use; configuration of 1M CaCl 2 ，CaCl 2 ·6H 2 Dissolving O2.191 g in 10ml deionized water, and autoclaving; then 5 XM 9 salt solution is prepared, na 2 PO 4 ·7H 2 O 12.8g、KH 2 PO 4 3.0g、NaCl 0.5g、NH 4 Cl 1.0g, 200ml deionized water is added for dissolution, and sterilization is carried out for 15min at 121 ℃; preparing 20% glucose solution, dissolving 4g glucose in 16ml deionized water, and filtering with 0.22 μm filter for sterilization; sterile Chamber 200ml of 5 XM 9 salt solution, 1M MgSO 4 2ml, 20% glucose solution 20ml, 1M CaCl 2 0.1ml of sterile deionized water was added to 1L.
Yeast powder and peptone were purchased from oxoid, and other reagents were purchased from national pharmaceutical systems chemical reagent Co.
1. Shake flask seed culture
50mL of LB culture medium is filled in a 250mL triangular flask, and a glycerol tube of escherichia coli recombinant bacteria (CGMCC 11059) is inoculated, and shake culture is carried out at 37 ℃ and 250r/min for 11h.
2. Seed tank culture test
Shake flask seeds were inoculated in 1.5l M9 liquid medium at an initial 5% inoculum size: first, 1M MgSO is configured 4 In the fermentation tank, the initial volume of the fermentation tank after inoculation is 25-60% of the working volume, the temperature is controlled at 37 ℃, the pH is maintained at 6.8-7.5, the dissolved oxygen is controlled at 20%, the ventilation is 0.1-2.0vvm, and the rotating speed is 200-1000r/min; when the cell concentration reached OD600 of 20, the cells were inoculated as seed liquid into a fermenter.
3. Fermentation test in fermenter
Seed liquid is inoculated into a fermentation tank containing an M9 liquid culture medium according to an inoculum size of 5%, the initial volume of the fermentation tank after inoculation is 25% -60% of the working volume, and fermentation is carried out according to a two-stage fermentation method (Tian Kangming et al, report of bioengineering, 29:111-114,2013). In the thallus growth stage of fermentation, the temperature is controlled at 37 ℃, the pH is maintained at 6.8-7.5, the ventilation is 0.1-2.0vvm, the rotating speed is 200-1000r/min, and the dissolved oxygen is controlled at 20%; when the concentration of the thalli reaches OD600 of 30, the thalli enters a lactic acid fermentation stage, the temperature is controlled at 42 ℃, the stirring rotation speed is adjusted to 200r/min, and 25wt% of calcium hydroxide suspension is fed in to maintain the pH value at 6.0-7.5. Glucose in the fermentation acid-producing stage adopts a continuous flow feeding or batch feeding mode, wherein in the batch feeding method, glucose solution with the final concentration of 6wt% is fed for four times, and the total feeding amount is 25% of the initial fermentation volume; in the fed-batch feeding method, the sugar concentration is maintained by controlling the acceleration of the sugar liquid flow, and the total sugar liquid amount is 25% of the initial fermentation volume.
4. Analysis of fermentation process
Sample preparation: taking 1mL of fermentation liquor to be detected, uniformly mixing the fermentation liquor with 50 mu L of 50% concentrated sulfuric acid, centrifuging for 10min at 8000r/min, absorbing a proper amount of supernatant, adding acetonitrile to a volume of 5mL, uniformly mixing, centrifuging for 5min at 10000r/min, diluting the supernatant with 5mM sulfuric acid, filtering the diluted supernatant by a 0.22 mu m organic microporous filter membrane, and analyzing and measuring related components.
(1) Glucose concentration determination: the glucose concentration of the sample was measured by using an SBA-40C type biosensor after the sample was diluted with deionized water, and the average value of the three parallel data was taken.
(2) OD detection, namely diluting fermentation liquor in a thallus growth stage by deionized water, OD600 detection, diluting fermentation liquor in a lactic acid fermentation stage by 1M hydrochloric acid, and OD600 detection.
(3) D-lactic acid, L-lactic acid and succinic acid content determination: HPLC was used and the chromatographic detection conditions were: the chromatographic column is an HPX-87H organic acid analysis column, the column temperature is 65 ℃, the detection wavelength is 210nm, the mobile phase is 5mM sulfuric acid solution, the flow rate is 0.8mL/min, and the sample injection amount is 10 mu L. All data are averages of 3 replicates.
Inhibition ratio = (N1-N2)/N1 × 100%
Wherein N1: succinic acid content of control group, N2: detecting the content of succinic acid
Example 1
Effect of methyl glycolate on lactic acid fermentation
Inoculating shake flask seed liquid of lactic acid production strain into 1.5L M9 liquid culture medium, culturing at 37deg.C for 7 hr/min, inoculating the culture liquid into M9 liquid culture medium with glucose concentration of 30g/L according to inoculum size of initial OD value of 0.3, controlling initial volume of 50L fermentation tank at 25L, temperature at 37deg.C, ventilation amount of 0.5-2.0vvm, stirring rotation speed of 200-1000r/min, controlling dissolved oxygen of 20%, and maintaining pH at 6.5-7.0 with ammonia water; when the cell concentration reaches to OD600 of 30, methyl glycolate with final concentration of 0-10mM is added, ventilation is closed, the stirring speed is 200r/min, the fermentation temperature is controlled to be 42 ℃, the total glucose (the total glucose added is about 240g/L based on the initial volume of fermentation) is added into a fermentation tank for 4 times, 25wt.% of calcium hydroxide suspension is fed into the fermentation tank to maintain pH at 7.0, and fermentation is finished after the concentration of residual sugar is lower than 0.2g/L before the fermentation is finished.
Under the fermentation condition, the total amount of fed-batch glucose is kept consistent, and the fermentation liquid is taken to detect the content of lactic acid and succinic acid.
TABLE 1 influence of methyl glycolate on lactic acid fermentation
| Concentration mM | Lactic acid g/L | Succinic acid content ppm | Inhibition rate% |
| 0 | 145 | 2000 | 0 |
| 1 | 145.33 | 1565 | 21.75 |
| 3 | 145.43 | 1435 | 28.25 |
| 5 | 145.76 | 1000 | 50 |
| 7 | 145.93 | 780 | 61 |
| 10 | 146.24 | 375 | 81.25 |
Example 2
Influence of dimethyl malonate on lactic acid fermentation
The lactic acid fermentation procedure was as in example 1, and when the cell concentration reached OD600 of 25, dimethyl malonate was added at a final concentration of 0-20 mM.
TABLE 2 influence of methyl malonate on lactic fermentation
| Concentration mM | Lactic acid g/L | Succinic acid content ppm | Inhibition rate% |
| 0 | 142 | 1820 | 0 |
| 10 | 142.43 | 1250 | 31.32 |
| 12 | 142.55 | 1105 | 39.29 |
| 15 | 142.83 | 732 | 59.78 |
| 18 | 142.90 | 645 | 64.56 |
| 20 | 142.96 | 560 | 69.23 |
Example 3
Effect of diethyl Hydroxymalonate on lactic fermentation
The lactic acid fermentation procedure was as in example 1, and when the cell concentration reached OD600 of 20, dimethyl malonate was added at a final concentration of 0-1 mM.
TABLE 3 Effect of diethyl Hydroxymalonate on lactic fermentation
| Concentration mM | Lactic acid g/L | Succinic acid content ppm | Inhibition rate% |
| 0 | 137.5 | 1750 | 0 |
| 0.2 | 137.90 | 1230 | 29.71 |
| 0.3 | 138.05 | 1025 | 41.43 |
| 0.5 | 138.30 | 700 | 60.00 |
| 0.7 | 138.56 | 355 | 79.71 |
| 1 | 138.57 | 345 | 80.29 |
Example 4
Effect of 3-nitropropionic acid on lactic acid fermentation
The lactic acid fermentation procedure was as in example 1, and when the cell concentration reached OD600 of 30, 0-0.1mM 3-nitropropionic acid was added as the final concentration.
TABLE 4 3 influence of nitropropionic acid on lactic acid fermentation
Example 5
Effects of mercuric chloride on lactic acid fermentation
The lactic acid fermentation procedure was as in example 1, and when the cell concentration reached an OD600 of 25, 0-0.1mM mercuric chloride was added as the final concentration.
TABLE 5 influence of mercuric chloride on lactic acid fermentation
| Concentration mM | Lactic acid g/L | Succinic acid content ppm | Inhibition rate% |
| 0 | 142 | 1820 | 0 |
| 0.01 | 142.13 | 1645 | 9.62 |
| 0.03 | 142.62 | 1005 | 44.78 |
| 0.05 | 142.85 | 705 | 61.26 |
| 0.07 | 142.91 | 630 | 65.38 |
| 0.1 | 142.99 | 522 | 71.32 |
Example 6
Effect of 5,5' -dithiobis (2-nitrobenzoic acid) on lactic acid fermentation
The lactic acid fermentation procedure was as in example 1, and when the cell concentration reached OD600 of 30, 5' -dithiobis (2-nitrobenzoic acid) was added at a final concentration of 0-0.1 mM.
TABLE 6 influence of 5,5' -dithiobis (2-nitrobenzoic acid) on lactic acid fermentation
| Concentration mM | Lactic acid g/L | Succinic acid content ppm | Inhibition rate% |
| 0 | 145 | 2000 | 0 |
| 0.01 | 145.48 | 1365 | 31.75 |
| 0.03 | 145.80 | 956 | 52.20 |
| 0.05 | 145.95 | 752 | 62.40 |
| 0.07 | 146.06 | 612 | 69.40 |
| 0.1 | 146.10 | 555 | 72.25 |
Example 7
Effect of combination C on lactic acid fermentation
The lactic acid fermentation procedure was as in example 1, and when the cell concentration reached an OD600 of 30, a final concentration of 0-0.05mM was added, combined with C, which was a mixture of ethyl 3-nitropropionate, mercuric chloride and 5,5' -dithiobis (2-nitrobenzoic acid) in a composition ratio of 5:3:2.
TABLE 7 influence of combination C on lactic acid fermentation
| Concentration mM | Lactic acid g/L | Succinic acid content ppm | Inhibition rate% |
| 0 | 145 | 2000 | 0 |
| 0.01 | 145.31 | 1593 | 20.35 |
| 0.02 | 145.58 | 1235 | 38.25 |
| 0.03 | 145.92 | 795 | 60.25 |
| 0.04 | 146.23 | 387 | 80.65 |
| 0.05 | 146.53 | 0 | 100 |
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art will appreciate that, in light of the principles of the present invention, improvements and modifications can be made without departing from the scope of the invention.
Claims (10)
1. A method for reducing succinic acid, a byproduct of lactic acid fermentation, comprising the steps of:
(1) Culturing lactic acid producing bacteria seed liquid, and inoculating the lactic acid producing bacteria seed liquid into a fermentation tank containing a culture medium for bacterial growth; (2) When the concentration of the thalli reaches an OD600 of 10-50, adding an isocitrate lyase specific inhibitor, and then entering an acidogenic stage for lactic acid fermentation; the specific inhibitor is one or a combination of methyl glycolate, dimethyl malonate, diethyl malonate, ethyl 3-nitropropionate, mercuric chloride or 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB).
2. The method of claim 1, wherein the combination of inhibitors is a mixture of methyl glycolate, dimethyl malonate, and mercuric chloride; or is a mixture of methyl glycolate, dimethyl malonate, diethyl malonate and ethyl 3-nitropropionate; a more preferred combination is a mixture of ethyl 3-nitropropionate, mercuric chloride, and 5,5' -dithiobis (2-nitrobenzoic acid).
3. The method according to claim 1 or 2, wherein the concentration of the specific inhibitor in step (2) is 0.01-20mM, preferably 0.03-15mM.
4. The method according to claim 2, wherein the mixture of the plurality of inhibitors ethyl 3-nitropropionate, mercuric chloride and 5,5' -dithiobis (2-nitrobenzoic acid) has a composition ratio of (5-7): 2-3): 1-2, at a concentration of 0.01-0.05mM, preferably 0.03-0.05mM.
5. The method of any one of claims 1-4, wherein the lactic acid producing species in step (1) is selected from one or more of the group consisting of escherichia coli recombinant bacteria, bacillus coagulans, bacillus subtilis, lactobacillus delbrueckii, lactobacillus bulgaricus, aspergillus niger, and yeast.
6. The method of any one of claims 1 to 5, wherein the temperature in the cell growth stage of step (1) is controlled to be 37-45 ℃, the pH is 6.8-7.5, the ventilation is 0.1-2.0vvm, the rotation speed is 200-1000r/min, and the dissolved oxygen is 10-30%.
7. The method of any one of claims 1 to 6, wherein the lactic acid fermentation process in step (2) further comprises a process of maintaining a pH of 6.0 to 7.5 by feeding an alkaline neutralizing agent; the alkaline neutralizer is one or more of calcium hydroxide, sodium hydroxide, magnesium hydroxide and ammonia water, preferably calcium hydroxide.
8. The method of any one of claims 1-7, wherein the fermentative acidogenic stage in step (2) further comprises a continuous fed-batch or fed-batch glucose process.
9. The method according to any one of claims 1 to 8, wherein the culture conditions in the acidogenic stage in step (2) are temperature controlled between 37 and 50 ℃, preferably between 37 and 45 ℃, more preferably between 40 and 45 ℃, and rotational speed controlled between 100 and 500rpm, more preferably between 100 and 300rpm.
10. The method of any one of claims 1-9, wherein the succinic acid-specific inhibitor is added in step (2) at an OD600 of 15-45, more preferably 20-30.
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