CN108034599B - One plant of Lactobacillus brevis for efficiently synthesizing γ-aminobutyric acid from brewed spirit system - Google Patents
One plant of Lactobacillus brevis for efficiently synthesizing γ-aminobutyric acid from brewed spirit system Download PDFInfo
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- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 title claims abstract description 101
- 229960003692 gamma aminobutyric acid Drugs 0.000 title claims abstract description 89
- 240000001929 Lactobacillus brevis Species 0.000 title claims abstract description 73
- 235000013957 Lactobacillus brevis Nutrition 0.000 title claims abstract description 71
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 title claims abstract description 12
- 230000002194 synthesizing effect Effects 0.000 title abstract description 7
- 230000001580 bacterial effect Effects 0.000 claims abstract description 51
- 238000000855 fermentation Methods 0.000 claims abstract description 47
- 230000004151 fermentation Effects 0.000 claims abstract description 47
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 26
- 235000013923 monosodium glutamate Nutrition 0.000 claims abstract description 26
- 229940073490 sodium glutamate Drugs 0.000 claims abstract description 25
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 17
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 17
- 230000012010 growth Effects 0.000 claims abstract description 13
- 235000013305 food Nutrition 0.000 claims abstract description 11
- 239000000758 substrate Substances 0.000 claims abstract description 11
- 244000005700 microbiome Species 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims abstract description 4
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 241000894006 Bacteria Species 0.000 claims description 33
- 239000003795 chemical substances by application Substances 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 13
- 230000000813 microbial effect Effects 0.000 claims description 11
- 239000012530 fluid Substances 0.000 claims description 10
- 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 8
- 239000003814 drug Substances 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 7
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 6
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 6
- 235000013922 glutamic acid Nutrition 0.000 claims description 6
- 239000004220 glutamic acid Substances 0.000 claims description 6
- 229940079593 drug Drugs 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 239000008101 lactose Substances 0.000 claims description 3
- 244000144972 livestock Species 0.000 claims description 3
- 239000002243 precursor Substances 0.000 claims description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- 230000003197 catalytic effect Effects 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- 239000002068 microbial inoculum Substances 0.000 claims description 2
- 244000144977 poultry Species 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 241000193830 Bacillus <bacterium> Species 0.000 claims 1
- 239000003054 catalyst Substances 0.000 claims 1
- 239000006071 cream Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 17
- 239000000047 product Substances 0.000 abstract description 6
- 238000012216 screening Methods 0.000 abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 3
- 150000004965 peroxy acids Chemical class 0.000 abstract description 3
- 239000006052 feed supplement Substances 0.000 abstract description 2
- 230000001186 cumulative effect Effects 0.000 abstract 1
- 230000002906 microbiologic effect Effects 0.000 abstract 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 32
- 239000004310 lactic acid Substances 0.000 description 16
- 235000014655 lactic acid Nutrition 0.000 description 16
- 239000002609 medium Substances 0.000 description 14
- 102000008214 Glutamate decarboxylase Human genes 0.000 description 10
- 108091022930 Glutamate decarboxylase Proteins 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- MKYPKZSGLSOGLL-LURJTMIESA-N 4-(L-gamma-glutamylamino)butanoic acid Chemical compound OC(=O)[C@@H](N)CCC(=O)NCCCC(O)=O MKYPKZSGLSOGLL-LURJTMIESA-N 0.000 description 3
- 102000003669 Antiporters Human genes 0.000 description 3
- 108090000084 Antiporters Proteins 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- OHVGNSMTLSKTGN-BTVCFUMJSA-N [C].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O Chemical compound [C].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O OHVGNSMTLSKTGN-BTVCFUMJSA-N 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 3
- 239000000796 flavoring agent Substances 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000011953 bioanalysis Methods 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000007979 citrate buffer Substances 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000000284 resting effect Effects 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000001694 spray drying Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 235000020097 white wine Nutrition 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 240000006024 Lactobacillus plantarum Species 0.000 description 1
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920001007 Nylon 4 Polymers 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 230000002378 acidificating effect Effects 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
- 229940124277 aminobutyric acid Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
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- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 229940072205 lactobacillus plantarum Drugs 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 235000021404 traditional food Nutrition 0.000 description 1
- 238000012549 training Methods 0.000 description 1
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Classifications
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- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
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- 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
- C12N1/20—Bacteria; Culture media therefor
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- 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
- C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/121—Brevis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
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- 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
- C12R2001/225—Lactobacillus
- C12R2001/24—Lactobacillus brevis
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
The invention discloses the Lactobacillus brevis for efficiently synthesizing γ-aminobutyric acid that one plant is originated from brewed spirit system, belong to food microorganisms technical field.Lactobacillus brevis bacterial strain D17 of the invention is isolated from traditional liquor brewing system, is preserved in China General Microbiological preservation administrative center, deposit number CGMCCNO.14385 on July 6th, 2017.Lactobacillus brevis D17 bacterial strain screening of the invention is in brewed spirit system, the environment such as the tolerable high alcohol of peracid, GAD system can be induced quickly by sodium glutamate, and the GABA synthesis of bacterial strain is coupled with growth, thallus itself can maintain vigour in the substrate and product of high concentration, therefore have the ability for efficiently synthesizing GABA.Using the bacterial strain, using sour feed supplement method batch fermentation is controlled, GABA cumulative concentration reaches 132.63g/L, production efficiency 3.16g/L/h.Lactobacillus brevis D17 bacterial strain in the present invention can be used for high-purity GABA preparation, feed grade GABA preparation etc. because of the source attribute of ability and pure natural bacterial strain with Efficient Conversion sodium glutamate synthesis GABA.
Description
Technical field
The present invention relates to the Lactobacillus brevis for efficiently synthesizing γ-aminobutyric acid that one plant is originated from brewed spirit system, belong to food
Product microorganisms technical field.
Background technique
γ-aminobutyric acid (GABA) is a kind of nonprotein amino acid, participates in the multinomial physiology of animal, plant and microorganism
Activity.As a kind of important inhibitory neurotransmitter, GABA, which has mammalogy, to calm the nerves, reduces blood pressure, improving sleep
Etc. different physiological roles, can be used as bioactive substance and be widely used in the industries such as food, medicine and feed, be furthermore alternatively arranged as
Synthesize the precursor of the chemical products such as 2-Pyrrolidone, nylon -4.Therefore, GABA has a vast market application prospect.
It is obtained currently, food-grade GABA multi-pass crosses two methods of natural product extraction or Microbe synthesis, wherein microorganism
Synthesis has broader practice prospect because can achieve higher generation efficiency and purity.Microbe fermentation method prepares GABA
Mainly completed by the glutamic acid decarboxylase enzyme system (GAD system) of microorganism.GAD system is mainly by glutamate decarboxylase
GAD and Glu-GABA antiporter protein is constituted, and in acid condition, Glu-GABA antiporter protein is by extracellular glutamic acid
It is transported to intracellular, sloughs α-carboxyl under glutamate decarboxylase (GAD) catalysis intracellular, form GABA, the GABA after synthesis is again
It is transported by Glu-GABA antiporter protein to extracellular, process consumption H+, the pH of cultivating system is gradually in GABA synthesis process
It increases.
Lactic acid bacteria is the important sources of food-grade production bacterial strain, with the food-safe attention of people, more and more
Researcher focus on utilizes lactic acid bacteria production GABA, it has now been found that the microorganism that can synthesize GABA include Lactobacillus brevis
With lactobacillus plantarum etc..According to reported Lactobacillus brevis (14 plants) genome analysis, 13 plants of Lactobacillus brevis have GAD system,
Only 1 plant, without GAD system, illustrates that Lactobacillus brevis has the potential for the cell factory for becoming GABA production.
Generally acknowledged high yield GABA Lactobacillus brevis is L.brevis NCL912 at present, and GABA yield is up to 103g/L, bacterial strain hair
Ferment 12h cell concentration reaches maximum, but with the addition of substrate and the generation of product, the growth of bacterial strain is heavily suppressed, nothing
Method realizes that bacterial strain continuously ferments production, and entire fermentation period is 48~60h, and production efficiency is not high；Lactobacillus brevis NPS-QW-145
Ferment 72h, and GABA yield is only 25.8g/L；Lactobacillus brevis K203 fermentation 72h, GABA yield are 44.4g/L, and GABA is produced
For raw bacterium, fermentation period is longer, and the combined coefficient of the GABA of bacterial strain is lower.Therefore, the bacterium of GABA is efficiently synthesized to obtain
Strain, the GABA synthesis capability for not requiring nothing more than bacterial strain is strong, while fermentation period will also reduce.Although the GABA of some lactic acid bacterias is closed
It can be coupled at growth, but its GABA combined coefficient is still very low.
It is, thus, sought for one plant of energy rapid induction GAD system, generates the lactic acid bacteria of GABA, and can be for a long time hypertonic
Survival in environment, continuously fermenting for it synthesizes GABA and provides condition, reduces fermentation time, bacterial strain is made to have high yield GABA ability
While, GABA production efficiency with higher.(Wu,Q.Shah,N.P."High gamma-aminobutyric acid
production from lactic acid bacteria:emphasis on Lactobacillus brevis as a
functional dairy starter”,Critical Review in Food Science and Nutrition,1,1-
46,2016.Haixing Li,Yusheng Cao,et al.“Production of gamma-aminobutyric acid
by Lactobacillus brevis NCL912 using fed-batch fermentation”Microbial Cell
Factories,9,1-7,2010.Wu,Q.,Shah,N.P.“Gas release-based prescreening combined
with reversed-phase HPLC quantitation for efficient selection of high-gamma-
aminobutyric acid(GABA)-producing lactic acid bacteria”,Journal of Dairy
Science,2015,98,790-797.)。
Traditional food of the white wine as China, is rich in lactic acid bacteria in fermentation system, and lactic acid bacteria strains can long-term surviving in
In the peracid (pH is 3.5 or so) of brewed spirit system, the extreme environment of high alcohol content (50-90g/L), therefore, it is necessary to from
Screening has food-borne bacterial strain that is resistance to hypertonic and efficiently synthesizing GABA in traditional fermented food (white wine), promotes microbe fermentation method
Synthesize the industrialization of GABA.
Summary of the invention
In view of the prior art difficult point and there are the problem of, the present invention provides one plant have efficiently synthesize GABA ability
Lactobacillus brevis, and it is applied to bioanalysis synthesis GABA.Lactobacillus brevis of the invention is from brewed spirit system (fermented grain)
Isolated, for the yield for carrying out controlling sour fed batch fermentation 42h synthesis GABA up to 132.63g/L, production efficiency is reachable
3.16g/L/h。
Have the first purpose of the invention is to provide one plant and efficiently synthesize the Lactobacillus brevis D17 of GABA ability, in
On July 6th, 2017 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC
NO.14385。
The Lactobacillus brevis is using lactic acid bacteria enriched medium (MRS culture medium), from brewed spirit system (fermented grain)
Isolated, Lactobacillus brevis (Lactobacillus brevis), strain number D17 are accredited as through 16s rDNA sequencing.
A second object of the present invention is to provide the microbial bacterias for containing the Lactobacillus brevis CGMCC NO.14385 bacterial strain
Agent.
In one embodiment of the invention, the microbial bacterial agent contains Lactobacillus brevis CGMCC NO.14385 thallus
Living cells, freeze-drying obtain Lactobacillus brevis CGMCC NO.14385 dry mycelium, immobilization Lactobacillus brevis CGMCC
The solid bacterium of NO.14385 cell, the liquid bacterial agent of Lactobacillus brevis CGMCC NO.14385, Lactobacillus brevis CGMCC NO.14385
Agent, or in the form of other are any existing for Lactobacillus brevis CGMCC NO.14385 bacterial strain.
In one embodiment of the invention, food, feeding also be can be applied to containing any in the microbial bacterial agent
The bacterial strain, such as bacillus licheniformis, saccharomyces cerevisiae, bacillus subtilis of any kind etc. of material, drug or its preparation.
In one embodiment of the invention, food, feed, medicine can be also used for containing any in the microbial bacterial agent
The carrier of object.
It is to utilize short lactic acid of the invention third object of the present invention is to provide the method for bioanalysis synthesis GABA a kind of
Bacterium CGMCC NO.14385 bacterial strain is produced.
In one embodiment of the invention, the method is with glucose, sucrose, lactose or arabinose for growth
Carbon source synthesizes γ-aminobutyric acid using glutamic acid or sodium glutamate as precursor.
In one embodiment of the invention, the method is carried out using Lactobacillus brevis CGMCC NO.14385 bacterial strain
Batch fermentation, fermentation process do not need to carry out pH value adjusting and anaerobism control.In one embodiment of the invention, specifically
It is: Lactobacillus brevis CGMCC NO.14385 is inoculated in the GYP liquid fermentation medium of different glucose carbon source, adds
The substrate that glutamic acid or sodium glutamate not higher than 50g/L are synthesized as GABA, wherein concentration of glucose is 10~50g/L, in
It is suitble to ferment under the condition of culture of Lactobacillus brevis.
In one embodiment of the invention, the method includes carrying out during the fermentation to fermentation system pH value
Control.
In one embodiment of the invention, the method is carried out using Lactobacillus brevis CGMCC NO.14385 bacterial strain
Fed-batch fermentation, fermentation process carry out pH value adjusting, do not need to carry out anaerobism control.In one embodiment of the present invention
In, specifically: using bioreactor, Lactobacillus brevis CGMCC NO.14385 is inoculated in the fermentation training of the GYP containing sodium glutamate
It supports in base, uses the sulfuric acid of 5mol/L concentration by the pH of fermentation liquid control for 5.0, in 37 DEG C × 100rpm stirring stuffiness condition
Under ferment.To avoid inhibition of the high concentration of glutamate sodium to thalli growth, in 12~for 24 hours between supplement sodium glutamate substrate and
Glucose carbon source.Preferably, sodium glutamate substrate and glucose addition are carried out in 6h, 12h, 18h and for 24 hours, GABA is in fermentation 42h
Concentration be 132.63g/L, production efficiency is up to 3.16g/L/h at this time.
In one embodiment of the invention, the method includes first being cultivated thallus, urged using thallus as full cell
Agent, using glutamic acid or sodium glutamate as substrate, carries out whole-cell catalytic and synthesizes γ-in suitable pH value conversion fluid
Aminobutyric acid.
In one embodiment of the invention, the method is that first culture acquisition Lactobacillus brevis CGMCC NO.14385 is thin
Born of the same parents recycle resting cell method.In one embodiment of the invention, specifically: picking Lactobacillus brevis CGMCC
NO.14385 is connected in the GYP liquid fermentation medium of sodium glutamate of the GYP containing 10g/L, and 37 DEG C × 200rpm cultivates 12h, is received
Collect thallus.Wet thallus is taken, the Na of the 0.2mol/LpH4.6 of 30~50g/L sodium glutamate is put into2HPO4In citrate buffer solution,
1.5~5h is reacted under the conditions of 30 DEG C × 200rpm.Preferably, conversion reaction system is 40mL, initial concentration of sodium glutamate
1g wet thallus is added in 30g/L, converts to obtain GABA 14.5g/L, and transformation efficiency is the wet bacterium of 9.7g/L/h/g.
Fourth object of the present invention is to provide a kind of preparation method of dry bacterium powder containing GABA, including the use of of the invention short
Lactic acid bacteria CGMCC NO.14385 bacterial strain.
In one embodiment of the invention, which comprises take out Lactobacillus brevis bacterial strain CGMCC NO.14385
Fermentation liquid (GABA content is not less than 20~25g/L) after fermentation, using 60~65 DEG C of 1~3h of concentration, total solid contains
It is spray-dried to get the dry bacterium powder containing GABA after amount reaches 30~40%.Spray drying condition are as follows: feeding temperature
30~40 DEG C, 140~150 DEG C of inlet temperature, 50~60 DEG C of outlet temperature, 0.25~0.3MPa of inlet pressure, centrifugal turntable turns
19000~20000r/min of speed, drying time are 5~15s.Product discharge moisture content≤7% is obtained, is sealed after cooling.
The present invention also provides the Lactobacillus brevis CGMCC NO.14385 in food, prepares drug or livestock and poultry cultivation field
Application.
Beneficial effects of the present invention:
In the fermented grain of Lactobacillus brevis CGMCC NO.14385 of the invention screened from fen-flavor type white spirit brewing system, have resistance to
By the characteristic of the brewing environment of peracid (pH be 3.5 or so), high alcohol content (50-90g/L) in fen-flavor type white spirit preparation process,
Growth can be kept for a long time in extreme environment, the high vigor microbial inoculum of preparation can be applied to the industries such as food, medicine, livestock culture；
The GAD system of bacterial strain can be induced quickly by sodium glutamate, and the GABA synthesis of bacterial strain is synchronous with thalli growth to be carried out, and highly concentrated
It maintains vigour in the environment of degree substrate, product and sulfuric acid, generates GABA for continuous culture bacterial strain and foundation is provided, GABA production can be improved
Shorten fermentation period while amount, further promotes the production efficiency of the GABA of bacterial strain.
Biomaterial preservation
Lactobacillus brevis D17, taxology are named as Lactobacillus brevis Lactobacillus brevis, protect on July 6th, 2017
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.14385.
Detailed description of the invention
The colony morphology characteristic figure of Fig. 1 Lactobacillus brevis D17；
The morphological features figure of Fig. 2 Lactobacillus brevis D17；
Fig. 3 sodium glutamate standard items and GABA standard items high performance liquid chromatography detection figure；
Fig. 4 bacterium fermentation liquid GABA high performance liquid chromatography detection figure；
Fig. 5 Lactobacillus brevis D17 bacterial strain GABA synthesis is coupled with growth；
Fig. 6 Lactobacillus brevis D17 fed batch fermentation efficiently synthesizes the strain growth of GABA, substrate utilizes and GABA generates song
Line.
Specific embodiment
Embodiment 1: the screening of Lactobacillus brevis
5g fen-flavor type white spirit fermented grain raw material is weighed, 50mL sterile saline is added and is shaken.Bacteria suspension is drawn, with nothing
Bacterium physiological saline carries out 10 times of gradient dilutions, and 100-200 μ L is taken to be coated on containing CaCO3With MC culture medium (the pancreas egg of sodium glutamate
White peptone 0.5%, beef extract 0.5%, yeast extract 0.5%, glucose 2%, lactose 2%, calcium carbonate 1%, sodium glutamate
1%, agar 2% is quality-volume fraction, pH6.8) or MRS culture medium (Sigma-Aldrich purchase) plate on, in 37
DEG C Anaerobic culturel 48h, when there is milky or light yellow bacterium colony, and occurring hydrolysis circle person preliminary judgement around it is lactic acid bacteria.
Picking single colonie is separated, after purification, and being inoculated with packing has in the test tube of MRS fluid nutrient medium, is inoculated in after being incubated overnight
In GYP liquid fermentation medium containing sodium glutamate, with the GABA content in HPLC method detection fermented supernatant fluid, to acquisition
GABA producing bacterial strain carries out guarantor bacterium.The results show that 65 strains of lactic acid bacteria are filtered out from brewed spirit system, and after the 48h that ferments, measurement
GABA concentration in fermentation liquid, wherein 9 strains of lactic acid bacteria GABA yield are greater than the bacterial strain of 5g/L.By GABA strain inoculated in GYP liquid
In body fermentation medium, every 2h sampling is filtered out using the GABA and sodium glutamate substrate content in HPLC analysis fermentation supernatant
Fermentation time is short, can efficiently synthesize the lactic acid bacteria of GABA, and screening obtains the Lactobacillus brevis bacterial strain that number is D17, fermentation medium
Middle starting concentration of sodium glutamate 10g/L, ferment 12h, and 6.5g/L GABA, production efficiency 0.542g/L/h can be obtained.
Obtained Lactobacillus brevis D17, colonial morphology and thalli morphology are as shown in Figure 1 and Figure 2.Lactobacillus brevis D17 in
On July 6th, 2017 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC
NO.14385。
Embodiment 2: acid synthesis GABA is not controlled using Lactobacillus brevis D17 bacterial strain
GYP culture medium: glucose 1%, yeast extract 1%, peptone 0.5%, sodium acetate 0.2%, magnesium sulfate 0.02%, sulphur
Sour manganese 0.01%, ferrous sulfate 0.01%, sodium chloride 0.01% are quality-volume fraction.2% is added in solid medium
The agar of concentration adds various concentration sodium glutamate in fermentation medium.
Lactobacillus brevis is oxygen tolerance type lactic acid bacteria, and growth under the conditions of supporting well is better than Anaerobic culturel, therefore recommends
Lactobacillus brevis D17 bacterial strain is cultivated under the conditions of supporting well.
Aseptically, the Lactobacillus brevis D17 bacterial strain -80 DEG C saved, lines on GYP solid plate, 37 DEG C quiet
Culture is set, from picking Lactobacillus brevis D17 single colonie on the GYP solid plate of activation after single colonie is grown, is inoculated in GYP seed
In culture medium, 37 DEG C of stationary cultures are for 24 hours.Seed culture fluid is taken, new GYP seed culture medium is inoculated in by 10% inoculum concentration
In, after 37 DEG C of stationary culture 12h, culture solution is as fermentation seed.In 250mL triangular flask be packed into 100mL addition 10~
The GYP fermentation medium of 50g/L glucose carbon source and 50g/L sodium glutamate, take culture 12h fermentation seed by 10% inoculation
Amount inoculation cultivates 48h in 37 DEG C × 200rpm.Fermented supernatant fluid is through high performance liquid chromatography detection GABA content.
Experimental result is as shown in figure 5, the GABA synthesis of Lactobacillus brevis D17 is coupled with growth.Using 30g/L glucose conduct
Carbon source, it is 27.6g/L, production efficiency 0.863g/L/h that Lactobacillus brevis, which synthesizes GABA yield,.
Embodiment 3: it carries out controlling sour fed batch fermentation synthesis GABA using Lactobacillus brevis D17 bacterial strain
The bacterial strain D17 that will be activated on inclined-plane, is inoculated in GYP fluid nutrient medium, and for 24 hours, first order seed is made in 37 DEG C of cultures
First order seed culture solution is pressed 10% inoculum concentration by culture solution, is connected to the GYP fermentation medium that pH is 5 sodium glutamates containing 74.8g/L
In, using bioreactor control 37 DEG C of temperature, 200rpm stirring, with 5mol/L sulfuric acid control pH be 5.0,6h, 12h,
18h, for 24 hours when each feed supplement (sodium glutamate) 37.3g, 74.8g, 37.3g, 37.3g；Simultaneously in 12h, 18h, for 24 hours when respectively add carbon
Source (glucose) 5g, 7.5g, 15g, ferment 48h.Using the GABA content of HPLC analysis detection fermented supernatant fluid.
As a result as shown in fig. 6, the GABA synthesis of Lactobacillus brevis D17 bacterial strain is coupled with growth, in entire fermentation process, bacterium
Strain has vigor always, and cell concentration is stepped up, and illustrates that bacterial strain can be kept in the substrate, product and acidic environment of high concentration
The ability for efficiently synthesizing GABA, when fermenting 42h, the concentration of GABA is 132.63g/L, and production efficiency is up to 3.16g/L/h.
Embodiment 4: using first cultivation, resting cell method synthesizes GABA to Lactobacillus brevis D17 bacterial strain again
From picking Lactobacillus brevis D17 single colonie in the GYP solid slope culture medium of activation, it is inoculated in GYP fluid nutrient medium
In, for 24 hours, primary seed solution is made in 37 DEG C of stationary cultures, and primary seed solution is inoculated in fresh GYP liquid by 10% inoculum concentration
In body culture medium, after 37 DEG C of stationary culture 12h, fermentation seed is made.500mL is packed into the shaking flask of 1L containing 1% sodium glutamate
GYP fluid nutrient medium takes the fermentation seed of culture 12h, is inoculated with, 37 DEG C × 200rpm by 10% inoculum concentration, cultivates 12h.It takes
Fermentation liquid out is centrifuged 10min in 4 DEG C × 6000rpm, collects thallus.1g wet thallus is resuspended in containing 30g/L sodium glutamate
The Na of 0.2M pH4.62HPO4Citrate buffer solution 40mL reacts 1.5h under the conditions of 30 DEG C × 200rpm.In final conversion fluid
GABA content be 14.448g/L, production efficiency be 9.7g/L/h/g wet thallus.
Embodiment 5: the dry bacterium powder containing GABA is prepared using Lactobacillus brevis D17
The fermentation liquid of Lactobacillus brevis D17 after fermentation is concentrated, concentration condition be 60 DEG C, 1.5h, concentrate into
Row spray drying processing, input concentration 40%, 35 DEG C of feeding temperature, 140 DEG C of inlet temperature, 55 DEG C of outlet temperature, inlet gas
Press 0.3MPa, centrifugal turntable revolving speed preservation 20000r/min.
Dry bacterium powder yield after drying containing GABA are as follows: 15%, moisture content 5%.Viable count is up to 6.0 × 109Cfu/g,
GABA content >=20%.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. the Lactobacillus brevis (Lactobacillus brevis) of one plant of synthesis γ-aminobutyric acid, in preservation on July 6 in 2017
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.14385.
2. a kind of microbial bacterial agent, which is characterized in that the microbial bacterial agent contains Lactobacillus brevis CGMCC NO.14385 thallus
Living cells, freeze-drying obtain Lactobacillus brevis CGMCC NO.14385 dry mycelium, immobilization Lactobacillus brevis CGMCC
The solid bacterium of NO.14385 cell, the liquid bacterial agent of Lactobacillus brevis CGMCC NO.14385, Lactobacillus brevis CGMCC NO.14385
Agent, or in the form of other are any existing for Lactobacillus brevis CGMCC NO.14385 bacterial strain.
3. microbial bacterial agent according to claim 2, which is characterized in that in the microbial bacterial agent also containing it is any can
Bacterium applied to food, feed, drug or any kind of its preparation.
4. microbial bacterial agent according to claim 2, which is characterized in that can also be used containing any in the microbial bacterial agent
In the carrier of food, feed or drug.
5. a kind of method for preparing γ-aminobutyric acid, which is characterized in that the method is to utilize short cream described in claim 1
Bacillus.
6. according to the method described in claim 5, it is characterized in that, the method is with glucose, sucrose, lactose or Arab
Sugar is growth carbon source, synthesizes γ-aminobutyric acid using glutamic acid or sodium glutamate as precursor.
7. according to the method described in claim 5, it is characterized in that, the method includes during the fermentation to fermentation system acid
Basicity is controlled.
8. according to the method described in claim 5, it is characterized in that, the method includes, first cultivate thallus, using thallus as
Whole-cell catalyst, using glutamic acid or sodium glutamate as substrate, carries out whole-cell catalytic in suitable pH value conversion fluid
Synthesize γ-aminobutyric acid.
9. a kind of application of the microbial inoculum containing claim 1 bacterial strain, which is characterized in that the application includes by Lactobacillus brevis
The dry powder of CGMCC NO.14385 is added in feed.
10. Lactobacillus brevis described in claim 1 in food, prepare the application of drug or livestock and poultry cultivation field.
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| CN110241061B (en) * | 2019-06-06 | 2021-03-02 | 江南大学 | Method for improving synthesis capacity of lactobacillus brevis gamma-aminobutyric acid and application thereof |
| CN112300955A (en) * | 2019-07-29 | 2021-02-02 | 台湾中油股份有限公司 | Gamma-aminobutyric acid high-yield lactic acid bacterial strain and application thereof |
| CN111471724A (en) * | 2020-05-09 | 2020-07-31 | 济南大学 | Method for whole-cell transformation production of food-grade gamma-aminobutyric acid |
| CN111778191B (en) * | 2020-07-17 | 2021-05-14 | 鲁东大学 | Lactobacillus brevis strain and culture method and application thereof |
| CN113444664B (en) * | 2021-07-01 | 2023-12-12 | 陕西广播电视大学（陕西工商职业学院） | Lactobacillus brevis for producing gamma-aminobutyric acid and application thereof |
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Application publication date: 20180515 Assignee: Guangdong Jianlibao Co.,Ltd. Assignor: Jiangnan University Contract record no.: X2024980001407 Denomination of invention: Efficient synthesis of a strain from Baijiu brewing system g- Lactobacillus brevis for aminobutyric acid Granted publication date: 20190917 License type: Common License Record date: 20240125 |