CN108034599A - One plant of Lactobacillus brevis for efficiently synthesizing γ-aminobutyric acid for being derived from brewed spirit system - Google Patents
One plant of Lactobacillus brevis for efficiently synthesizing γ-aminobutyric acid for being derived from brewed spirit system Download PDFInfo
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- CN108034599A CN108034599A CN201711275417.9A CN201711275417A CN108034599A CN 108034599 A CN108034599 A CN 108034599A CN 201711275417 A CN201711275417 A CN 201711275417A CN 108034599 A CN108034599 A CN 108034599A
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- lactobacillus brevis
- gaba
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- aminobutyric acid
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- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 title claims abstract description 102
- 229960003692 gamma aminobutyric acid Drugs 0.000 title claims abstract description 90
- 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 13
- 230000002194 synthesizing effect Effects 0.000 title abstract description 6
- 230000001580 bacterial effect Effects 0.000 claims abstract description 51
- 238000000855 fermentation Methods 0.000 claims abstract description 40
- 230000004151 fermentation Effects 0.000 claims abstract description 40
- 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 25
- 229940073490 sodium glutamate Drugs 0.000 claims abstract description 20
- 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
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims abstract description 9
- 244000005700 microbiome Species 0.000 claims abstract description 9
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims abstract description 4
- 241000894006 Bacteria Species 0.000 claims description 37
- 239000012530 fluid Substances 0.000 claims description 16
- 239000003795 chemical substances by application Substances 0.000 claims description 15
- 230000000813 microbial effect Effects 0.000 claims description 12
- 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
- 239000007788 liquid Substances 0.000 claims description 7
- 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
- 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
- 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
- 210000000481 breast Anatomy 0.000 claims 1
- 239000003054 catalyst Substances 0.000 claims 1
- 230000003197 catalytic effect Effects 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 17
- 239000000047 product Substances 0.000 abstract description 6
- 238000002360 preparation method Methods 0.000 abstract description 5
- 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
- 102000008214 Glutamate decarboxylase Human genes 0.000 description 10
- 108091022930 Glutamate decarboxylase Proteins 0.000 description 10
- 235000015097 nutrients Nutrition 0.000 description 7
- 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
- 239000002609 medium Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 239000002054 inoculum Substances 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
- 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
- 238000011081 inoculation 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
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 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
- 238000005516 engineering process Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 239000001963 growth medium Substances 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
- 238000011218 seed culture Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 235000002639 sodium chloride Nutrition 0.000 description 2
- 238000001694 spray drying 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
- 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
- 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
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000007036 catalytic synthesis reaction Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 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
- 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
- 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
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001629 suppression Effects 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
- 230000009466 transformation Effects 0.000 description 1
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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- 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
- C12P13/005—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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- 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
- C12R2001/225—Lactobacillus
- C12R2001/24—Lactobacillus brevis
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- Life Sciences & Earth Sciences (AREA)
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- Engineering & Computer Science (AREA)
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- Microbiology (AREA)
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- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Polymers & Plastics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Nutrition Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses the Lactobacillus brevis for efficiently synthesizing gamma aminobutyric acid that one plant is derived from brewed spirit system, belong to food microorganisms technical field.The Lactobacillus brevis bacterial strain D17 of the present 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.The Lactobacillus brevis D17 bacterial strain screenings of the present invention are in brewed spirit system, the environment such as the high alcohol of tolerable peracid, GAD systems can be induced quickly by sodium glutamate, and the GABA synthesis of bacterial strain is coupled with growth, thalline itself can maintain vigour in the substrate and product of high concentration, therefore with the ability for efficiently synthesizing GABA.Using the bacterial strain, using the sour feed supplement method batch fermentation of control, GABA cumulative concentrations reach 132.63g/L, production efficiency 3.16g/L/h.Lactobacillus brevis D17 bacterial strains in the present invention are because of ability and the source attribute of pure natural bacterial strain with Efficient Conversion sodium glutamate synthesis GABA, available for high-purity GABA preparations, feed grade GABA preparations etc..
Description
Technical field
The present invention relates to the Lactobacillus brevis for efficiently synthesizing γ-aminobutyric acid that one plant is derived from brewed spirit system, belong to food
Product microbial technology field.
Background technology
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, the industries such as food, medicine and feed can be widely used in as bioactive substance, be alternatively arranged as in addition
Synthesize the precursor of the chemical products such as 2-Pyrrolidone, nylon -4.Therefore, GABA has a vast market application prospect.
At present, food-grade GABA is more is obtained by two methods of natural product extraction or Microbe synthesis, wherein microorganism
Synthesize because higher generation efficiency and purity can be reached, there is broader practice prospect.Microbe fermentation method prepares GABA
Mainly completed by the glutamic acid decarboxylase enzyme system (GAD systems) of microorganism.GAD systems are mainly by glutamate decarboxylase
GAD and Glu-GABA antiporter proteins are formed, and in acid condition, Glu-GABA antiporter proteins are by extracellular glutamic acid
Intracellular is transported to, α-carboxyl is sloughed under glutamate decarboxylase (GAD) catalysis of intracellular, forms GABA, the GABA after synthesis is again
Transported by Glu-GABA antiporter proteins to extracellular, process consumption H+, the pH of cultivating system is gradual in GABA building-up processes
Rise.
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 Lactobacillus brevis (14 plants) genome analysis reported, 13 plants of Lactobacillus brevis have GAD systems,
Only 1 plant, without GAD systems, illustrates that Lactobacillus brevis has the potential for the cell factory for becoming GABA productions.
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 concentrations reach 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 whole 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 is 44.4g/L, is produced for GABA
For raw bacterium, fermentation period is longer, and the combined coefficient of the GABA of bacterial strain is relatively low.Therefore, the bacterium of GABA is efficiently synthesized to obtain
Strain, it is strong not require nothing more than the GABA synthesis capabilities of bacterial strain, while fermentation period will also reduce.Although the GABA of some lactic acid bacterias is closed
It can be coupled into growth, but its GABA combined coefficient is still very low.
It is, thus, sought for one plant can rapid induction GAD systems, produce the lactic acid bacteria of GABA, and can be for a long time hypertonic
Survival in environment, provides condition for its synthesis GABA that continuously ferments, reduces fermentation time, bacterial strain is had high yield GABA abilities
While, there is higher GABA production efficiencys.(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.
The content of the invention
For existing technological difficulties and there are the problem of, the present invention provides one plant have efficiently synthesize GABA abilities
Lactobacillus brevis, and it is applied to bioanalysis synthesis GABA.The Lactobacillus brevis of the present invention is from brewed spirit system (fermented grain)
Isolated, the yield for control sour fed batch fermentation 42h synthesis GABA is reachable up to 132.63g/L, production efficiency
3.16g/L/h。
First purpose of the present invention, which is to provide one plant and has, efficiently synthesizes the Lactobacillus brevis D17 of GABA abilities, 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 to utilize lactic acid bacteria enriched medium (MRS culture mediums), from brewed spirit system (fermented grain)
It is isolated, it is Lactobacillus brevis (Lactobacillus brevis) through 16s rDNA sequencing identifications, strain number D17.
Second object of the present invention is to provide the microbial bacteria containing the Lactobacillus brevis CGMCC NO.14385 bacterial strains
Agent.
In one embodiment of the invention, the microbial bacterial agent contains Lactobacillus brevis CGMCC NO.14385 thalline
Living cells, freeze-drying obtained Lactobacillus brevis CGMCC NO.14385 dry myceliums, the Lactobacillus brevis CGMCC of immobilization
NO.14385 cells, the liquid bacterial agent of Lactobacillus brevis CGMCC NO.14385, the solid bacterium of Lactobacillus brevis CGMCC NO.14385
Agent, or Lactobacillus brevis CGMCC NO.14385 bacterial strains existing in the form of other are any.
In one embodiment of the invention, food, feeding also be can be applied to containing any in the microbial bacterial agent
The bacterial strain of material, medicine or its any kind prepared, such as bacillus licheniformis, saccharomyces cerevisiae, bacillus subtilis etc..
In one embodiment of the invention, food, feed, medicine can be also used for containing any in the microbial bacterial agent
The carrier of thing.
Third object of the present invention is to provide a kind of method of bioanalysis synthesis GABA, is the short lactic acid using the present invention
Bacterium CGMCC NO.14385 bacterial strains are produced.
In one embodiment of the invention, the method is using glucose, sucrose, lactose or arabinose as growth
Carbon source, γ-aminobutyric acid is synthesized 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 strains
Batch fermentation, fermentation process need not carry out acid-base value adjusting and anaerobism control.In one embodiment of the invention, specifically
It is：Lactobacillus brevis CGMCC NO.14385 are inoculated in the GYP liquid fermentation mediums of different glucose carbon source, are added
The substrate that glutamic acid or sodium glutamate not higher than 50g/L are synthesized as GABA, wherein concentration of glucose are 10~50g/L, in
It is adapted to ferment under the condition of culture of Lactobacillus brevis.
In one embodiment of the invention, the described method includes fermentation system acid-base value is carried out during the fermentation
Control.
In one embodiment of the invention, the method is carried out using Lactobacillus brevis CGMCC NO.14385 bacterial strains
Fed-batch ferments, and fermentation process carries out acid-base value adjusting, it is not necessary to carries out anaerobism control.In one embodiment of the present invention
In, it is specifically：Using bioreactor, Lactobacillus brevis CGMCC NO.14385 are inoculated in the GYP fermentation trainings containing sodium glutamate
Support in base, use the sulfuric acid of 5mol/L concentration to control the pH of zymotic fluid for 5.0, stuffiness condition is stirred in 37 DEG C × 100rpm
Under ferment.To avoid suppression of the high concentration of glutamate sodium to thalli growth, between 12~24h supplement sodium glutamate substrate and
Glucose carbon source.Preferably, sodium glutamate substrate and glucose addition are carried out in 6h, 12h, 18h and 24h, 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 described method includes first cultivated thalline, urged by the use of thalline as full cell
Agent, in suitable acid-base value conversion fluid, using glutamic acid or sodium glutamate as substrate, carry out whole-cell catalytic synthesis γ-
Aminobutyric acid.
In one embodiment of the invention, the method is that first culture acquisition Lactobacillus brevis CGMCC NO.14385 are thin
Born of the same parents, recycle resting cell method.In one embodiment of the invention, it is specifically：Picking Lactobacillus brevis CGMCC
NO.14385 is connected in the GYP liquid fermentation mediums of sodium glutamates of the GYP containing 10g/L, and 37 DEG C × 200rpm culture 12h, are received
Collect thalline.Wet thallus is taken, is put into the Na of the 0.2mol/LpH4.6 of 30~50g/L sodium glutamates2HPO4In-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
30g/L, adds 1g wet thallus, converts to obtain GABA 14.5g/L, 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, using the short of the present invention
Lactic acid bacteria CGMCC NO.14385 bacterial strains.
In one embodiment of the invention, the described method includes：Take out Lactobacillus brevis bacterial strain CGMCC NO.14385
Zymotic fluid (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 after amount reaches 30~40%, up to the dry bacterium powder containing GABA.Spray drying condition is：Feeding temperature
30~40 DEG C, 140~150 DEG C of inlet temperature, 50~60 DEG C, 0.25~0.3MPa of inlet pressure of outlet temperature, centrifugal turntable turn
19000~20000r/min of speed, drying time are 5~15s.Product discharge moisture≤7% is obtained, is sealed after cooling.
The present invention also provides the Lactobacillus brevis CGMCC NO.14385 in food, prepare medicine or livestock and poultry cultivation field
Application.
Beneficial effects of the present invention：
In fermented grains of the Lactobacillus brevis CGMCC NO.14385 of the present invention screened from fen-flavor type white spirit brewing system, have resistance to
The characteristic of the brewing environment of peracid (pH is 3.5 or so), high alcohol content (50-90g/L) in by fen-flavor type white spirit preparation process,
It can keep growth 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 systems of bacterial strain quickly can be subject to sodium glutamate to induce, and the GABA synthesis of bacterial strain is synchronous with thalli growth to be carried out, and highly concentrated
Maintain vigour in the environment of degree substrate, product and sulfuric acid, producing GABA for continuous culture bacterial strain provides foundation, can improve GABA productions
Shorten fermentation period while amount, further lift the production efficiency of the GABA of bacterial strain.
Biomaterial preservation
Lactobacillus brevis D17, taxology are named as Lactobacillus brevis Lactobacillus brevis, are protected on July 6th, 2017
China Committee for Culture Collection of Microorganisms's common micro-organisms center is hidden in, deposit number is CGMCC NO.14385.
Brief description of the drawings
The colony morphology characteristic figure of Fig. 1 Lactobacillus brevis D17；
The morphological features figure of Fig. 2 Lactobacillus brevis D17；
Fig. 3 sodium glutamates standard items and GABA standard items high performance liquid chromatography detection figures；
Fig. 4 bacterium zymotic fluid GABA high performance liquid chromatography detection figures；
Fig. 5 Lactobacillus brevis D17 bacterial strains GABA synthesis is coupled with growth；
The strain growth, substrate utilization and GABA that Fig. 6 Lactobacillus brevis D17 fed batch fermentations efficiently synthesize GABA produce song
Line.
Embodiment
Embodiment 1：The screening of Lactobacillus brevis
5g fen-flavor type white spirit fermented grain raw materials are weighed, 50mL sterile salines is added and is shaken.Bacteria suspension is drawn, with nothing
Bacterium physiological saline carries out 10 times of gradient dilutions, takes 100-200 μ L to be coated on containing CaCO3With MC culture mediums (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 mediums (Sigma-Aldrich purchases) tablet on, in 37
DEG C Anaerobic culturel 48h, when there is milky or light yellow bacterium colony, and it is lactic acid bacteria to occur hydrolysis circle person preliminary judgement around it.
Picking single bacterium colony, is separated, after purification, is inoculated with the test tube that packing has MRS fluid nutrient mediums, is inoculated in after being incubated overnight
In GYP liquid fermentation mediums containing sodium glutamate, the GABA content in fermented supernatant fluid is detected with HPLC methods, to acquisition
GABA producing bacterial strains carry 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, measure
GABA concentration in zymotic fluid, wherein 9 strains of lactic acid bacteria GABA yield are more than the bacterial strain of 5g/L.By GABA inoculations in GYP liquid
In body fermentation medium, sampled per 2h, GABA and sodium glutamate substrate content in fermentation supernatant are analyzed using HPLC, is filtered out
Fermentation time is short, can efficiently synthesize the lactic acid bacteria of GABA, and screening obtains the Lactobacillus brevis bacterial strain that numbering is D17, fermentation medium
Middle starting concentration of sodium glutamate 10g/L, ferment 12h, can obtain 6.5g/L GABA, production efficiency 0.542g/L/h.
Obtained Lactobacillus brevis D17, its 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 strains
GYP culture mediums：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%, is quality-volume fraction.2% is added in solid medium
The agar of concentration, various concentrations sodium glutamate is added in fermentation medium.
Lactobacillus brevis is oxygen tolerance type lactic acid bacteria, its growth under the conditions of supporting well is better than Anaerobic culturel, therefore recommends
Lactobacillus brevis D17 bacterial strains are cultivated under the conditions of supporting well.
Aseptically, by the Lactobacillus brevis D17 bacterial strains of -80 DEG C of preservations, line on GYP solid plates, 37 DEG C quiet
Culture is put, from picking Lactobacillus brevis D17 single bacterium colonies on the GYP solid plates of activation after single bacterium colony is grown, is inoculated in GYP seeds
In culture medium, 37 DEG C of quiescent culture 24h.Seed culture fluid is taken, new GYP seed culture mediums are inoculated in by 10% inoculum concentration
In, after 37 DEG C of quiescent culture 12h, nutrient solution is as fermentation seed.In 250mL triangular flasks load 100mL addition 10~
The GYP fermentation mediums of 50g/L glucose carbon sources and 50g/L sodium glutamates, take culture 12h fermentation seed by 10% inoculation
Amount inoculation, 48h is cultivated 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 conducts
Carbon source, Lactobacillus brevis synthesis GABA yield is 27.6g/L, production efficiency 0.863g/L/h.
Embodiment 3：Carry out controlling sour fed batch fermentation synthesis GABA using Lactobacillus brevis D17 bacterial strains
The bacterial strain D17 that will be activated on inclined-plane, is inoculated in GYP fluid nutrient mediums, and 37 DEG C of culture 24h, are made first order seed
Nutrient solution, 10% inoculum concentration is pressed by first order seed nutrient solution, is connected to the GYP fermentation mediums that pH is 5 sodium glutamates containing 74.8g/L
In, using bioreactor control 37 DEG C of temperature, 200rpm stirring, pH is controlled as 5.0 using 5mol/L sulfuric acid, 6h, 12h,
Each feed supplement (sodium glutamate) 37.3g, 74.8g, 37.3g, 37.3g when 18h, 24h；At the same time carbon is respectively added in 12h, 18h, 24h
Source (glucose) 5g, 7.5g, 15g, ferment 48h.The GABA content of detection fermented supernatant fluid is analyzed using HPLC.
The results are shown in Figure 6, and the GABA synthesis of Lactobacillus brevis D17 bacterial strains is coupled with growth, in whole fermentation process, bacterium
Strain has vigor all the time, and cell concentration is stepped up, and illustrates that bacterial strain can be kept in the substrate, product and sour environment of high concentration
The ability of GABA is efficiently synthesized, the concentration of GABA is 132.63g/L when fermenting 42h, 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 strains again
From picking Lactobacillus brevis D17 single bacterium colonies in the GYP solid slope culture mediums of activation, GYP fluid nutrient mediums are inoculated in
In, 37 DEG C of quiescent culture 24h, are made primary seed solution, and primary seed solution is inoculated in fresh GYP liquid by 10% inoculum concentration
In body culture medium, after 37 DEG C of quiescent culture 12h, fermentation seed is made.Load 500mL in the shaking flask of 1L and contain 1% sodium glutamate
GYP fluid nutrient mediums, take the fermentation seed of culture 12h, are inoculated with by 10% inoculum concentration, 37 DEG C × 200rpm, cultivate 12h.Take
Go out zymotic fluid, centrifuge 10min in 4 DEG C × 6000rpm, collect thalline.1g wet thallus is resuspended in containing 30g/L sodium glutamates
The Na of 0.2M pH4.62HPO4- citrate 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 is 9.7g/L/h/g wet thallus.
Embodiment 5：The dry bacterium powder containing GABA is prepared using Lactobacillus brevis D17
The zymotic fluids of Lactobacillus brevis D17 after fermentation are 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 rotating speed preservation 20000r/min.
The dry bacterium powder yield containing GABA is after drying：15%, moisture 5%.Viable count is up to 6.0 × 109Cfu/g,
GABA content >=20%.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
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, it is characterised in that the microbial bacterial agent contains Lactobacillus brevis CGMCC NO.14385 thalline
Living cells, freeze-drying obtained Lactobacillus brevis CGMCC NO.14385 dry myceliums, the Lactobacillus brevis CGMCC of immobilization
NO.14385 cells, the liquid bacterial agent of Lactobacillus brevis CGMCC NO.14385, the solid bacterium of Lactobacillus brevis CGMCC NO.14385
Agent, or Lactobacillus brevis CGMCC NO.14385 bacterial strains existing in the form of other are any.
3. microbial bacterial agent according to claim 2, it is characterised in that in the microbial bacterial agent also containing it is any can
Bacterium applied to food, feed, medicine or its any kind prepared.
4. microbial bacterial agent according to claim 2, it is characterised in that can also be used containing any in the microbial bacterial agent
In the carrier of food, feed or medicine.
A kind of 5. method for preparing γ-aminobutyric acid, it is characterised in that the method is to utilize the short breast 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, and γ-aminobutyric acid is synthesized as precursor using glutamic acid or sodium glutamate.
7. according to the method described in claim 5, it is characterized in that, the described 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 described method includes, first cultivate thalline, by the use of thalline as
Whole-cell catalyst, in suitable acid-base value conversion fluid, using glutamic acid or sodium glutamate as substrate, carries out whole-cell catalytic
Synthesize γ-aminobutyric acid.
9. a kind of application of the microbial inoculum containing claim 1 bacterial strain, it is characterised in that the application is included 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 in medicine or livestock and poultry cultivation field.
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