CN116804173A - Enterococcus avium with high gamma-aminobutyric acid yield and screening method and application thereof - Google Patents
Enterococcus avium with high gamma-aminobutyric acid yield and screening method and application thereof Download PDFInfo
- Publication number
- CN116804173A CN116804173A CN202310029834.4A CN202310029834A CN116804173A CN 116804173 A CN116804173 A CN 116804173A CN 202310029834 A CN202310029834 A CN 202310029834A CN 116804173 A CN116804173 A CN 116804173A
- Authority
- CN
- China
- Prior art keywords
- aminobutyric acid
- gamma
- enterococcus
- yield
- yeast
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 title claims abstract description 154
- 229960003692 gamma aminobutyric acid Drugs 0.000 title claims abstract description 79
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 title claims abstract description 75
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000012216 screening Methods 0.000 title claims abstract description 28
- 241001468179 Enterococcus avium Species 0.000 title claims abstract description 22
- 239000000843 powder Substances 0.000 claims abstract description 18
- 241000194033 Enterococcus Species 0.000 claims abstract description 16
- 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 15
- 235000013923 monosodium glutamate Nutrition 0.000 claims abstract description 15
- 229940073490 sodium glutamate Drugs 0.000 claims abstract description 15
- 235000013305 food Nutrition 0.000 claims abstract description 10
- 238000004321 preservation Methods 0.000 claims abstract description 5
- RQFCJASXJCIDSX-UUOKFMHZSA-N guanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 claims abstract description 3
- 230000001580 bacterial effect Effects 0.000 claims description 25
- 239000001963 growth medium Substances 0.000 claims description 25
- 238000000855 fermentation Methods 0.000 claims description 24
- 230000004151 fermentation Effects 0.000 claims description 24
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 12
- 238000009630 liquid culture Methods 0.000 claims description 8
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- 238000001694 spray drying Methods 0.000 claims description 6
- 239000000796 flavoring agent Substances 0.000 claims description 5
- 230000000813 microbial effect Effects 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 125000003118 aryl group Chemical group 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 244000144972 livestock Species 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 3
- 239000008176 lyophilized powder Substances 0.000 claims description 3
- 239000006872 mrs medium Substances 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 230000002906 microbiologic effect Effects 0.000 claims description 2
- 244000144977 poultry Species 0.000 claims description 2
- 238000002798 spectrophotometry method Methods 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- 229940124277 aminobutyric acid Drugs 0.000 claims 1
- 244000005700 microbiome Species 0.000 abstract description 6
- 241000186660 Lactobacillus Species 0.000 abstract description 3
- 229940039696 lactobacillus Drugs 0.000 abstract description 3
- 238000009629 microbiological culture Methods 0.000 abstract description 3
- 239000000758 substrate Substances 0.000 abstract description 3
- 235000001497 healthy food Nutrition 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 13
- 102000008214 Glutamate decarboxylase Human genes 0.000 description 7
- 108091022930 Glutamate decarboxylase Proteins 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 235000015203 fruit juice Nutrition 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 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 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 2
- 241001101522 Chiococca Species 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 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 description 2
- 235000008708 Morus alba Nutrition 0.000 description 2
- 240000000249 Morus alba Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000002337 anti-port Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 235000019219 chocolate Nutrition 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 229940099596 manganese sulfate Drugs 0.000 description 2
- 235000007079 manganese sulphate Nutrition 0.000 description 2
- 239000011702 manganese sulphate Substances 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000002068 microbial inoculum Substances 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 238000004816 paper chromatography Methods 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 235000013618 yogurt Nutrition 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 102000003669 Antiporters Human genes 0.000 description 1
- 108090000084 Antiporters Proteins 0.000 description 1
- FRPHFZCDPYBUAU-UHFFFAOYSA-N Bromocresolgreen Chemical compound CC1=C(Br)C(O)=C(Br)C=C1C1(C=2C(=C(Br)C(O)=C(Br)C=2)C)C2=CC=CC=C2S(=O)(=O)O1 FRPHFZCDPYBUAU-UHFFFAOYSA-N 0.000 description 1
- 206010010075 Coma hepatic Diseases 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 230000006518 acidic stress Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000006520 extracellular acidic stress Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 239000002515 guano Substances 0.000 description 1
- 239000006458 gyp medium Substances 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 201000001059 hepatic coma Diseases 0.000 description 1
- 208000007386 hepatic encephalopathy Diseases 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 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
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- KVNYFPKFSJIPBJ-UHFFFAOYSA-N ortho-diethylbenzene Natural products CCC1=CC=CC=C1CC KVNYFPKFSJIPBJ-UHFFFAOYSA-N 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 229930182852 proteinogenic amino acid Natural products 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- 239000001393 triammonium citrate Substances 0.000 description 1
- 235000011046 triammonium citrate Nutrition 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of food microorganisms, and discloses enterococcus avium capable of producing gamma-aminobutyric acid in high yield, and a screening method and application thereof. The enterococcus guani G5 is screened from white spirit Daqu powder and preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 25992 in the period of 10 and 28 of 2022. The screening method is efficient and safe, the obtained enterococcus guanylate G5 strain has the capability of high-yield gamma-aminobutyric acid, 50G/L sodium glutamate is used as a substrate, and the GABA yield can reach 27.7G/L, so that the method is the highest yield of the current enterococcus GABA. Meanwhile, the enterococcus avium G5 strain is applied to lactobacillus products, is an internationally recognized safe and healthy food, and has good market prospect.
Description
Technical Field
The invention belongs to the technical field of food microorganisms, and particularly relates to a bird intestine strain for high-yield gamma-aminobutyric acid, a screening method and application thereof.
Background
Gamma-aminobutyric acid (GABA) is a non-proteinogenic amino acid, a white powder widely found in nature, readily soluble in water, slightly soluble in hot ethanol, insoluble in cold ethanol, diethyl ether and benzene. GABA can reduce blood ammonia, promote brain metabolism, and regulate in vivo metabolism balance; can be used as neurotransmitter signal inhibitor, can regulate sleep, treat physiological diseases such as headache, hepatic coma, hypertension, etc., and can also be used for synthesizing 2-pyrrolidone and 4-polyamide nylon, and can be used in food and industrial production.
And the application field of the gamma-aminobutyric acid is wide, so that the market demand is greatly increased. Currently, synthetic gamma-aminobutyric acid produced by fermentation of lactic acid bacteria can be added to beverages, chocolate and chocolate products, candies, baked foods, puffed foods, and the like. With the importance of food safety, more and more researchers focus on the production of gamma-aminobutyric acid using lactic acid bacteria, and thus, it is very important to obtain safe and industrially valuable gamma-aminobutyric acid producing bacteria.
The biological method mainly comprises a biological fermentation method and a biological conversion method, and the bacterial strain utilizes sodium glutamate as a substrate to synthesize GABA, and is mainly realized by a glutamic acid decarboxylase system (GAD system). The GAD system includes glutamate decarboxylase (GAD) and glutamate-GABA antiporters. When a microorganism is subjected to acid stress, H+ leaks into cells, the microorganism utilizes Glu-GABA antiport protein to transport extracellular glutamic acid into cells, alpha-carboxyl groups are catalyzed and removed by intracellular glutamate decarboxylase (GAD) to generate GABA, and meanwhile, the generated GABA is further transported to the outside of the cells by the Glu-GABA antiport protein. The whole system generates 1 molecule of GABA and consumes 1 molecule of intracellular H+, so as to stabilize the intracellular pH and relieve the extracellular acid stress.
Although the GABA yield can be obviously improved through culture medium and fermentation process optimization, in the actual production process, the laboratory-scale efficient production process can not meet the requirements of low cost and high profit of industrial production, and compared with the production strain with high yield, the production strain has more practical production significance. Therefore, screening for strains that produce GABA efficiently remains the basis for pushing the industrialized application of GABA.
At present, screening methods of GABA producing bacteria are mainly based on paper chromatography, thin layer chromatography and high performance liquid chromatography for primary screening or secondary screening. The paper chromatography method is simple in operation and low in cost, but takes a long time; the thin layer chromatography is convenient to detect and has high sensitivity, but takes a long time; the high performance liquid chromatography has high sensitivity, but has complicated operation and high cost, and is not suitable for being used as a primary screening method.
Enterococcus avium can produce GABA, but its yield is low, how to obtain enterococcus avium with high GABA yield is a process necessary for solving the industrial application of the strain.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the guano intestinal strain with high gamma-aminobutyric acid yield, and the screening method and application thereof.
In order to solve the problems in the prior art, the invention adopts the following technical scheme:
enterococcus guani G5 with high yield of gamma-aminobutyric acid is preserved in China General Microbiological Collection Center (CGMCC) with a preservation number of 25992 at 10-28 of 2022, and is named enterococcus guani in taxonomyEnterococcus avium。
As an improvement, the enterococcus guani with high gamma-aminobutyric acid yield is a milky white colony, the colony diameter is 0.1+/-0.05 mm, the edge is neat, and the enterococcus guani is gram positive, free of spores and free of movement.
The screening method of the enterococcus avium with high gamma-aminobutyric acid yield comprises the following steps:
step 1, preparation of enrichment medium
MRS culture medium, GYP culture medium, MRS culture medium with sodium glutamate added with different concentrations, GYP culture medium;
step 2, enrichment method
Weighing crushed white spirit distiller's yeast, putting the white spirit distiller's yeast into a conical flask, adding a sterile MRS culture medium or GYP liquid culture medium, shaking the mixture uniformly, standing the mixture at 37 ℃ for culturing 48 h, inoculating fermentation liquor into the MRS or GYP liquid culture medium containing sodium glutamate, and culturing 48 h at 200 rpm;
and 3, selecting single bacterial colony, inoculating the single bacterial colony into GYP liquid fermentation medium containing sodium glutamate, taking fermentation liquor with pH of more than 5 as a primary screening standard, further separating and purifying, transferring the fermentation liquor into a test tube with MRS medium, preserving at 4 ℃, re-screening the bacterial strain by utilizing a spectrophotometry and an HPLC method, detecting the synthesis of gamma-aminobutyric acid, and purifying by a flat plate to obtain the bacterial strain with high yield of gamma-aminobutyric acid.
The improvement is that the distiller's yeast of the white spirit in the step 2 is aromatic white spirit distiller's yeast, strong aromatic white spirit distiller's yeast, maotai-flavor white spirit distiller's yeast or sesame-flavor white spirit distiller's yeast.
The gamma-aminobutyric acid-containing freeze-dried bacterial powder is prepared by using the enterococcus avium CZ1 fermentation broth, and the water content of discharged materials of the obtained freeze-dried bacterial powder is less than or equal to 10 percent, and the freeze-dried bacterial powder is sealed after cooling; the method comprises the following specific steps: taking enterococcus avium G5 for fermentation, concentrating at 60-65 ℃ to 1-3 h when the content of gamma-aminobutyric acid in fermentation broth is not less than 20-25G/L, and spray drying after the total solid content reaches 30-40%, thus obtaining the freeze-dried bacterial powder containing GABA.
As an improvement, the spray drying conditions are: the feeding temperature is 40-50 ℃, the inlet temperature is 100-120 ℃, the outlet temperature is 60-70 ℃, the inlet air pressure is 0.4-0.5 MPa, the rotating speed of the centrifugal turntable is 18500-19000rpm, and the drying time is 10-15s.
A microbial agent contains enterococcus guanylate G5 with high yield of gamma-aminobutyric acid or lyophilized powder.
The enterococcus guani G5, the freeze-dried bacterial powder or the microbial agent with high gamma-aminobutyric acid yield can be applied to the preparation of foods, medicines or livestock and poultry cultivation.
The beneficial effects are that:
compared with the prior art, the method takes the starter Daqu for brewing the white spirit as a screening resource, and ensures that the GABA producing strain is highly enriched in a microorganism enrichment and GABA producing strain double enrichment mode; the method combines a mode of rapidly detecting pH, and simultaneously combines colorimetry re-screening and flat plate purification, so that GABA producing bacteria can be efficiently and rapidly screened.
In addition, the lactobacillus which is screened by the method and has high yield of gamma-aminobutyric acid has high yield of GABA, and the prepared high-activity microbial inoculum can be applied to industries such as food, medicine, livestock breeding and the like, and provides technical support for promoting industrial application of enterococcus avium. Can provide a new idea for the source of GABA producing bacteria and prepare fruit juice and yoghurt containing high gamma-aminobutyric acid products.
Drawings
FIG. 1 shows a GABA producer screening plate for Daqu liquor
FIG. 2 shows the primary screening of GABA producing bacteria in Daqu liquor;
FIG. 3 shows the ability of high performance liquid chromatography to detect GABA producing bacteria.
FIG. 4 is a colony morphology profile of strain G5;
FIG. 5 is a gram stain and cell morphology of strain G5;
FIG. 6 is a 16S rRNA amplification electrophoretogram of enterococcus avium G5.
Detailed Description
EXAMPLE 1 screening of lactic acid bacteria producing gamma-aminobutyric acid
1. Culture medium
MRS medium: glucose 2%, yeast extract 0.5%, peptone 1%, beef extract 1%, dipotassium hydrogen phosphate 0.2%, triammonium citrate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.058%, manganese sulfate 0.025%, tween 80 1 mL, pH 6.2, all mass-volume fractions.
GYP medium: glucose 1%, yeast extract 1%, peptone 0.5%, sodium acetate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.01%, ferrous sulfate 0.01% and sodium chloride 0.01% by mass-volume fraction.
Agar of 2% concentration was added to the solid medium.
2. Method of
Pulverizing Daqu liquor, sieving with 100 mesh sieve, and making into Daqu liquor powder.
Adopting a gradient dilution separation method, weighing 0.5. 0.5 g of Daqu powder of white spirit (Fenjiu), adding into 5 mL of liquid culture medium of MRS or GYP, and standing and culturing at 37 ℃ for 48 h to prepare first-stage enriched seed liquid.
Absorbing the first-stage enriched seed liquid, inoculating in 5 mL liquid culture medium containing 10-50 g/L MRS or GYP according to 10% inoculum size, culturing at 37deg.C and 200 rpm for 48 h, and making into second-stage enriched seed liquid.
Absorbing the 0.1 mL second-level enriched seed liquid, and carrying out gradient dilution (10 -1 -10 -5 ) Taking the concentration of 0.1 mL to be 10 -4 ，10 -5 Is spread on GYP and MRS culture medium plates containing 50 g/L sodium glutamate and bromocresol green, respectively, and is subjected to anaerobic culture at 37 ℃ for 48 h.
Single colonies (figure 1) with yellow colors around the colonies on the plates are respectively picked for enrichment and purification, and the colonies generated by enrichment on MRS are inoculated into GYP liquid seed culture medium, and are subjected to anaerobic culture at 37 ℃ for 48 h.
Seed solution was inoculated into 1% sodium glutamate GYP liquid medium at 37℃for anaerobic culture at 60 h at an inoculum size of 10%, and the strain with pH higher than 5 was initially selected by pH detection (FIG. 2).
Diluting the bacterial liquid of GABA high-yield strain obtained by re-screening to 10 -5 Then coating on MRS plate, standing at 37deg.C for culturing 48 h, picking 3-5 single colonies, inoculating into MRS liquid culture medium containing 50 g/L sodium glutamate, culturing 48 h at 37deg.C and 200 rpm, detecting pH, and selecting pH and primary screeningSimilar bacteria, which are single strains after purification and the content of gamma-aminobutyric acid in the fermentation supernatant was detected by HPLC.
The results showed that 15 strains with GABA yield greater than 1G/L were obtained by screening, wherein the G5 with the highest GABA yield (FIG. 3) was 27.7G/L.
EXAMPLE 2 identification of gamma-aminobutyric acid high-yielding strain G5
And (5) carrying out 16S rDNA identification on the high-yield gamma-aminobutyric acid strain obtained by screening. The bacterial liquid obtained by culturing 24 h with GYP liquid culture medium at 37 ℃ is used for extracting genome DNA by a conventional bacterial genome extraction method.
Taking the genome as a template, and 27F/1492R as an amplification primer, wherein the sequence of the amplification primer is as follows: 27F: AGAGTTTGATCMTGGCTCAG;1492R: GGTTACCTTGTTACGACTT, takara primerstar MIX DNA polymerase was used for PCR. The sample adding proportion is as follows: 1. Mu.L of template, 0.5. Mu.L of primer each, 25. Mu.L of DNA polymerase, and 23. Mu.L of sterilized ultrapure water. PCR conditions were 98℃for 40 s, 98℃for 10 s, 55℃for 15s annealing, 72℃for 90 s,30 cycles, 72℃for 10 min. The electrophoretogram after amplification is shown in FIG. 6.
The PCR product is purified and then sent to a sequencing process, the sequencing result shows that the length of the PCR product is 1452 and bp, and the homology of the 16S rDNA of the strain and enterococcus avium is 99% through Blast comparison analysis, so that the strain is identified as enterococcus avium @Enterococcus avium) The mixture was designated as enterococcus avium G5, and the taxonomic designation was enterococcus aviumEnterococcus aviumThe microbial strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 10 months and 28 days in 2022, wherein the preservation address is CGMCC No.25992, which is the national institute of microbiology, national academy of sciences, no. 3, beijing, chaoyang, and the preservation address is CGMCC No. 1.
EXAMPLE 3 preparation of GABA-containing Dry bacterial powder Using enterococcus avium G5
Enterococcus avium G5 was inoculated into MRS liquid medium with 50G/L sodium glutamate as substrate, and cultured at 37℃and 200 rpm for 48 h. And concentrating the whole fermentation broth after fermentation, wherein the concentration condition is 65 ℃ and 0.5-h. Spray drying the concentrated solution to obtain dry powder containing GABA.
The spray drying conditions were: the feeding concentration is 50%, the feeding temperature is 40 ℃, the inlet temperature is 120 ℃, the outlet temperature is 60 ℃, the inlet air pressure is 0.4 MPa, and the rotational speed of the centrifugal turntable is 19000 r/min; the yield of the dry bacterial powder containing GABA after drying is 20%, the moisture content is 7%, and the number of viable bacteria reaches 4.0X10 9 cfu/g, GABA content is more than or equal to 20 percent.
EXAMPLE 4 preparation of Gamma-aminobutyric acid-containing dairy product and fruit juice drink by enterococcus avium G5 fermentation
Selecting a proper amount of enterococcus avium G5 from the activated GYP solid slant culture medium, inoculating the enterococcus avium G5 into liquid GYP seed liquid, and standing and culturing at 37 ℃ for 48 h; inoculating 48 h seed culture solution into MRS culture medium containing 10 g/L sodium glutamate according to 10% of inoculation amount, and standing at 37deg.C for culturing 48 h as fermentation seed; filling 5 mL pure milk or mulberry juice (50% pure water is added) into a 10 mL test tube, adding 10 g/L sodium glutamate, taking fermentation seeds of culture 48 h, removing culture medium, re-suspending with physiological saline, inoculating 10% of inoculating amount into pure milk and mulberry juice respectively, culturing 48 h at 37deg.C, detecting fermentation broth by high performance liquid chromatography, wherein gamma-aminobutyric acid content is 169 mg/L and 376 mg/L respectively, and viable count is 2.5X10 respectively 9 cfu/mL and 1.66×10 8 cfu/mL。
In summary, the invention takes the starter Daqu for brewing the white spirit as a screening resource, and the GABA producing strain is highly enriched in a mode of microorganism enrichment and GABA producing strain double enrichment; the method combines a mode of rapidly detecting pH, and simultaneously combines colorimetry re-screening and flat plate purification, so that GABA producing bacteria can be efficiently and rapidly screened. The lactobacillus which is screened by the method and has high-yield gamma-aminobutyric acid has high-yield GABA capability, and the prepared high-activity microbial inoculum can be applied to industries such as food, medicine, livestock breeding and the like, and provides technical support for promoting the industrial application of enterococcus avium. The fruit juice and the yoghurt containing the high gamma-aminobutyric acid product are prepared, and a new idea is provided for the source of GABA producing bacteria.
Claims (8)
1. High yield of gammaEnterococcus avium G5 of aminobutyric acid is preserved in China General Microbiological Collection Center (CGMCC) at 10 and 28 days 2022, with preservation number of CGMCC NO.25992, and taxonomy named enterococcus aviumEnterococcus avium。
2. The high-yield gamma-aminobutyric acid enterococcus G5 according to claim 1, wherein the high-yield gamma-aminobutyric acid enterococcus is a milky white colony with a colony diameter of 0.1+/-0.05 mm, and has clean edges, gram positive, spore-free and motionless.
3. The screening method of enterococcus guani G5 based on high-yield gamma-aminobutyric acid according to claim 1, comprising the steps of:
step 1, preparation of enrichment medium
MRS culture medium, GYP culture medium, MRS culture medium with sodium glutamate added with different concentrations, GYP culture medium;
step 2, enrichment method
Weighing crushed white spirit distiller's yeast, putting the white spirit distiller's yeast into a conical flask, adding a sterile MRS culture medium or GYP liquid culture medium, shaking the mixture uniformly, standing the mixture at 37 ℃ for culturing 48 h, inoculating fermentation liquor into the MRS or GYP liquid culture medium containing sodium glutamate, and culturing 48 h at 200 rpm;
and 3, selecting single bacterial colony, inoculating the single bacterial colony into GYP liquid fermentation medium containing sodium glutamate, taking fermentation liquor with pH of more than 5 as a primary screening standard, further separating and purifying, transferring the fermentation liquor into MRS medium containing sodium glutamate, re-screening the bacterial strain by utilizing a spectrophotometry and an HPLC method, detecting the synthesis of gamma-aminobutyric acid, and purifying by a flat plate to obtain the bacterial strain with high yield of gamma-aminobutyric acid.
4. The method for screening G5 of high-yield gamma-aminobutyric acid according to claim 3, wherein the distiller's yeast of white spirit in step 2 is aromatic white spirit distiller's yeast, strong aromatic white spirit distiller's yeast, maotai-flavor white spirit distiller's yeast, sesame-flavor white spirit distiller's yeast, or double-flavor white spirit distiller's yeast.
5. The gamma-aminobutyric acid-containing freeze-dried bacterial powder is characterized in that the gamma-aminobutyric acid-containing freeze-dried bacterial powder is prepared by using the enterococcus avium G5 fermentation broth of claim 1, and the water content of discharged materials of the obtained freeze-dried bacterial powder is less than or equal to 10 percent, and the freeze-dried bacterial powder is sealed after cooling; the method comprises the following specific steps: taking enterococcus avium G5 for fermentation, concentrating at 60-65 ℃ to 1-3 h when the content of gamma-aminobutyric acid in fermentation broth is not less than 20-25G/L, and spray drying after the total solid content reaches 30-40%, thus obtaining the freeze-dried bacterial powder containing GABA.
6. The gamma-aminobutyric acid-containing lyophilized powder of claim 5, wherein the spray drying conditions are: the feeding temperature is 40-50 ℃, the inlet temperature is 100-120 ℃, the outlet temperature is 60-70 ℃, the inlet air pressure is 0.4-0.5 MPa, the rotating speed of the centrifugal turntable is 18500-19000rpm, and the drying time is 10-15s.
7. A microbial agent comprising the enterococcus guanylate G5 having a high yield of gamma-aminobutyric acid according to claim 1 or a lyophilized powder according to claim 5.
8. Use of enterococcus guani G5 based on the high-yield gamma-aminobutyric acid according to claim 1, the freeze-dried bacterial powder according to claim 5, or the microbial agent according to claim 7 for preparing food, medicines or livestock and poultry cultivation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310029834.4A CN116804173A (en) | 2023-01-09 | 2023-01-09 | Enterococcus avium with high gamma-aminobutyric acid yield and screening method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310029834.4A CN116804173A (en) | 2023-01-09 | 2023-01-09 | Enterococcus avium with high gamma-aminobutyric acid yield and screening method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116804173A true CN116804173A (en) | 2023-09-26 |
Family
ID=88078626
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310029834.4A Pending CN116804173A (en) | 2023-01-09 | 2023-01-09 | Enterococcus avium with high gamma-aminobutyric acid yield and screening method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116804173A (en) |
-
2023
- 2023-01-09 CN CN202310029834.4A patent/CN116804173A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109182171B (en) | Mutagenic strain for high yield of gamma-aminobutyric acid and biological preparation thereof | |
| CN101074426B (en) | Method for producing bean-dregs feed containing conjugated linolic acid by plant lactobacillin fermentation | |
| JP5910978B2 (en) | Non-protein amino acid-producing lactic acid bacteria and their uses | |
| CN110734880B (en) | Lactobacillus plantarum Bama06 derived from Guangxi Bama and having high vitamin B yield and application thereof | |
| CN113061558B (en) | Composite probiotics and feed additive containing bacillus coagulans HALO178 | |
| CN110129234B (en) | Mutagenized bacillus subtilis strain with high natural vitamin K2 yield and application thereof | |
| JPWO2007097374A1 (en) | Lactic acid bacteria having the ability to produce γ-aminobutyric acid | |
| KR20130063253A (en) | The alcohol resistant strain of lactic acid bacteria, pediococcus acidilactici and its use | |
| CN111748512A (en) | Nitrogen source suitable for efficiently proliferating bifidobacterium adolescentis and application thereof | |
| CN109136129B (en) | Lactobacillus acidophilus NCU426 | |
| JP2004357535A (en) | NEW LACTOBACILLUS HAVING IMMUNOPOTENTIATIVE ACTIVITY AND gamma-AMINOBUTYRIC ACID-PRODUCING ABILITY AND UTILIZATION THEREOF | |
| CN115141860A (en) | Method for producing gamma-aminobutyric acid and fermentation culture prepared by same | |
| CN106119166B (en) | One plant of Switzerland lactic acid bacteria and its application | |
| CN115895974B (en) | Lactobacillus plantarum rich in selenium and high in gamma-aminobutyric acid yield and application thereof | |
| CN114958694B (en) | Lactobacillus rhamnosus for co-production of conjugated linoleic acid and gamma-aminobutyric acid and application thereof | |
| CN116004423B (en) | Bacillus bailii and application thereof | |
| KR100631857B1 (en) | -3 Lactobacillus Brevis OPK-3 Having a High Production Ability of Gamma-Aminobutyric Acid | |
| CN116804173A (en) | Enterococcus avium with high gamma-aminobutyric acid yield and screening method and application thereof | |
| CN112391297B (en) | Candida utilis for degrading patulin, biological preparation and application thereof | |
| CN109423467A (en) | A kind of lactobacillus plantarum of lactic acid high yield and its purposes in food and field of fodder | |
| CN107118885A (en) | A kind of method that the fermented wine containing GABA is produced using the piece of resistance to ethanol coccus | |
| CN116948915B (en) | Bacillus sojae and application thereof | |
| CN114181874B (en) | Deep-sea micro bacillus and application thereof in enhancing flavor of aquatic seasoning | |
| CN116286513B (en) | Lactobacillus johnsonii FR-1012 and method for industrially producing gamma-aminobutyric acid by same | |
| CN115386525B (en) | Bacillus subtilis, microbial inoculum, application and method for preparing tetramethylpyrazine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |