CN110218675A - The streptococcus thermophilus 210202 of one plant of highly producing gamma-aminobutyric acid and its application - Google Patents
The streptococcus thermophilus 210202 of one plant of highly producing gamma-aminobutyric acid and its application Download PDFInfo
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- streptococcus thermophilus
- 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 139
- 229960003692 gamma aminobutyric acid Drugs 0.000 title claims abstract description 98
- 241000194020 Streptococcus thermophilus Species 0.000 title claims abstract description 91
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 235000020183 skimmed milk Nutrition 0.000 claims abstract description 24
- 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 20
- 235000013923 monosodium glutamate Nutrition 0.000 claims abstract description 20
- 229940073490 sodium glutamate Drugs 0.000 claims abstract description 20
- 235000021262 sour milk Nutrition 0.000 claims abstract description 20
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 19
- 230000001580 bacterial effect Effects 0.000 claims abstract description 15
- 235000013365 dairy product Nutrition 0.000 claims abstract description 15
- 238000000855 fermentation Methods 0.000 claims abstract description 11
- 230000004151 fermentation Effects 0.000 claims abstract description 11
- 244000005700 microbiome Species 0.000 claims abstract description 8
- 239000000843 powder Substances 0.000 claims abstract description 8
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- 241000894006 Bacteria Species 0.000 claims description 29
- 238000002360 preparation method Methods 0.000 claims description 14
- 239000002253 acid Substances 0.000 claims description 5
- 239000006071 cream Substances 0.000 claims description 5
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- 239000008267 milk Substances 0.000 claims description 4
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- 238000005238 degreasing Methods 0.000 claims description 2
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- 238000004519 manufacturing process Methods 0.000 abstract description 8
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- 235000021107 fermented food Nutrition 0.000 abstract description 4
- 238000013459 approach Methods 0.000 abstract description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 24
- 239000000047 product Substances 0.000 description 13
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 5
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- 201000004384 Alopecia Diseases 0.000 description 2
- 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 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
- 241000192125 Firmicutes Species 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
- 102000008214 Glutamate decarboxylase Human genes 0.000 description 2
- 108091022930 Glutamate decarboxylase Proteins 0.000 description 2
- 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 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 235000020167 acidified milk Nutrition 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 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 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000007872 degassing Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 235000013376 functional food Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 229940054441 o-phthalaldehyde Drugs 0.000 description 2
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
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- 102000004169 proteins and genes Human genes 0.000 description 2
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- 235000009566 rice Nutrition 0.000 description 2
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
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- 201000010538 Lactose Intolerance Diseases 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
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- 238000007796 conventional method Methods 0.000 description 1
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- 230000007812 deficiency Effects 0.000 description 1
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- 239000011724 folic acid Substances 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
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- 235000021474 generally recognized As safe (food) Nutrition 0.000 description 1
- 235000021473 generally recognized as safe (food ingredients) Nutrition 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
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- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 235000021110 pickles Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000004144 purine metabolism Effects 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000000926 separation method 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
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical class OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1238—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using specific L. bulgaricus or S. thermophilus microorganisms; using entrapped or encapsulated yoghurt bacteria; Physical or chemical treatment of L. bulgaricus or S. thermophilus cultures; Fermentation only with L. bulgaricus or only with S. thermophilus
-
- 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
-
- 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
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/249—Thermophilus
-
- 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
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
Abstract
The invention discloses the streptococcus thermophilus 210202 of one plant of highly producing gamma-aminobutyric acid and its applications.The streptococcus thermophilus 210202 was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on October 15th, 2018, and deposit number is CGMCC No.16585.The present invention filters out the strains of streptococcus thermophilus 210202 that high yield GABA can be metabolized in cow's milk from natural fermented food, and to its identification in terms of having carried out morphology, metabolism and science of heredity, is accredited as streptococcus thermophilus strain.The bacterial strain can the content of high yield GABA, GABA be 0.3273g/L in the fermentation system containing sodium glutamate, skimmed milk powder reconstituted milk.The present invention provides new approach to prepare the ferment agent for sour milk for being rich in γ-aminobutyric acid or dairy products, significant to the sour milk product of industrialized production high-content GABA.
Description
Technical field
The present invention relates to technical field of microbial fermentation, and in particular, to the thermophilus of one plant of highly producing gamma-aminobutyric acid
Bacterium 210202 and its application.
Background technique
γ-aminobutyric acid (γ-aminobutyric acid, GABA) is most important in mammalian central nervous system
Inhibitory neurotransmitter, GABA almost has inhibiting effect to all neurons in brain.Lacking GABA in human body is to lead
The main reason for causing the body and mind malaise symptoms of people.And the importance for supplementing GABA be that can releive and inhibition be overexcited and
Fierce nerve information conduction, thus make one to stabilize it is gentle get off, stress can be effectively relieved.
Lactic acid bacteria is widely used in the food such as Yoghourt, cheese and pickles as a kind of microorganism of aliment security level (GRAS)
Production in, had a lot research shows that lactic acid bacteria has glutamate decarboxylase activity, glutamic acid decarboxylase generation can be catalyzed
GABA.Traditional fermented food is to produce the main separation source of GABA lactic acid bacteria, and acid environment is rich in substrate glutamic acid as cream
Sour bacterium synthesis GABA provides advantageous condition.The bacterial strain of high yield GABA is obtained from lactic acid bacteria, and is applied to and is prepared function
It in energy property food or isolates and purifies to obtain GABA preparation, there is good application prospect.
Although many in relation to producing the report of GABA lactic acid bacteria so far, carry out to the lactic acid bacteria of high yield GABA
Separate, identify, using etc. research for food industry still to be important, it is stable, safe, efficient in the hope of obtaining
GABA is used to research and develop the functional food rich in GABA.For being rich in the exploitation Japanology of γ-aminobutyric acid functional food
Work it is more, have it is some realize merchandized handling, such as Japan Jia Yelong tea, Ao Lizha company production be rich in GABA
Rice germ extracts, the germination sugar rice of Fa Kaier company, chlorella yakult sour milk beverage containing GABA etc..And China is in this respect
Starting stage, currently, China has beneficial ten thousand Bioisystech Co., Ltd in Zhejiang successfully to produce rich in GABA meters of plumules, good fortune height is carried out in Anhui
The GABA food additives etc. of limited liability company, section production.But the country GABA is applied to it is less in dairy products, especially with
High yield GABA lactic acid bacteria be strain participate in fermenting fermented dairy product obtained almost without.Therefore, pass through the cream to high yield GABA
Sour bacteria strain is screened, and as ferment agent for sour milk for developing the dairy products rich in GABA with important society
Meaning and economic value.
Streptococcus thermophilus (Streptococcus thermophilus) is also referred to as saliva chain coccus thermophilous subspecies, is one
Kind gram-positive bacterium, amphimicrobian.Its do not generate cytochromes, oxidizing ferment and and negative catalase, it is immovable
Gemma can not be formed.Streptococcus thermophilus be it is fimbriate, optimum growth temperature be 35~42 DEG C.Streptococcus thermophilus is also classified
For lactic acid bacteria, and it is widely used in fermented milk products industry.Under normal conditions, streptococcus thermophilus and lactobacillus bulgaricus
(Lactobacillus bulgaricus) cooperative fermentation in cow's milk, streptococcus thermophilus may be provided to lactobacillus bulgaricus
Its required folic acid and formic acid, both acid play an important role in the purine metabolism of lactobacillus bulgaricus.USDA
Data are shown, in 1998, horse Soviet Union's lira cheese and 600,000,000 kilograms of Yoghourt more than 1,000,000,000 kilograms were sent out with streptococcus thermophilus
Ferment production.Although streptococcus contains some pathogenic bacteria, food service industry thinks that streptococcus thermophilus will than other streptococcus
Safer, somebody thinks that the cheese containing streptococcus thermophilus living and Yoghourt are beneficial to health, thermophilus living
The digestion dairy products that bacterium can help the crowd of lactose intolerance to be more easier.There is correlative study to confirm streptococcus thermophilus tool
The standby ability for producing GABA, but the content of GABA is relatively low.Therefore, it is necessary to filter out the streptococcus thermophilus that a plant height produces GABA.
Summary of the invention
The purpose of the invention is to overcome the above-mentioned deficiency of the prior art, the thermophilic of one plant of highly producing gamma-aminobutyric acid is provided
Hot streptococcus (Streptococcus thermophilus) 210202.
Another object of the present invention is to provide the streptococcus thermophilus 210202 of above-mentioned highly producing gamma-aminobutyric acid to prepare
Application in γ-aminobutyric acid.
It is rich in preparation that another object of the present invention is to provide the streptococcus thermophilus 210202 of above-mentioned highly producing gamma-aminobutyric acid
Application in ferment agent for sour milk containing γ-aminobutyric acid.
Another object of the present invention is to provide the streptococcus thermophilus 210202 of above-mentioned highly producing gamma-aminobutyric acid in preparation cream
Application in product.
Another object of the present invention is to provide a kind of ferment agent for sour milk rich in γ-aminobutyric acid.
Another object of the present invention is to provide a kind of preparation methods of dairy products rich in γ-aminobutyric acid
To achieve the goals above, the present invention is achieved by following scheme:
The present invention filters out the strains of streptococcus thermophilus that high yield GABA can be metabolized in cow's milk from natural fermented food,
And to its identification in terms of having carried out morphology, metabolism and science of heredity, API and 16S rRNA qualification result verifies it as hammer
Pseudomonas (Streptococcus) thermophilus strain (S.thermophilus).By to its product γ-aminobutyric acid (GABA)
Identified and detected, demonstrate its ability for producing GABA in the skimmed milk powder reconstituted milk (abbreviation skimmed milk), and with it is of the same race
Other bacterial strains are compared, the results showed that the ability that the bacterial strain produces GABA is higher than other lactic acid bacterias.
Therefore, the streptococcus thermophilus (Streptococcus of one plant of highly producing gamma-aminobutyric acid is claimed in the present invention
Thermophilus) 210202, it is commonly micro- that China Committee for Culture Collection of Microorganisms is deposited on October 15th, 2018
Bio-Centers (CGMCC), deposit number are CGMCC No.16585.Preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, Institute of Microorganism, Academia Sinica.
Streptococcus thermophilus 210202 of the present invention has following form and physiology, biochemical characteristic:
(1) morphological feature
The grown under anaerobic conditions on M17 Agar Plating of streptococcus thermophilus 210202 is good, and cultivating can within 48h
Form apparent bacterium colony, bacterium colony is creamy white, the circle of diameter 0.5mm~1mm, opaque；Surface is wet in elliposoidal protrusion
Moisten, is smooth, outer rim is neat.
Under microscope, through Gram's staining, streptococcus thermophilus 210202 is in Gram-positive, and size is 0.8~1 μm of diameter
Almost spherical, it is single, in pairs or in short chain arrangement.
(2) physio-biochemical characteristics
Streptococcus thermophilus 210202 is gram-positive bacteria, and atrichia is irremovable, cannot generate gemma, amphimicrobian,
Suitable growth temperature is 41~48 DEG C, lacks surface mucin, therefore do not have pathogenic bacteria feature.It produces a small amount of extracellular more
Sugar can be fermented with metabolising lactose, glucose in cow's milk and be generated lactic acid, and decomposition of protein ability is weak.With the presence of sodium glutamate
In the case where, the ability that metabolism generates γ-aminobutyric acid is significantly higher than other strains of streptococcus thermophilus.
The 16S rRNA sequence of the streptococcus thermophilus 210202 is as shown in SEQ ID NO:1.
The streptococcus thermophilus 210202 that the highly producing gamma-aminobutyric acid is also claimed in the present invention is preparing gamma-amino fourth
Application in acid.
The streptococcus thermophilus 210202 that the highly producing gamma-aminobutyric acid is also claimed in the present invention is rich in γ-ammonia in preparation
Application in the ferment agent for sour milk of base butyric acid.
The streptococcus thermophilus 210202 of the highly producing gamma-aminobutyric acid is also claimed in preparing dairy products in the present invention
Using.
Preferably, the dairy products are Yoghourt.
It is highly preferred that the dairy products are the Yoghourt rich in γ-aminobutyric acid.
A kind of ferment agent for sour milk rich in γ-aminobutyric acid is also claimed in the present invention, contains in the ferment agent for sour milk
The streptococcus thermophilus 210202 of above-mentioned highly producing gamma-aminobutyric acid.
Preferably, the content of streptococcus thermophilus 210202 is 1 × 10 in the ferment agent for sour milk9Cfu/g~1 ×
1012cfu/g。
It is highly preferred that the content of streptococcus thermophilus 210202 is 1 × 10 in the ferment agent for sour milk11cfu/g。
A kind of preparation method of dairy products rich in γ-aminobutyric acid is also claimed in the present invention, includes the following steps:
The amount that the bacterium solution of above-mentioned streptococcus thermophilus 210202 is pressed to 1%~6% (v/v), is added to containing 2~5g/L paddy ammonia
Sour sodium, in the fermentation system of 8%~12% (w/w) skimmed milk powder reconstituted milk, degreasing raw milk or full-cream raw milk；In 32~43
24~48h of stationary culture is under the conditions of DEG C to get the dairy products for being rich in γ-aminobutyric acid；The bacterium of the streptococcus thermophilus 210202
Living bacteria count is 1 × 10 in liquid8Cfu/mL~1 × 1010cfu/mL。
Preferably, the additive amount of 210202 bacterium solution of streptococcus thermophilus is 3% (v/v)；Contain 3g/L in fermentation system
Sodium glutamate, 10% (w/v) skimmed milk powder reconstituted milk.
Preferably, the temperature of the stationary culture be 37 DEG C, time 48h.
Compared with prior art, the invention has the following advantages:
The present invention filters out the strains of streptococcus thermophilus that high yield GABA can be metabolized in cow's milk from natural fermented food
210202, and to its identification in terms of having carried out morphology, metabolism and science of heredity, it is accredited as streptococcus streptococcus thermophilus
Kind.The bacterial strain can the content of high yield GABA, GABA be in the fermentation system containing sodium glutamate, skimmed milk powder reconstituted milk
0.3273g/L.The present invention provides new approach to prepare the ferment agent for sour milk for being rich in γ-aminobutyric acid or dairy products, to work
The sour milk product that industry metaplasia produces high-content GABA is significant.
Detailed description of the invention
Fig. 1 is that the 16S rRNA of 210202 streptococcus thermophilus of bacterial strain (Streptococcus thermophilus) identifies knot
Fruit.
Fig. 2 is growthform of 210202 streptococcus thermophilus of bacterial strain on culture medium.Wherein, a is streptococcus thermophilus
210202 growing state on M17 Agar Plating, b is for streptococcus thermophilus 210202 micro- after Gram's staining
Form under mirror.
Fig. 3 is the content map of γ-aminobutyric acid standard items.Wherein, a is figure of GABA mark product concentration when being 10 μ g/mL
Spectrum, b are map of GABA mark product concentration when being 50 μ g/mL, the map that c is GABA mark product concentration when being 100 μ g/mL, d GABA
Map when mark product concentration is 150 μ g/mL, e are map of GABA mark product concentration when being 250 μ g/mL.
Fig. 4 is the fermentating metabolism product content map of strains of streptococcus thermophilus 210202.
Fig. 5 is the fermentating metabolism product content map of strains of streptococcus thermophilus 210201.
Specific embodiment
With reference to the accompanying drawings of the specification and specific embodiment is made the present invention and is further elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
The screening and identification of 1 streptococcus thermophilus of embodiment
It screens and obtains from the Yoghourt obtained in Xinjiang Sayram Lake side Kazak resident's yurt, take the fresh milk sample
1g, using fresh milk acquired by sterile saline 1:10 gradient dilution, then from the sample of -5, -6, -7 three dilution gradients
The middle sample for taking 100 μ L respectively is coated on MRS plating medium, is placed in anaerobic environment, is placed at 37 DEG C and is cultivated 48h.Later
On the plate of -5 dilution gradients, picking single colonie carries out plate streaking purifying, then is placed in Anaerobic culturel 48h at 37 DEG C, repeats
Three times, bacterial strain 210202 is obtained.
Taxology identification is carried out to the bacterial strain using the technologies such as API50 reagent strip and 16S rRNA, is accredited as streptococcus
Thermophilus strain (as shown in Figure 1).Then the bacterial strain is saved using 10% (m/v) skimmed milk, it is spare in -80 DEG C of cold storage.
The streptococcus thermophilus 210202 has following form and physiology, biochemical characteristic:
(1) morphological feature
The grown under anaerobic conditions on M17 Agar Plating of streptococcus thermophilus 210202 is good, and cultivating can within 48h
Form apparent bacterium colony, bacterium colony is creamy white, the circle of diameter 0.5mm~1mm, opaque；Surface is wet in elliposoidal protrusion
Moisten, is smooth, outer rim is neat (as shown in Figure 2 a).
Under microscope, through Gram's staining, streptococcus thermophilus 210202 is in Gram-positive, and size is 0.8~1 μm of diameter
Almost spherical, it is single, in pairs or in short chain arrangement (as shown in Figure 2 b).
(2) physio-biochemical characteristics
Streptococcus thermophilus 210202 is gram-positive bacteria, and atrichia is irremovable, cannot generate gemma, amphimicrobian,
Suitable growth temperature is 41-45 DEG C, lacks surface mucin, therefore do not have pathogenic bacteria feature.A small amount of exocellular polysaccharide is produced,
It can ferment with metabolising lactose, glucose in cow's milk and generate lactic acid, decomposition of protein ability is weak.Having existing for sodium glutamate
In the case of, the ability that metabolism generates γ-aminobutyric acid is significantly higher than other strains of streptococcus thermophilus.
The 16S rRNA sequence of the streptococcus thermophilus 210202 is as shown in SEQ ID NO:1.
The screening technique of the strains of streptococcus thermophilus of 2 high yield GABA of embodiment
Other 74 plants of streptococcus thermophilus in streptococcus thermophilus 210202 obtained in embodiment 1 and own strain library are existed
Carry out producing the comparison of GABA amount under same culture conditions.Specific step is as follows:
1, the production of hybrid seeds
It uses MRS plate streaking to activate respectively the 75 plants of bacterial strains of streptococcus thermophilus frozen, then picks them separately single colonie
Using M17 broth bouillon 37 DEG C of stationary cultures for 24 hours, after being repeated twice, that is, seed liquor is made.
2, primary dcreening operation
The additive amount that seed liquor is pressed to 3% (v/v), draws activated seed liquor and is inoculated in M17 synthetic media, set
At 37 DEG C after stationary culture 72h, GABA content in its fermentation liquid is measured, completes the primary dcreening operation to high yield GABA bacterium.
3, secondary screening
The additive amount that seed liquor is pressed to 3% (v/v) draws activated seed liquor and is inoculated in the de- of the sodium glutamate containing 3g/L
In fat milk powder reconstituted milk (10%, w/v) culture medium, be placed at 37 DEG C after stationary culture 48h, to GABA content in its acidified milk into
Row measurement, completes the secondary screening to high yield GABA bacterium.
GABA content in 3 high performance liquid chromatography detection skimmed milk of embodiment
The present embodiment uses high performance liquid chromatography, detects commercially available GABA sterling, establishes the standard curve of peak area and content,
And determine retention time；Then the skimmed milk sample after streptococcus thermophilus fermentation 48h is detected, the peak area of specific time is calculated,
Calculate the GABA content in sample, the production GABA amount of more different streptococcus thermophilus.
One, the preparation of detection reagent
1,5% (v/v) acetum, the sodium hydroxide solution of 2mol/L, 5% (w/v) solution of trichloroacetic acid.
2, borate buffer
2.47g boric acid is weighed, 80mL distilled water is dissolved in, is then adjusted to pH 10.2 with the sodium hydroxide solution of 2mol/L.
3, the preparation of derivating agent
0.1g o-phthalaldehyde (OPA) is weighed in 10mL brown volumetric flask, 1mL methanol (chromatographic grade) is added to sufficiently dissolve
Afterwards, 160 μ L beta -mercaptoethanols are added, the borate buffer constant volume 10mL of pH 10.2 is then used.
4, the preparation of mobile phase
Mobile phase A: weighing 2.0412g sodium acetate, is dissolved in 1000mL pure water, 0.22mL triethylamine is then added, and with 5%
(v/v) acetum is adjusted to pH 7.2, is eventually adding 5mL tetrahydrofuran, crosses spare after 0.45 μm of water system filter membrane, ultrasonic degassing.
Mobile phase B: 500mL acetonitrile (chromatographic grade) and 500mL methanol (chromatographic grade) are mixed in equal volume, ultrasonic degassing standby
With.
Two, HPLC analysis method
1, sample pre-treatments: taking appropriate acidified milk/fermentation liquid to be placed in centrifuge tube, respectively in 7830rpm/12000rpm, 4 DEG C
Under the conditions of be centrifuged 3min, then take suitable supernatant, mixed in equal volume with 5% (w/v) solution of trichloroacetic acid, and in
12000rpm, 15min is centrifuged under the conditions of 4 DEG C.
2, derivation process: taking the sample in right amount after pre-treatment is complete, mixes in equal volume with derivating agent, the derivative 2min of room temperature
Afterwards, upper machine analysis.Deriving method and each parameter are as shown in table 1.
1 HPLC analytical parameter setup of table
Three, testing result
HPLC analyzes result as shown in table 2, table 3.
The retention time and peak area of 2 various concentration GABA of table mark product
| GABA marks product concentration (μ g/mL) | Retention time (min) | Peak area |
| 10 | 22.521 | 102745 |
| 50 | 22.360 | 652944 |
| 100 | 22.436 | 1326068 |
| 150 | 22.367 | 2063380 |
| 250 | 22.331 | 3500344 |
3 bacterial strain 210202 of table is compared with other streptococcus thermophilus produce GABA amount
| Strain number | Bacterial strain type | Condition of culture | GABA content (g/L) |
| 210201 | Streptococcus thermophilus | 48h, 37 DEG C, 10% skimmed milk, 3g/L sodium glutamate | 0.0102 |
| 210222 | Streptococcus thermophilus | 48h, 37 DEG C, 10% skimmed milk, 3g/L sodium glutamate | 0.0106 |
| 210202 | Streptococcus thermophilus | 48h, 37 DEG C, 10% skimmed milk, 3g/L sodium glutamate | 0.3273 |
| 210207 | Streptococcus thermophilus | 48h, 37 DEG C, 10% skimmed milk, 3g/L sodium glutamate | 0.0721 |
| 220209 | Streptococcus thermophilus | 48h, 37 DEG C, 10% skimmed milk, 3g/L sodium glutamate | 0.0828 |
| 220221 | Streptococcus thermophilus | 48h, 37 DEG C, 10% skimmed milk, 3g/L sodium glutamate | 0.0106 |
| 210001 | Streptococcus thermophilus | 48h, 37 DEG C, 10% skimmed milk, 3g/L sodium glutamate | 0.0181 |
| 210002 | Streptococcus thermophilus | 48h, 37 DEG C, 10% skimmed milk, 3g/L sodium glutamate | 0.0156 |
| 210101 | Streptococcus thermophilus | 48h, 37 DEG C, 10% skimmed milk, 3g/L sodium glutamate | 0.0103 |
| 210102 | Streptococcus thermophilus | 48h, 37 DEG C, 10% skimmed milk, 3g/L sodium glutamate | 0.0213 |
| 210106 | Streptococcus thermophilus | 48h, 37 DEG C, 10% skimmed milk, 3g/L sodium glutamate | 0.0276 |
| 210200 | Streptococcus thermophilus | 48h, 37 DEG C, 10% skimmed milk, 3g/L sodium glutamate | 0.0188 |
| 210201 | Streptococcus thermophilus | 48h, 37 DEG C, 10% skimmed milk, 3g/L sodium glutamate | 0.0097 |
The above result shows that compared to other lactic acid bacterias, 210202 energy high yield GABA of streptococcus thermophilus, therefore in 2018
October 15 was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and deposit number is
CGMCC No.16585.Preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
Ferment agent for sour milk or dairy products rich in γ-aminobutyric acid can be prepared using the bacterial strain, industrialized production height is contained
The sour milk product for measuring GABA is significant.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of shield range can also be made on the basis of above description and thinking for those of ordinary skill in the art
Other various forms of variations or variation, there is no necessity and possibility to exhaust all the enbodiments.It is all of the invention
Made any modifications, equivalent replacements, and improvements etc., should be included in the protection of the claims in the present invention within spirit and principle
Within the scope of.
Sequence table
<110>Green's Bioengineering (Shenzhen) Co., Ltd.
The streptococcus thermophilus 210202 of<120>one plants of highly producing gamma-aminobutyric acids and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1296
<212> DNA
<213>streptococcus thermophilus (Streptococcus thermophilus)
<400> 1
ggcccgggaa cgtattcacc gcggcgtgct gatccgcgat tactagcgat tccgacttca 60
tgtaggcgag ttgcagccta caatccgaac tgagattggc tttaagagat tagctcgccg 120
tcaccgactc gcaactcgtt gtaccaacca ttgtagcacg tgtgtagccc aggtcataag 180
gggcatgatg atttgacgtc atccccacct tcctccggtt tattaccggc agtctcgcta 240
gagtgcccaa ctgaatgatg gcaactaaca ataggggttg cgctcgttgc gggacttaac 300
ccaacatctc acgacacgag ctgacgacaa ccatgcacca cctgtcaccg atgtaccgaa 360
gtaactttct atctctagaa atagcatcgg gatgtcaaga cctggtaagg ttcttcgcgt 420
tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc ccccgtcaat tcctttgagt 480
ttcaaccttg cggtcgtact ccccaggcgg agtgcttaat gcgttagctg cggcactgaa 540
tcccggaaag gatccaacac ctagcactca tcgtttacgg cgtggactac cagggtatct 600
aatcctgttc gctccccacg ctttcgagcc tcagcgtcag ttacagacca gagagccgct 660
ttcgccaccg gtgttcctcc atatatctac gcatttcacc gctacacatg gaattccact 720
ctccccttct gcactcaagt ttgacagttt ccaaagcgaa ctatggttga gccacagcct 780
ttaacttcag acttatcaaa ccgcctgcgc tcgctttacg cccaataaat ccggacaacg 840
ctcgggacct acgtattacc gcggctgctg gcacgtagtt agccgtccct ttctggtaag 900
ctaccgtcac agtgtgaact ttccactctc acacccgttc ttgacttaca acagagcttt 960
acgatccgaa aaccttcttc actcacgcgg cgttgctcgg tcagggttgc ccccattgcc 1020
gaagattccc tactgctgcc tcccgtagga gtctgggccg tgtctcagtc ccagtgtggc 1080
cgatcaccct ctcaggtcgg ctatgtatcg tcgcctaggt gagccattac ctcacctact 1140
agctaataca acgcaggtcc atcttgtagt ggagcaattg cccctttcaa ataaatgaca 1200
tgtgtcatcc attgttatgc ggtattagct atcgtttcca atagttatcc cccgctacaa 1260
ggcaggttac ctacgcgtta ctcacccgtt cgcaac 1296
Claims (10)
1. the streptococcus thermophilus (Streptococcus thermophilus) 210202 of one plant of highly producing gamma-aminobutyric acid, special
Sign is that the bacterial strain was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms on October 15th, 2018
The heart (CGMCC), deposit number are CGMCC No.16585.
2. the streptococcus thermophilus 210202 of highly producing gamma-aminobutyric acid described in claim 1 answering in preparation γ-aminobutyric acid
With.
3. the acid that the streptococcus thermophilus 210202 of highly producing gamma-aminobutyric acid described in claim 1 is rich in γ-aminobutyric acid in preparation
Application in milk leavening.
4. the streptococcus thermophilus 210202 of highly producing gamma-aminobutyric acid described in claim 1 is preparing the application in dairy products.
5. a kind of ferment agent for sour milk rich in γ-aminobutyric acid, which is characterized in that contain claim in the ferment agent for sour milk
The streptococcus thermophilus 210202 of 1 highly producing gamma-aminobutyric acid.
6. ferment agent for sour milk according to claim 5, which is characterized in that streptococcus thermophilus 210202 in the ferment agent for sour milk
Content be 1 × 109Cfu/g~1 × 1012cfu/g。
7. ferment agent for sour milk according to claim 6, which is characterized in that streptococcus thermophilus 210202 in the ferment agent for sour milk
Content be 1 × 1011cfu/g。
8. a kind of preparation method of the dairy products rich in γ-aminobutyric acid, which comprises the steps of:
The amount that the bacterium solution of streptococcus thermophilus 210202 described in claim 1 is pressed to 1%~6% (v/v), is added to containing 2~5g/
L-Glu sodium, in the fermentation system of 8%~12% (w/w) skimmed milk powder reconstituted milk, degreasing raw milk or full-cream raw milk；In
24~48h of stationary culture is under the conditions of 32~43 DEG C to get the dairy products for being rich in γ-aminobutyric acid；The streptococcus thermophilus
Living bacteria count is 1 × 10 in 210202 bacterium solution8Cfu/mL~1 × 1010cfu/mL。
9. preparation method according to claim 8, which is characterized in that the additive amount of 210202 bacterium solution of streptococcus thermophilus is
3% (v/v)；Contain 3g/L sodium glutamate, 10% (w/v) skimmed milk powder reconstituted milk in fermentation system.
10. preparation method according to claim 8, which is characterized in that the temperature of the stationary culture is 37 DEG C, the time is
48h。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113272418A (en) * | 2021-03-17 | 2021-08-17 | 微康益生菌(苏州)股份有限公司 | Streptococcus thermophilus for producing gamma-aminobutyric acid and application thereof |
| CN113817632A (en) * | 2021-08-24 | 2021-12-21 | 杭州娃哈哈科技有限公司 | Streptococcus thermophilus, sleep-aiding fermented milk base material rich in GABA (gamma-aminobutyric acid), lactobacillus beverage and preparation method |
| CN114437971A (en) * | 2022-01-24 | 2022-05-06 | 浙江一鸣食品股份有限公司 | Streptococcus thermophilus capable of highly producing gamma-aminobutyric acid and efficiently converting sodium glutamate as well as culture method and application of streptococcus thermophilus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113272418A (en) * | 2021-03-17 | 2021-08-17 | 微康益生菌(苏州)股份有限公司 | Streptococcus thermophilus for producing gamma-aminobutyric acid and application thereof |
| WO2022193163A1 (en) * | 2021-03-17 | 2022-09-22 | 微康益生菌(苏州)股份有限公司 | STREPTOCOCCUS THERMOPHILUS PRODUCING γ-AMINOBUTYRIC ACID AND APPLICATION THEREOF |
| CN113817632A (en) * | 2021-08-24 | 2021-12-21 | 杭州娃哈哈科技有限公司 | Streptococcus thermophilus, sleep-aiding fermented milk base material rich in GABA (gamma-aminobutyric acid), lactobacillus beverage and preparation method |
| CN113817632B (en) * | 2021-08-24 | 2023-12-05 | 杭州娃哈哈科技有限公司 | Streptococcus thermophilus, sleep-aiding fermented milk base material rich in GABA (gamma-amino acid residues), lactobacillus beverage and preparation method |
| CN114437971A (en) * | 2022-01-24 | 2022-05-06 | 浙江一鸣食品股份有限公司 | Streptococcus thermophilus capable of highly producing gamma-aminobutyric acid and efficiently converting sodium glutamate as well as culture method and application of streptococcus thermophilus |
| CN114437971B (en) * | 2022-01-24 | 2023-08-08 | 浙江一鸣食品股份有限公司 | Streptococcus thermophilus capable of producing gamma-aminobutyric acid in high yield and converting sodium glutamate in high efficiency, and culture method and application thereof |
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