CN109280714A - A kind of screening technique of the interior lactic acid bacteria strains for producing GABA of chickpea milk - Google Patents
A kind of screening technique of the interior lactic acid bacteria strains for producing GABA of chickpea milk Download PDFInfo
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- CN109280714A CN109280714A CN201811247079.2A CN201811247079A CN109280714A CN 109280714 A CN109280714 A CN 109280714A CN 201811247079 A CN201811247079 A CN 201811247079A CN 109280714 A CN109280714 A CN 109280714A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
The invention discloses a kind of screening techniques for the lactic acid bacteria strains that GABA is produced in chickpea milk, and steps are as follows: 1) chickpea milk sample pre-treatments；2) paper chromatography amino acid separation；3) the first judgement of the lactic acid bacteria of GABA is produced；4) the secondary judgement of the lactic acid bacteria of GABA is produced；5) Morphological Identification of the lactic acid bacteria of GABA is produced；6) physiological and biochemical test of the lactic acid bacteria of GABA is produced；7) 16SrDNA Sequence Identification produces the bacterial strain of the lactic acid bacteria of GABA.The present invention passes through when high performance liquid chromatograph detects No. 2 mixed liquors, No. 2 solution are divided into two parts, first examine the solution of a portion that data record is got off, another part mixed liquor is detected again after standing 20-24h, data record is got off, the data that comparison front and back is detected, GABA amount in solution after standing obviously increases, so that it is determined that there is the bacterial strain for the lactic acid bacteria for producing GABA inside chickpea milk, experimental error is reduced, is laid the foundation to develop the functional food rich in GABA.
Description
Technical field
The present invention relates to the lactic acid that GABA is produced in a kind of screening technique of lactic acid bacteria strains more particularly to a kind of chickpea milk
The screening technique of bacteria strain.
Background technique
Chick-pea, for Papilionaceae herbaceous plant, alias peach your beans, chicken beans, heart beans etc. are India and Pakistani
One of important five cereals eat chick-pea also very universal and Uygur medicine medicinal herbs most in use in Europe.Chick-pea is because of its face shape
It is peculiar, point such as olecranon, therefore claim this name.Chick-pea is leguminous herbaceous plant, originating from western part of Asia and Near East area.It is in the world
The biggish bean of cultivated area, about 100,015,000 mu, wherein the cultivated area of India and Pakistani two countries accounts for full generation
80% or more of boundary, the only fragmentary distribution of China.The starch of chick-pea has Chinese chestnut fragrance.Chick-pea is made into chickpea milk can
It is the nutraceutical of baby and the elderly to be easy to assimilate, includes GABA, Chinese gamma-amino inside chickpea milk
Butyric acid is called amino acid injection-800, is a kind of natural amino acid of nonprotein composition, is most important in mammalian central nervous system
Inhibitory neurotransmitter, have important physiological function: such as hypotensive activity；Improve brain function, extends memory；Calm mind
Through angst resistance effect；Improve liver function；Renal function is activated, cholesterol is reduced.GABA is in addition to above-mentioned physiological action, also
There is adjusting hormone secretion, improve climacteric syndrome, promotes alcohol metabolism, smelly eliminating, high-efficient fat reducing, induce human spermatogoa acrosome, promote
The functions such as the disease-resistant and growth of precession object.In addition, numerous studies prove, the shortage of GABA will lead to the generation of epileptics, therefore
Suitably supplement GABA has certain prevention effect to epileptics caused by the shortage due to GABA in the diet.Due to its special physiological
Function, GABA have become a kind of new function sex factor, are just being widely used in the row such as health care of food, medicine, chemical industry and agricultural
Industry has broad application prospects.
Produced in traditional chickpea milk the lactic acid bacteria strains of GABA screening only simply extract sub-fraction sample into
Row test for identification, which goes out, contains GABA inside chickpea milk, can not determine whether there is the lactic acid bacteria strains for producing GABA, and tradition
Identification mode do not pass through repeated detection, cause identification it is not scientific.
Summary of the invention
The object of the present invention is to provide a kind of screening techniques for the lactic acid bacteria strains that GABA is produced in chickpea milk, solve tradition
Method identification is not scientific, can not determine the lactic acid bacteria strains that there are problems that producing GABA inside chickpea milk.
In order to achieve the above object, the present invention is achieved by the following technical solutions:
A kind of screening technique of the interior lactic acid bacteria strains for producing GABA of chickpea milk, includes the following steps:
1) mixed liquor of chickpea milk block and water is prepared；
2) using the amino acid in paper chromatography separation mixed liquor；
3) lactic acid bacteria that GABA is produced in mixed liquor is judged using high performance liquid chromatograph；
4) Morphological Identification is carried out to the lactic acid bacteria for producing GABA in mixed liquor；
5) physiological and biochemical test is carried out to the lactic acid bacteria for producing GABA in mixed liquor；
6) using the bacterial strain for the lactic acid bacteria for producing GABA in 16SrDNA Sequence Identification mixed liquor；
7) step 2) to testing result 6) is compared and analyzed, filters out the lactic acid bacteria bacterium for producing GABA in mixed liquor
Strain.
Preferably, in step 1), chickpea milk block is mixed with water, and after mixing evenly, in 25~40 DEG C of environment
Stand 10min.
Preferably, step 2) specific steps include: firstly, cut an aperture in about 1 centimetre of filter paper upper end-face edge of center,
Filter paper item is lain against on clean paper, is gently drawing a horizontal line with pencil at the 25mm of filter paper lower end, and in the center of horizontal line
Draw a diameter be 2mm small circle, with capillary aspiration step 1) in mixed liquor, the point sample in the small circle of filter paper lower end,
Dry Boiling tube is taken, is added by water saturated phenol solution, pin is worn at away from filter paper lower end 12.5mm, from filter paper upper end
Wear cotton thread at aperture and divide right and left, adjust line height make filter paper be vertically suspended from test tube and it is lower it is rapid immerse solution 12.5mm,
Solvent can be seen at this time to move up, when solvent is raised to certain altitude, carefully paper slip is taken out, solvent is marked with pencil and is risen
Forward position one layer of 0.2% indenes three with pin nail on small wooden frame, is equably sprayed into filter paper both ends with sprayer after drying
Then ketone ethanol solution again dries up filter paper item, i.e., it can be seen that showing each color dot on paper, each color dot represents a kind of amino
Acid.
Preferably, step 3) includes:
3.1) it produces the first judgement of the lactic acid bacteria of GABA: being taken out using the mixed liquor that high performance liquid chromatograph prepares step 1)
Enter sampling valve, brought into chromatographic column by mobile phase, after each aminoacid ingredient in column is separated, sequentially enter detector into
Row detects, and the recording device inside high performance liquid chromatograph records all data of GABA by comparative analysis；
3.2) it produces the secondary judgement of the lactic acid bacteria of GABA: taking mixed liquor in step 1) to stand 20-24h, reuse efficiently
Liquid chromatograph is detected, and all data of GABA is recorded, and is increased by the amount that correlation data observes GABA.
Preferably, step 4) includes: that the mixed liquor prepared in step 1) is taken to be put under optical microscopy, is reflected by morphology
Fixed, observation cultural characteristic, cellular morphology and its dyeing property, special eucaryotic cell structure, motility carry out pair with conventional GABA
Than identifying there is the bacterial strain for the lactic acid bacteria for producing GABA in mixed liquor.
Preferably, step 5) includes: that the mixed liquor prepared in step 1) is taken to carry out sugar, alcohol fermentation test, by different micro-
Biological decomposition has very big difference using the ability of carbohydrate, to identify GABA, then extraction step 1) in prepare mixed liquor carry out it is resistance to
Salt test, last extraction step 1) in prepare mixed liquor carry out different temperatures test, determine produce GABA lactic acid bacteria presence.
Preferably, step 6) includes: the mixed liquor for taking and preparing in step 1), is extracted using bacteria total DNA extracts kit
Genomic DNA carries out PCR amplification using the 16S rDNA universal primer of bacterium, after PCR, draws PCR product 5 μ L and 1 μ L
Loading buffer mixing, with 1.0% agarose gel electrophoresis, after electrophoresis with ethidium bromide EB dyeing 20~
30min, sequencing after PCR product is purified are homologous by 16S rDNA sequence and the nucleotide sequence progress in GenBank database
Property analysis, to produce GABA lactic acid bacteria identify.
Preferably, voltage needed for the agarose gel electrophoresis is 100V.
Beneficial effects of the present invention: the present invention takes out equivalent sample in chickpea milk and is divided into 4 parts, respectively 1,2,3,4
Number mixed liquor, environment locating for every portion of mixed liquor is identical, passes through paper chromatography, high performance liquid chromatography to each mixed liquor respectively
Instrument, Morphological Identification, physiological and biochemical test and 16SrDNA Sequence Identification mode determine in the mixed solution of chickpea milk preparation
Containing the lactic acid bacteria for producing GABA, the method detected in several ways in this way makes the detection for producing the lactic acid bacteria of GABA more quasi-
Really, when and detecting No. 2 mixed liquors by high performance liquid chromatograph again, No. 2 solution is divided into two parts, first examine it
The solution of middle a part gets off data record, and another part mixed liquor stands 20-24h and detected again, by data record
Get off, the GABA amount in the solution after standing obviously increases, so that it is determined that there is the lactic acid bacteria for producing GABA inside chickpea milk
Bacterial strain lays the foundation to develop the functional food rich in GABA.
Specific embodiment
With reference to embodiment, the specific embodiment of the present invention is further described.Following embodiment is only used for more
Add and clearly demonstrate technical solution of the present invention, and not intended to limit the protection scope of the present invention.
Embodiment 1
A kind of screening technique of the interior lactic acid bacteria strains for producing GABA of chickpea milk, specifically includes the following steps:
1) chickpea milk sample pre-treatments: pocket knife cuts 12g chickpea milk block, and successively equivalent is put into four culture mediums,
Label 1,2,3,4, each culture medium are added 10ml clear water, chickpea milk are stirred clockwise by glass bar, at 25~40 DEG C
10min is stood in environment；
2) it paper chromatography amino acid separation: firstly, cutting an aperture in about 1 centimetre of filter paper upper end-face edge of center, will filter
Paper slip is lain against on clean paper, is gently drawing a horizontal line with pencil at the 25mm of filter paper lower end, and draw one in the center of horizontal line
Diameter is the small circle of 2mm, with capillary aspiration step 1) in mixed liquor inside No. 1 culture medium, in the small of filter paper lower end
Point sample in circle takes dry Boiling tube, is added by water saturated phenol solution 10ml, wears major part at away from filter paper lower end 12.5mm
Needle wears cotton thread from the aperture of filter paper upper end and divides right and left, and the height for adjusting line is suspended from filter paper vertically in test tube and lower rapids
Solution 12.5mm is immersed, solvent can be seen at this time and move up, when solvent is raised to certain altitude, carefully take out paper slip, with
Pencil marks the forward position of solvent rising, by filter paper both ends, with pin nail on small wooden frame, after drying equably with sprayer
One layer of 0.2% ethanol solution of ninhydrin is sprayed, is then again dried up filter paper item, i.e., it can be seen that showing each color dot, Mei Gese on paper
Point represents a kind of amino acid；
3) produce the first judgement of the lactic acid bacteria of GABA: No. 2 mixed liquors for step 1) being prepared using high performance liquid chromatograph
It is pumped into 6ml sampling valve, is brought into chromatographic column by mobile phase, after each aminoacid ingredient in column is separated, sequentially enters inspection
It surveys device to be detected, the recording device inside high performance liquid chromatograph records all data of GABA by comparative analysis
Come；
4) it produces the secondary judgement of the lactic acid bacteria of GABA: remaining No. 2 mixed liquors of step 3) being stood into 20-24h, are reused
High performance liquid chromatograph is detected, and all data of GABA is recorded, and is increased by the amount that correlation data observes GABA.
5) it produces the Morphological Identification of the lactic acid bacteria of GABA: No. 3 mixed liquor 2ml prepared in step 1) being taken to be put into optical microphotograph
Under mirror, by Morphological Identification, cultural characteristic, cellular morphology and its dyeing property, special eucaryotic cell structure, motility are observed,
It is compared with conventional GABA, identifies there is the bacterial strain for the lactic acid bacteria for producing GABA in mixed liquor；
6) it produces the physiological and biochemical test of the lactic acid bacteria of GABA: No. 3 mixed liquor 5ml prepared in step 1) being taken to carry out sugar, alcohol
Fermentation test, being decomposed by different microorganisms has very big difference using the ability of carbohydrate, to identify GABA, then extracts No. 3 mixing
Liquid 5ml carries out Salt tolerance, finally extracts No. 3 mixed liquor 4ml and carries out different temperatures test, determines depositing for the lactic acid bacteria for producing GABA
?；
7) 16SrDNA Sequence Identification produces the bacterial strain of the lactic acid bacteria of GABA: taking No. 4 mixed liquors prepared in step 1), uses
Bacteria total DNA extracts kit extracts genomic DNA, carries out PCR amplification, PCR knot using the 16S rDNA universal primer of bacterium
Shu Hou draws 5 μ L of PCR product and mixes with 1 μ L loading buffer, and with 1.0% agarose gel electrophoresis, electrophoresis terminates
20~30min is dyed with ethidium bromide EB afterwards, sequencing after PCR product is purified, by 16S rDNA sequence and GenBank database
In nucleotide sequence carry out homology analysis, to produce GABA lactic acid bacteria identify；
It prevents solution to be stained on test tube wall in step 2), first test tube is vertically fixed on rack for test tube, immersed in filter paper item
When mixed solution, it should be noted that the point of point sample cannot touch mixed solution, filter paper item can not contact test tube wall.
Mobile phase in step 3) is liquid, and when flowing through chromatographic column, the resistance being subject to is larger, in order to run through chromatography
Column, it is necessary to add high pressure to carrier fluid.
Voltage needed for agarose gel electrophoresis in step 7) is 100V.
Preliminary detection and secondary inspection are carried out to the production GABA lactic acid bacteria strains inside chickpea milk in step 3) and step 4)
It surveys, obtained mixed liquor GABA content such as the following table 1:
According to table 1 it can be seen that the content of the mixed liquor GABA after the standing in culture medium significantly rises, thus really
Determine the lactic acid bacteria strains that the package inside chickpea milk produces GABA, and the bacterial strain has the ability for producing GABA.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (8)
1. producing the screening technique of the lactic acid bacteria strains of GABA in a kind of chickpea milk, which comprises the steps of:
1) mixed liquor of chickpea milk block and water is prepared；
2) using the amino acid in paper chromatography separation mixed liquor；
3) lactic acid bacteria that GABA is produced in mixed liquor is judged using high performance liquid chromatograph；
4) Morphological Identification is carried out to the lactic acid bacteria for producing GABA in mixed liquor；
5) physiological and biochemical test is carried out to the lactic acid bacteria for producing GABA in mixed liquor；
6) using the bacterial strain for the lactic acid bacteria for producing GABA in 16SrDNA Sequence Identification mixed liquor；
7) step 2) to testing result 6) is compared and analyzed, filters out the lactic acid bacteria strains for producing GABA in mixed liquor.
2. producing the screening technique of the lactic acid bacteria strains of GABA in a kind of chickpea milk according to claim 1, feature exists
In in step 1), chickpea milk block being mixed with water, and after mixing evenly, stand 10min in 25~40 DEG C of environment.
3. producing the screening technique of the lactic acid bacteria strains of GABA in a kind of chickpea milk according to claim 1, feature exists
It include: to lay flat filter paper item firstly, cut an aperture in about 1 centimetre of filter paper upper end-face edge of center in, step 2) specific steps
In on clean paper, a horizontal line is gently being drawn with pencil at the 25mm of filter paper lower end, and drawing a diameter in the center of horizontal line is 2mm
Small circle, with capillary aspiration step 1) in mixed liquor, the point sample in the small circle of filter paper lower end takes dry Boiling tube,
It is added by water saturated phenol solution, wears pin at away from filter paper lower end 12.5mm, wear cotton thread from the aperture of filter paper upper end
And divide right and left, adjust line height make filter paper be vertically suspended from test tube and it is lower it is rapid immerse solution 12.5mm, at this time i.e. it can be seen that
Solvent moves up, and when solvent is raised to certain altitude, carefully takes out paper slip, and the forward position of solvent rising is marked with pencil, will be filtered
One layer of 0.2% ethanol solution of ninhydrin is equably sprayed with pin nail on small wooden frame in paper slip both ends with sprayer after drying,
Then filter paper item is dried up again, i.e., it can be seen that showing each color dot on paper, each color dot represents a kind of amino acid.
4. producing the screening technique of the lactic acid bacteria strains of GABA in a kind of chickpea milk according to claim 1, feature exists
In step 3) includes:
3.1) it produces the first judgement of the lactic acid bacteria of GABA: being drawn into mixed liquor prepared by step 1) using high performance liquid chromatograph
Sample valve, is brought into chromatographic column by mobile phase, after each aminoacid ingredient in column is separated, is sequentially entered detector and is examined
It surveys, the recording device inside high performance liquid chromatograph records all data of GABA by comparative analysis；
3.2) it produces the secondary judgement of the lactic acid bacteria of GABA: taking mixed liquor in step 1) to stand 20-24h, reuse efficient liquid phase
Chromatograph is detected, and all data of GABA is recorded, and is increased by the amount that correlation data observes GABA.
5. producing the screening technique of the lactic acid bacteria strains of GABA in a kind of chickpea milk according to claim 1, feature exists
In step 4) includes: that the mixed liquor prepared in step 1) is taken to be put under optical microscopy, passes through Morphological Identification, observation culture
Feature, cellular morphology and its dyeing property, special eucaryotic cell structure, motility, compare with conventional GABA, identify mixed liquor
The middle bacterial strain that there is the lactic acid bacteria for producing GABA.
6. producing the screening technique of the lactic acid bacteria strains of GABA in a kind of chickpea milk according to claim 1, feature exists
In step 5) includes: that the mixed liquor prepared in step 1) is taken to carry out sugar, alcohol fermentation test, is decomposed and is utilized by different microorganisms
The ability of carbohydrate has very big difference, to identify GABA, then extraction step 1) in prepare mixed liquor carry out Salt tolerance, finally mention
It takes the mixed liquor prepared in step 1) to carry out different temperatures test, determines the presence for producing the lactic acid bacteria of GABA.
7. producing the screening technique of the lactic acid bacteria strains of GABA in a kind of chickpea milk according to claim 1, feature exists
In step 6) includes: the mixed liquor for taking and preparing in step 1), extracts genomic DNA using bacteria total DNA extracts kit, adopts
PCR amplification is carried out with the 16S rDNA universal primer of bacterium, after PCR, draws 5 μ L of PCR product and 1 μ L loading
Buffer mixing dyes 20~30min with ethidium bromide EB after electrophoresis with 1.0% agarose gel electrophoresis, and PCR is produced
Nucleotide sequence in 16S rDNA sequence and GenBank database is carried out homology analysis, to production by sequencing after object is purified
The lactic acid bacteria of GABA is identified.
8. producing the screening technique of the lactic acid bacteria strains of GABA in a kind of chickpea milk according to claim 7, feature exists
In voltage needed for the agarose gel electrophoresis is 100V.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948767A (en) * | 2010-08-18 | 2011-01-19 | 南京农业大学 | Method for screening lactic acid bacteria producing gamma-amino butyric acid |
| CN102174449A (en) * | 2011-03-04 | 2011-09-07 | 天津科技大学 | Method for producing high-yield gamma-propalanine and application thereof |
-
2018
- 2018-10-25 CN CN201811247079.2A patent/CN109280714A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948767A (en) * | 2010-08-18 | 2011-01-19 | 南京农业大学 | Method for screening lactic acid bacteria producing gamma-amino butyric acid |
| CN102174449A (en) * | 2011-03-04 | 2011-09-07 | 天津科技大学 | Method for producing high-yield gamma-propalanine and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 李文等: "发酵鹰嘴豆乳产γ-氨基丁酸乳酸菌的复合诱变选育", 《食品科学》 * |
| 田蕊等: "产γ -氨基丁酸乳酸菌的筛选鉴定及其紫外诱变研究", 《食品工业科技》 * |
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