CN107957454A - The detection method of fatty acid component and application in a kind of alctasin strain - Google Patents
The detection method of fatty acid component and application in a kind of alctasin strain Download PDFInfo
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- CN107957454A CN107957454A CN201710990671.0A CN201710990671A CN107957454A CN 107957454 A CN107957454 A CN 107957454A CN 201710990671 A CN201710990671 A CN 201710990671A CN 107957454 A CN107957454 A CN 107957454A
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- fatty acid
- alctasin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
Detection method and application, specific steps the present invention provides fatty acid component in a kind of alctasin strain include：The collection and drying of thalline, methyl esterification of fatty acid, the condition for determining gaschromatographic mass spectrometry detection aliphatic acid, fatty acid component in alctasin strain is tested and analyzed by gaschromatographic mass spectrometry, according to the original spectral data of acquisition, the relative amount for detecting each fatty acid component in sample is calculated.The detection method of fatty acid component and application in a kind of alctasin strain of the present invention, propose a kind of alctasin bacterium identification method, the esterification method and step is simple, without heating, esterification yield is high, and detection time is short, easy analysis, by analyzing content of fatty acid and composition, sequence analysis and base chiller are carried out to production strain in the level of kind, are of great significance for the quality and security for improving galactenzyme raw cook.
Description
Technical field
The invention belongs to bioassay technique field, more particularly, to a kind of detection of fatty acid component in alctasin strain
Method and application.
Background technology
Alctasin (alias：Biofermin) it is lactobacillus preparation, it is a kind of digestant, draws for treating enteral abnormal fermentation
The intestinal tympanites that rises, diarrhea caused by indigestion etc..Now record in《Pharmacopoeia of People's Republic of China》2015 editions two, it is produced
Process should meet the requirement under Tiny ecosystem viable bacteria product introduction item, and production need to carry out determination of fatty acid with strain.Aliphatic acid is raw
One of basic structure component of object, most aliphatic acid exist with combining form in cell, and composition has important physiology
The lipid of function, they are the important substances for forming biomembrane.Modern microbiology research shows：It is general in the eucaryotic cell structure of bacterium
There is the homology of height all over the fatty acid composition that contains and the DNA of bacterium, the bacterium of different genera, the composition of its aliphatic acid and
Content shows different degrees of difference, and this species diversity is more stable, and various bacteriums have the cell of its characteristic
Aliphatic acid finger-print, and fatty acid analysis can distinguish genus and species and be clustered, its result and 16S rRNA gene orders point
Analysis has preferable uniformity.It there is no the method that fatty acid component is detected in alctasin strain, and strain fatty acid component at present
It is complicated, it is difficult to separate, conventional method complex steps, the problems such as required sample size is big, method is not suitable for promoting and applying.
The content of the invention
It is an object of the invention to propose the detection method of fatty acid component and application in a kind of alctasin strain, in kind
Level is upper to carry out production strain sequence analysis and base chiller, this method have be not required probe, it is easy to operate, when detecting
Between short, easy analysis.
To reach above-mentioned purpose, the technical proposal of the invention is realized in this way：
The detection method of fatty acid component, comprises the following steps in a kind of alctasin strain：
(1) collection and drying of thalline
Transfer by reference culture and by the isolated bacterial strain of galactenzyme raw cook in 100mL MRS culture mediums, 37 DEG C of cultures
48h~72h, in 4 000rmin-110min is centrifuged, 5~15 DEG C of centrifuging temperature removes supernatant, washed repeatedly with deionized water
Wash 2~5 times, obtained thalline is dried in vacuo 3h in 60 DEG C, and dry thalline is placed in drier, spare；
(2) methyl esterification of fatty acid
Precision weighs dry each 50mg of thalline and aliphatic acid reference substance respectively in screw-cap test tube, and 14% 3 is added to test tube
Boron fluoride-methanol or 1% sulfuric acid-methanol 1~5mL of solution, are filled with N immediately2Seal and carry out 5~15min of ultrasonic oscillation, put
In 60~80 DEG C of 25~40min of water-bath, 2mL n-hexanes are added after taking-up, shake 2~5min, take supernatant, then with 1mL just oneself
Alkane washs, and merges supernatant, with 0.22 μm of membrane filtration in clean sample injection bottle, for gas chromatography-mass spectrometry analysis；
(3) condition of gas chromatography-mass spectrum
Chromatographic condition：Chromatographic column is non-polar column, and carrier gas is helium, 250 DEG C of injector temperature, and sample size is 1 μ l, sample introduction
Split ratio 10:1, flow 1.0mLmin-1, temperature programming is 120 DEG C of holding 5min, then with 6 DEG C of min-1It is warming up to 240
DEG C, 10min is kept, then with 10 DEG C of min-1260 DEG C are warming up to, keeps 2min；
Mass Spectrometry Conditions：Ion gun EI, electron energy 70eV；Solvent delay 3min；EMV patterns are gain coefficient 1, scanning
Mode is full scan mode, and m/z is 35~450；Sample frequency is 2；
(4) data analysis and sample identification
After step (3), according to the original spectral data of acquisition, each fatty acid component is opposite in calculating detection sample
Content.
Further, step (1) the Plays bacterial strain for enterococcus faecalis 140623, (study by Chinese food drug assay
Institute, alctasin production are used), enterococcus faecalis CGMCC1.2135, Hai Shi enterococcus CGMCC1.595, enterococcus faecium CGMCC1.131
(China General Microbiological culture presevation administrative center), enterococcus faecium CICC21605, enterococcus faecalis CICC23658 (Chinese industrials
Microbiological Culture Collection administrative center), galactenzyme raw cook 20 batches, is respectively derived from 6 manufacturing enterprises.
Further, it is as follows the step of galactenzyme raw cook isolated strains in the step (1)：
Sample 10g is taken, adds 0.9% aseptic sodium chloride solution that 100ml is made, adds appropriate sterile glass beads, shakes up, be made 1：
10 suspension, with 0.9% aseptic sodium chloride solution gradient dilution.The suspension 1ml of 3 continuous acceptable diluent degree is taken, puts nothing
In bacterium plate, level at least two plate is often diluted.Inject 1% calcium carbonate of 15~20ml temperature no more than 45 DEG C and contain sugared beef fine jade
Fat culture medium, mixes, and solidifies, and is inverted, and when 37 DEG C of cultures 48 are small, the single bacterium colony that picking has transparent circle draws tablet, is separately cultured
Obtain aimed strain.
Further, 14% boron trifluoride-methanol or 1% sulfuric acid-methanol solution addition are 2mL in the step (2).
Further, aliphatic acid reference substance is C in the step (2)4~C24Fatty acid methyl ester mixing reference substance.
Further, chromatographic column is HP-5MS chromatographic columns in the step (3).
The detection method of fatty acid component is in alctasin strain sequence analysis and base chiller in a kind of alctasin strain
In application.
Relative to the prior art, the detection method of fatty acid component and application in a kind of alctasin strain of the present invention
Have the advantage that：
(1) in a kind of alctasin strain of the present invention fatty acid component detection method, it is proposed that a kind of alctasin
Bacterium identification method, the esterification method and step is simple, and without heating, esterification yield is high, and detection time is short, analysis side
Just；
(2) detection method of fatty acid component and application in a kind of alctasin strain of the present invention, by analyzing fat
Fat acid content and composition, carry out sequence analysis and base chiller, for improving alctasin in the level of kind to production strain
The quality and security of piece are of great significance.
Brief description of the drawings
The attached drawing for forming the part of the present invention is used for providing a further understanding of the present invention, schematic reality of the invention
Apply example and its explanation is used to explain the present invention, do not form inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 is the fatty acid methyl ester hybrid standard product total ion current gas chromatogram described in the embodiment of the present invention；
Fig. 2 is the reference culture total ion current gas chromatogram described in the embodiment of the present invention
Wherein A is 140623 total ion current gas chromatogram of enterococcus faecalis, and B is enterococcus faecalis
CGMCC1.2135 total ion current gas chromatograms, C are enterococcus faecium CGMCC1.131 total ion current gas-chromatographies
Figure, D is Hai Shi enterococcus CGMCC1.595 total ion current gas chromatograms, and E is enterococcus faecium CICC21605 total ion current gas phases
Chromatogram, F are enterococcus faecalis CICC23658 total ion current gas chromatograms；
Fig. 3 is the LH galactenzyme raw cook bacterial strain total ion current gas chromatograms described in the embodiment of the present invention
Wherein A is 10090903 total ion current gas chromatogram of lot number, and B is 10091403 total ion current gas-chromatography of lot number
Figure；
Fig. 4 is the GL galactenzyme raw cook bacterial strain total ion current gas chromatograms described in the embodiment of the present invention
Wherein A is 100601 total ion current gas chromatogram of lot number, and B is 101102 total ion of lot number (batch number)
Flow gas chromatogram；
Fig. 5 is the HB galactenzyme raw cook bacterial strain total ion current gas chromatograms described in the embodiment of the present invention
Wherein A is 20101012 total ion current gas chromatogram of lot number, and B is 20101101 total ion current gas-chromatography of lot number
Figure, C is 20101016 total ion current gas chromatogram of lot number, and D is 20101110 total ion current gas chromatogram of lot number；
Fig. 6 is the LY galactenzyme raw cook bacterial strain total ion current gas chromatograms described in the embodiment of the present invention
A is 101008 total ion current gas chromatogram of lot number, and B is 110302 total ion current gas chromatogram of lot number；
Fig. 7 is the SD galactenzyme raw cook bacterial strain total ion current gas chromatograms described in the embodiment of the present invention
A is 20100922 total ion current gas chromatogram of lot number, and B is 20101001 total ion current gas chromatogram of lot number, C
For 20101124 total ion current gas chromatogram of lot number, D is 20101212 total ion current gas chromatogram of lot number；
Fig. 8 is the SMX galactenzyme raw cook bacterial strain total ion current gas chromatograms described in the embodiment of the present invention
A is 20100922 total ion current gas chromatogram of lot number, and B is 20101001 total ion current gas chromatogram of lot number, C
For 20101124 total ion current gas chromatogram of lot number, D is 20101212 total ion current gas chromatogram of lot number.
Embodiment
With reference to embodiment and attached drawing, the present invention will be described in detail.
Embodiment 1
The detection method of fatty acid component, comprises the following steps in a kind of alctasin strain：
(1) collection and drying of thalline
Transfer by reference culture and by the isolated bacterial strain of galactenzyme raw cook in 100mL MRS culture mediums, 37 DEG C of cultures
48h~72h, in 4000rmin-110min is centrifuged, 8 DEG C of centrifuging temperature, removes supernatant, is washed repeatedly with deionized water 3 times,
Obtained thalline is dried in vacuo 3h in 60 DEG C, and dry thalline is placed in drier, spare；
(2) methyl esterification of fatty acid
Precision weighs dry thalline or aliphatic acid reference substance 50mg in screw-cap test tube, and it is borontrifluoride to add 14% to test tube
Boron-methanol or 1% sulfuric acid-methanol solution 2mL, are filled with N immediately2Seal and carry out ultrasonic oscillation 10min, be placed in 60 DEG C of water-baths
30min, adds 2mL n-hexanes after taking-up, shake 3min, take supernatant, then is washed with 1mL n-hexanes, merges supernatant, uses
0.22 μm of membrane filtration is in clean sample injection bottle, for gas chromatography-mass spectrometry analysis；
(3) condition of gas chromatography-mass spectrum
Chromatographic condition：Chromatographic column is HP-5MS chromatographic columns, and carrier gas is helium, and 250 DEG C of injector temperature, sample size is 1 μ l,
Sample introduction split ratio 10:1, flow 1.0mLmin-1, temperature programming is 120 DEG C of holding 5min, then with 6 DEG C of min-1It is warming up to
240 DEG C, 10min is kept, then with 10 DEG C of min-1260 DEG C are warming up to, keeps 2min；
Mass Spectrometry Conditions：Ion gun EI, electron energy 70eV；Solvent delay 3min；EMV patterns are gain coefficient 1, scanning
Mode is full scan mode, and m/z is 35~450；Sample frequency is 2.
(4) data analysis and sample identification
After step (3), according to the original spectral data of acquisition, each fatty acid component is opposite in calculating detection sample
Content.
Further, step (1) the Plays bacterial strain for enterococcus faecalis 140623, (study by Chinese food drug assay
Institute, alctasin production are used), enterococcus faecalis CGMCC1.2135, Hai Shi enterococcus CGMCC1.595, enterococcus faecium CGMCC1.131
(China General Microbiological culture presevation administrative center), enterococcus faecium CICC21605, enterococcus faecalis CICC23658 (Chinese industrials
Microbiological Culture Collection administrative center), galactenzyme raw cook 20 batches, is respectively derived from 6 manufacturing enterprises.
It is as follows the step of galactenzyme raw cook isolated strains in the step (1)：
Sample 10g is taken, adds 0.9% aseptic sodium chloride solution that 100ml is made, adds appropriate sterile glass beads, shakes up, be made 1：
10 suspension, with 0.9% aseptic sodium chloride solution gradient dilution.The suspension 1ml of 3 continuous acceptable diluent degree is taken, puts nothing
In bacterium plate, level at least two plate is often diluted.Inject 1% calcium carbonate of 15~20ml temperature no more than 45 DEG C and contain sugared beef fine jade
Fat culture medium, mixes, and solidifies, and is inverted, and when 37 DEG C of cultures 48 are small, the single bacterium colony that picking has transparent circle draws tablet, is separately cultured
Obtain aimed strain.
Aliphatic acid reference substance is C in the step (2)4~C24Fatty acid methyl ester mixing reference substance.
Fatty acid methyl ester mixing (C4~C24) standard items have 40 components.Under the conditions of GC-MS, retention time is by 6min
To totally 33 fatty acid components, and be respectively provided with higher abundance of isolated C10~C24 between 35min (see Fig. 1).Wherein
The content highest of C18 components, each main fatty acid component and the matching degree in mass spectrometric data storehouse (are shown in Table 1) more than 95%.Examination
Test and show, which can identify the fatty acid component of C10~C24.
1 fatty acid methyl ester of table mixes the retention time and relative amount of main fatty acid component in mark
Note (note)：ΔExpression double bond [Δdouble bond]
(4) the GC-MS detections of bacterial strain aliphatic acid
After reference culture and the galactenzyme raw cook bacterial strain methyl esterification of fatty acid of 6 manufacturing enterprises, GC-MS analyses, knot are carried out
Fruit sees Fig. 2-8.The results show that occur ten in 6 reference cultures and the alctasin bacterial strain gas chromatogram of 6 manufacturing enterprises
The main methyl esters peak such as four carbon alkanoic acids, hexadecanoic acid, gaidic acid, octadecanoic acid and octadecenoic acid, and and fat
Fatty acid methyl esters hybrid standard product it is consistent；Meanwhile also having the characteristic peak of nonadecane acid, the matching degree in mass spectrometric data storehouse exists
More than 94%.The gas chromatogram and each fat of enterococcus faecium CGMCC1.131 and enterococcus faecalis 140623 in 6 reference cultures
The ratio of acid constituents is close, and has larger difference with other 4 reference cultures.Hai Shi enterococcus CGMCC1.595 and SMX companies
Lot number is similar with the gas chromatogram of 20101001 alctasin bacterial strain for 20100922；Enterococcus faecalis 140623 and 6 productions
The gas chromatogram of other alctasin bacterial strains of enterprise is similar, and calculating each fatty acid component percentage with area normalization method contains
Amount, the ratio of obtained each fatty acid component is close, is shown in Table 2.
The relative amount A (%) of 2 reference culture of table and galactenzyme raw cook bacterial strain fatty acid component
Note (note)：CGMCC 1.595、CGMCC 1.131、CGMCC 1.2135、CICC 23658、CICC 21605、
140623 number for reference culture, remaining is each producer's galactenzyme raw cook lot number [CGMCC 1.595, CGMCC 1.131, CGMCC
1.2135,CICC 23658,CICC 21605]
(5) examination of method stability
The stability of instrument takes 5 parts of the fatty acid methyl ester hybrid standard product of same batch same concentrations, parallel sample introduction.With
As benchmark, Average residence time 11.437min, RSD value are 0.01% (n=5) at three peaks；Average peak area is 258
799 329, RSD value are 2.07% (n=5).Illustrate that instrument has preferable stability.
The repeatability of formicester method takes the enterococcus faecalis 140623 of phase homogenous quantities to dry 5 parts of thalline, carries out 14% trifluoro
Change the parallel laboratory test that boron-methanol carries out esterification.Using the constituent content ratio at the 3rd peak and first peak as benchmark, they
Average value be that 2.864, RSD values are 1.30% (n=5).Illustrate that esterification method has preferable repeatability.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
With within principle, any modification, equivalent replacement, improvement and so on, should all be included in the protection scope of the present invention god.
Claims (7)
- A kind of 1. detection method of fatty acid component in alctasin strain, it is characterised in that：Comprise the following steps：(1) collection and drying of thallineTransfer by reference culture and by the isolated bacterial strain of galactenzyme raw cook in 100mL MRS culture mediums, 37 DEG C of culture 48h~ 72h, in 4 000rmin-110min is centrifuged, 5~15 DEG C of centrifuging temperature, removes supernatant, 2~5 are washed repeatedly with deionized water Secondary, obtained thalline is dried in vacuo 3h in 60 DEG C, and dry thalline is placed in drier, spare；(2) methyl esterification of fatty acidPrecision weighs dry each 50mg of thalline and aliphatic acid reference substance respectively in screw-cap test tube, adds 14% 3 to test tube respectively Boron fluoride-methanol or 1% sulfuric acid-methanol 1~5mL of solution, are filled with N immediately2Seal and carry out 5~15min of ultrasonic oscillation, put In 60~80 DEG C of 25~40min of water-bath, 2mL n-hexanes are added after taking-up, shake 2~5min, take supernatant, then with 1mL just oneself Alkane washs, and merges supernatant, with 0.22 μm of membrane filtration in clean sample injection bottle, for gas chromatography-mass spectrometry analysis；(3) condition of gas chromatography-mass spectrumChromatographic condition：Chromatographic column is non-polar column, and carrier gas is helium, and 250 DEG C of injector temperature, sample size is 1 μ l, and sample introduction shunts Than 10:1, flow 1.0mLmin-1, temperature programming is 120 DEG C of holding 5min, then with 6 DEG C of min-1240 DEG C are warming up to, is protected 10min is held, then with 10 DEG C of min-1260 DEG C are warming up to, keeps 2min；Mass Spectrometry Conditions：Ion gun EI, electron energy 70eV；Solvent delay 3min；EMV patterns are gain coefficient 1, scan mode For full scan mode, m/z is 35~450；Sample frequency is 2；(4) data analysis and sample identificationAfter step (3), according to the original spectral data of acquisition, calculate the opposite of each fatty acid component in detection sample and contain Amount.
- 2. the detection method of fatty acid component in a kind of alctasin strain according to claim 1, it is characterised in that：It is described Step (1) Plays bacterial strain is enterococcus faecalis 140623, enterococcus faecalis CGMCC1.2135, Hai Shi enterococcus CGMCC1.595, dung Enterococcus CGMCC1.131, enterococcus faecium CICC21605, enterococcus faecalis CICC23658.
- 3. the detection method of fatty acid component in a kind of alctasin strain according to claim 1, it is characterised in that：It is described It is as follows the step of galactenzyme raw cook isolated strains in step (1)：Sample 10g is taken, adds 0.9% aseptic sodium chloride solution that 100ml is made, adds appropriate sterile glass beads, shakes up, be made 1：10 Suspension, with 0.9% aseptic sodium chloride solution gradient dilution, takes the suspension 1ml of 3 continuous acceptable diluent degree, puts sterile flat In ware, level at least two plate is often diluted, 1% calcium carbonate of 15~20ml of injection temperature no more than 45 DEG C contains sugared beef agar training Base is supported, is mixed, is solidified, is inverted, when 37 DEG C of cultures 48 are small, the single bacterium colony that picking has transparent circle draws tablet, is separately cultured to obtain Aimed strain.
- 4. the detection method of fatty acid component in a kind of alctasin strain according to claim 1, it is characterised in that：It is described 14% boron trifluoride-methanol or 1% sulfuric acid-methanol solution addition are 2mL in step (2).
- 5. the detection method of fatty acid component in a kind of alctasin strain according to claim 1, it is characterised in that：It is described Aliphatic acid reference substance is C in step (2)4~C24Fatty acid methyl ester mixing reference substance.
- 6. the detection method of fatty acid component in a kind of alctasin strain according to claim 1, it is characterised in that：It is described Chromatographic column is HP-5MS chromatographic columns in step (3).
- 7. if the detection method of fatty acid component in a kind of alctasin strain of claim 1-6 any one of them is in alctasin bacterium Application in kind sequence analysis and base chiller.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110146638A (en) * | 2019-05-22 | 2019-08-20 | 遵义医科大学 | A method of fatty acid species in measurement actinomyces novel species M8JJ-5 |
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Application publication date: 20180424 |