CN109613139A - The method that parting is carried out to beta lactoglobulin based on high performance liquid chromatography - Google Patents
The method that parting is carried out to beta lactoglobulin based on high performance liquid chromatography Download PDFInfo
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- CN109613139A CN109613139A CN201910004121.6A CN201910004121A CN109613139A CN 109613139 A CN109613139 A CN 109613139A CN 201910004121 A CN201910004121 A CN 201910004121A CN 109613139 A CN109613139 A CN 109613139A
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- 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
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Abstract
The present invention relates to a kind of methods for carrying out parting to beta lactoglobulin based on high performance liquid chromatography, include the following steps: step S1: utilizing freezing milk sample preparation parting sample；Step S2: to performance liquid chromatographic column sample introduction；Step S3: respectively in different time points with the elution sample of different volumes；Step S4: by beta lactoglobulin parting being A- beta lactoglobulin and B- beta lactoglobulin according to the retention time of appearance after parting sample elution and peak area parameter.The method provided by the invention for carrying out parting to beta lactoglobulin based on high performance liquid chromatography, favorable reproducibility, accuracy is high, can be realized the batch detection to sample, greatly reduces analysis time, and method is simple and easy, convenient and reliable.
Description
Technical field
The present invention relates to macromolecule liquid phase separation techniques fields, specifically design one kind based on high performance liquid chromatography to β milk-globule
The method of albumen progress parting.
Background technique
Beta lactoglobulin (β-lactglob μ lin, β-Lg) is a kind of peculiar whey that secretion is synthesized by galactophore epithelial cell
Albumen is one kind of protein in fresh milk, accounts for the 7%-12% of fresh milk protein, is primarily present in ox and other ruminants cream
In albumin.From the discovery beta lactoglobulin such as Aschaffenb μ rg there are since two kinds of genetic variants of A, B, researcher is to cream
The genetic polymorphism of middle main protein has made extensive and intensive studies.
On the one hand result of study discloses the type and existing difference of different mammalian milk proteins genetic variants,
Affiliation, evolution etc. between research bio-diversity, species provide valuable data；Lactoprotein is also shown simultaneously
There are certain correlations for certain economic characters of genetic polymorphism and animal, such as milk production and its curdled milk performance of composition, cream
With processing characteristics etc..Therefore, the research of lactoprotein genetic polymorphism just has theory significance and practical application value.
Mao (1992), Sacch (1993) are waited and are found β-Lg genotype and butterfat percnetage in the research to holstein cow
It is related；Berg (1992) Nian Faxian β-Lg genotype in Holstein cow is related to casein total amount in cream.
Summary of the invention
In order to need to select the lactoprotein of respective type according to real economy, the present invention provides one kind to be based on efficient liquid phase
The method that chromatography carries out parting to beta lactoglobulin, includes the following steps:
Step S1: parting sample is prepared using freezing milk sample；
Step S2: to performance liquid chromatographic column sample introduction；
Step S3: respectively in different time points with the elution sample of different volumes；
Step S4: it is by beta lactoglobulin parting according to the retention time of appearance after parting sample elution and peak area parameter
A- beta lactoglobulin and B- beta lactoglobulin.
Wherein, the step S1 includes:
Step S11: the first isometric working solution is added into the freezing milk sample of packing, oscillation 10s is mixed after sample melts
It is even, it is stored at room temperature 1 hour；
Step S12: by mixed liquor obtained by step S11 under 4 degrees Celsius, with 16000g speed centrifugation 5 minutes；
Step S13: removing skim-coat butterfat, bottom solution is placed in new centrifuge tube；
Step S14: the second working solution is added with dilution step S13 resulting solution with the volume of 1:3；
Step S15: filtered dilutions must be intended to the sample of parting.
Wherein, first working solution by following molar concentration solution mixing system at: 0.1mol/L it is bis--trihydroxy methyl ammonia
Methylmethane buffer, 6mol/L guanidine hydrochloride, 5.37mol/L sodium citrate and 19.5mol/L dithiothreitol (DTT).
Wherein, second working solution is the guanidine hydrochloride solution that molar concentration is 4.5mol/L, and the second working solution is molten
Liquid is consistent with the first eluent when elution.
Wherein, the Loading sequence in the step S2 includes:
Step S21: carrying out 60 minutes initial balances before sample introduction, guarantees detection line and pressure line in steady state；
Step S22: one needle standard sample of detection；
Step S23: blank control is carried out into first eluent of needle, it is ensured that carry out the inspection of normal sample under normal circumstances
It surveys；
Step S24: sample introduction product, also, every three sample needle carries out a needle blank control.
Wherein, in the step S3, flow velocity when elution is 0.5mL/min, and column temperature absorbs wave between 40-50 degrees Celsius
For length between 210-230nm, sampling volume is 10 μ L.
Wherein, in the step S3, the first eluent is the trifluoroacetic acid aqueous solution that mass concentration is 0.1%, and second washes
De- liquid is the trifluoroacetic acid solution that mass concentration is 0.1%, and solution system is acetonitrile.
Wherein, in the step S3, the volume ratio of the first eluent and the second eluent is between 1:3-3:1.
Wherein, in the step S3, the volume fraction of the first eluent rises to 67% by 57%, the volume of the second eluent
Score is down to 33% by 43%.
The method provided by the invention that parting is carried out to beta lactoglobulin based on high performance liquid chromatography, favorable reproducibility, accuracy
Height can be realized the batch detection to sample, greatly reduce analysis time, and method is simple and easy, convenient and reliable.
Detailed description of the invention
Fig. 1: the present invention separates situation its appearance period under different eluting liquid fractions.
Fig. 2: the present invention is in the identical situation of other separation conditions, the separation figure of different chromatographic columns.
Fig. 3: the liquid chromatogram of standard items A- beta lactoglobulin.
Fig. 4: the liquid chromatogram of standard items B- beta lactoglobulin.
Fig. 5: the liquid chromatogram of the separated standard items mixed type AB- beta lactoglobulin of the present invention.
Specific embodiment
In order to have further understanding to technical solution of the present invention and beneficial effect, it is described in detail with reference to the accompanying drawing
Technical solution of the present invention and its beneficial effect of generation.
In a preferred embodiment of the invention, the method that parting is carried out to beta lactoglobulin by high performance liquid chromatography, specifically
Operating method and quality control process it is as follows:
One, the preparation of sample
The first isometric working solution is added into the 500 μ L of freezing milk sample of packing, oscillation 10s is mixed after sample melts,
It is stored at room temperature 1h.By mixed liquor at 4 DEG C 16000g be centrifuged 5min, remove surface layer butterfat, take 300 μ L of bottom solution in it is new from
In heart pipe, be added the second working solution dilution (volume ratio 1:3), mix, 0.45 μm of organic system membrane filtration into sample injection bottle to
It surveys.
Wherein, the first working solution be 0.1mol/L BisTris, 6mol/L guanidine hydrochloride, 5.37mmol/L sodium citrate and
The mixed solution of 19.5mmol/L DTT.
Second working solution is 4.5mol/L guanidine hydrochloride solution, the first eluent when solution is elution.
Two, efficient liquid phase testing conditions
1, the mobile phase eluent needed for preparing, with suitable 0.45 μm of membrane filtration, ultrasonic degassing 15 minutes.
The aqueous solution of first eluent A:0.1%TFA；
The acetonitrile solution of second eluent B:0.1%TFA.
2, as follows according to method setting each parameter of instrument:
Flow velocity: 0.5mL/min
Column temperature: 45 DEG C
Absorbing wavelength: 214nm
Sampling volume: 10 μ L
Specifically, inventor is optimizing the experimental stage, under identical separation condition, under 40-45 degrees Celsius of column temperature
The separation of lactoglobulin has been carried out, discovery column temperature influences less chromatogram, but when column temperature raising, various beta lactoglobulin
Chromatographic peak quality makes moderate progress, therefore selects column temperature for 45 degrees Celsius.
The gradient of liquid phase elution is shown in Table 1, wherein A indicates that the first eluent, B indicate that the second eluent, percentage indicate body
Fraction.
Specifically, Fig. 1 is that the present invention separates situation its appearance period under different eluting liquid fractions, such as Fig. 1 institute
Show, present invention is generally directed to the appearance periods of 32-35 minutes various beta lactoglobulins to optimize: the first eluent A phase liquid by
57% rises to 67%, when the second eluent B phase liquid is down to 33% by 43%, the chromatographic peak separation of the beta lactoglobulin of tri- type of A, B, C
Preferably, in other ratios, each beta lactoglobulin cannot meet the requirement all preferably separated in addition to c-type simultaneously, and it is fixed to influence
Amount.
1 liquid chromatogram gradient table of table
3, efficient liquid phase system
Liquid phase systems are Agilent 1260, and chromatographic column is 3.5 μm of C8,100×4.6I.D.(Zorbax
300SB-C8RP, Agilent Technologies).
The autosampler of liquid chromatograph is furnished with the quantitative loop of 100 μ L.
Specifically, Fig. 2 is the present invention in the identical situation of other separation conditions, the separation figure of different chromatographic columns, wherein
The corresponding RX-C8 separation figure in figure top, the corresponding 300SB-C8 separation figure in figure lower section, as shown in Figure 2: RX-C8 is to beta lactoglobulin without guarantor
It stays or retention time is too long, better separating effect can be obtained using 300SB-C8 biochemical measurement chromatographic column.
4, quality controls
60min initial balance is carried out before every batch of sample feeding, guarantees detection line (baseline) and pressure line in steady state
Sample introduction, first detects a needle standard sample, then carries out blank elution into first eluent of needle blank, it is ensured that in normal situation into
The detection of row normal sample.Since sample matrix is complex, before sample introduction be provided with sample introduction needle outer wall washing, i.e., every needle into
Sample introduction needle is rinsed three times after complete sample；In addition, every three samples carry out needle blank elution, every 15 samples or so add one
Standard items detection, to avoid the drift of retention time.
5, the preparation of standard items
β-Lactoglob μ lin B from bovine milk (Sigma-Alorich, >=90% (PAGE).
It prepares beta lactoglobulin standard solution: accurately weighing beta lactoglobulin standard items 10.00mg in 1ml and have plug scale
In test tube, the standard items stock solution for the 10g/L being made into, 4 DEG C are saved backup.
Three, testing result
Fig. 3 to Fig. 5 is respectively standard items A- beta lactoglobulin, Type B-beta lactoglobulin and separated standard items of the invention
The liquid chromatogram of mixed type AB- beta lactoglobulin.As shown in Figure 3-Figure 5, according to retention time corresponding in standard items and sample
With the peak area of various protein, so that it may carry out qualitative and quantitative, standard to A- beta lactoglobulin, B- beta lactoglobulin in sample
The parameters such as sample retention time and peak area are shown in Table 2.Through comparing, A- beta lactoglobulin, B- beta lactoglobulin peak sequence are as follows: B- β
Lactoglobulin, A- beta lactoglobulin.
It is calculated using area normalization method known to after the content of A- beta lactoglobulin, B- beta lactoglobulin: two kinds of β-milk-globule egg
White separating degree can reach R=3.3, and result favorable reproducibility.
The parameters such as 2 standard sample retention time of table and peak area
Beneficial effects of the present invention are as follows:
1, the present invention establishes a kind of method for carrying out parting to A- beta lactoglobulin, B- beta lactoglobulin based on HPLC method.
2, favorable reproducibility of the present invention, accuracy is high, can be realized the batch detection to sample, greatly reduces analysis time,
And method is simple and easy, and it is convenient and reliable.
Although the present invention is illustrated using above-mentioned preferred embodiment, the protection model that however, it is not to limit the invention
It encloses, anyone skilled in the art are not departing within the spirit and scope of the present invention, and opposite above-described embodiment carries out various changes
It is dynamic still to belong to the range that the present invention is protected with modification, therefore protection scope of the present invention subjects to the definition of the claims.
Claims (9)
1. a kind of method for carrying out parting to beta lactoglobulin based on high performance liquid chromatography, which comprises the steps of:
Step S1: parting sample is prepared using freezing milk sample；
Step S2: to performance liquid chromatographic column sample introduction；
Step S3: respectively in different time points with the elution sample of different volumes；
Step S4: by beta lactoglobulin parting being A- β according to the retention time of appearance after parting sample elution and peak area parameter
Lactoglobulin and B- beta lactoglobulin.
2. the method for carrying out parting to beta lactoglobulin based on high performance liquid chromatography as described in claim 1, which is characterized in that
The step S1 includes:
Step S11: the first isometric working solution is added into the freezing milk sample of packing, oscillation 10s is mixed after sample melts, room
Temperature stands 1 hour；
Step S12: by mixed liquor obtained by step S11 under 4 degrees Celsius, with 16000g speed centrifugation 5 minutes；
Step S13: removing skim-coat butterfat, bottom solution is placed in new centrifuge tube；
Step S14: the second working solution is added with dilution step S13 resulting solution with the volume of 1:3；
Step S15: filtered dilutions must be intended to the sample of parting.
3. the method for carrying out parting to beta lactoglobulin based on high performance liquid chromatography as claimed in claim 2, which is characterized in that
First working solution by following molar concentration solution mixing system at: 0.1mol/L it is bis--TRIS buffer,
6mol/L guanidine hydrochloride, 5.37mol/L sodium citrate and 19.5mol/L dithiothreitol (DTT).
4. the method for carrying out parting to beta lactoglobulin based on high performance liquid chromatography as claimed in claim 2, which is characterized in that
Second working solution is the guanidine hydrochloride solution that molar concentration is 4.5mol/L, and when the solution of the second working solution and elution
One eluent is consistent.
5. the method for carrying out parting to beta lactoglobulin based on high performance liquid chromatography as described in claim 1, which is characterized in that
Loading sequence in the step S2 includes:
Step S21: carrying out 60 minutes initial balances before sample introduction, guarantees detection line and pressure line in steady state；
Step S22: one needle standard sample of detection；
Step S23: blank control is carried out into first eluent of needle, it is ensured that carry out the detection of normal sample under normal circumstances；
Step S24: sample introduction product, also, every three sample needle carries out a needle blank control.
6. the method for carrying out parting to beta lactoglobulin based on high performance liquid chromatography as described in claim 1, it is characterised in that:
In the step S3, flow velocity when elution is 0.5mL/min, and column temperature is between 40-50 degrees Celsius, and absorbing wavelength is between 210-
230nm, sampling volume are 10 μ L.
7. the method for carrying out parting to beta lactoglobulin based on high performance liquid chromatography as described in claim 1, it is characterised in that:
In the step S3, the first eluent is the trifluoroacetic acid aqueous solution that mass concentration is 0.1%, and the second eluent is mass concentration
For 0.1% trifluoroacetic acid solution, and solution system is acetonitrile.
8. the method for carrying out parting to beta lactoglobulin based on high performance liquid chromatography as described in claim 1, it is characterised in that:
In the step S3, the volume ratio of the first eluent and the second eluent is between 1:3-3:1.
9. the method for carrying out parting to beta lactoglobulin based on high performance liquid chromatography as described in claim 1, it is characterised in that:
In the step S3, the volume fraction of the first eluent rises to 67% by 57%, and the volume fraction of the second eluent is down to by 43%
33%。
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5756680A (en) * | 1994-01-05 | 1998-05-26 | Sepragen Corporation | Sequential separation of whey proteins and formulations thereof |
| CN101613408A (en) * | 2009-08-06 | 2009-12-30 | 浙江贝因美科工贸股份有限公司 | The separation of whey-protein and measuring method |
| CN102539575A (en) * | 2012-01-17 | 2012-07-04 | 上海德诺产品检测有限公司 | Method for detecting content of beta-lactoglobulin in dairy product |
| CN106793797A (en) * | 2014-08-15 | 2017-05-31 | 预层析股份有限公司 | The method for separating alpha-lactalbumin and beta lactoglobulin |
| CN108072725A (en) * | 2016-11-09 | 2018-05-25 | 北京奶牛中心 | The method to beta-casein parting in dairy products based on high performance liquid chromatography |
-
2019
- 2019-01-03 CN CN201910004121.6A patent/CN109613139A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5756680A (en) * | 1994-01-05 | 1998-05-26 | Sepragen Corporation | Sequential separation of whey proteins and formulations thereof |
| CN101613408A (en) * | 2009-08-06 | 2009-12-30 | 浙江贝因美科工贸股份有限公司 | The separation of whey-protein and measuring method |
| CN102539575A (en) * | 2012-01-17 | 2012-07-04 | 上海德诺产品检测有限公司 | Method for detecting content of beta-lactoglobulin in dairy product |
| CN106793797A (en) * | 2014-08-15 | 2017-05-31 | 预层析股份有限公司 | The method for separating alpha-lactalbumin and beta lactoglobulin |
| CN108072725A (en) * | 2016-11-09 | 2018-05-25 | 北京奶牛中心 | The method to beta-casein parting in dairy products based on high performance liquid chromatography |
Non-Patent Citations (4)
| Title |
|---|
| DING, XIAOJING 等: "Analysis of α-lactalbumin, β-lactoglobulin A and B in whey protein powder, colostrum, raw milk, and infant formula by CE and LC", 《DAIRY SCIENCE & TECHNOLOGY》 * |
| 周立 等: "《仪器分析技术》", 30 November 2018 * |
| 王浩: "乳品中七种主要蛋白质分离与定量的新方法研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 董爱军 等: "高效液相色谱法分离测定牦牛乳中的8种蛋白质", 《分析测试学报》 * |
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Application publication date: 20190412 |