CN110221007A - Adulterated detection method in a kind of identification buffalo's milk - Google Patents
Adulterated detection method in a kind of identification buffalo's milk Download PDFInfo
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Abstract
The invention discloses detection methods adulterated in a kind of identification buffalo's milk, the following steps are included: first sample acquisition and instrument prepare, the mixed solution of BisTris, guanidine hydrochloride, sodium citrate and DTT are prepared again as working solution I, prepare guanidine hydrochloride solution as working solution II；Then fetch water respectively cow's milk and milk sample to be measured is appropriate and perform the following operation: isometric working solution I is added, is centrifuged and skims upper-layer fat again after vortex mixing, bottom solution is taken to be placed in new centrifuge tube, the mixing of working solution II is added through membrane filtration；It is subsequently placed in test in efficient liquid phase automatic sampling chromatograph and obtains fingerprint chromatogram；Fingerprint chromatogram is compared.The present invention provides the detection methods that black-and-white flower cow's milk whether is adulterated in a kind of quickly detection buffalo's milk, this method has good reproducibility and specificity, the representative finger-print of sample room can be obtained, can analyze whether mix He Sitan milk supply in buffalo milk to fast and stable.
Description
Technical field
The present invention relates to technical field of food detection, in particular to adulterated detection method in a kind of identification buffalo's milk.
Background technique
Buffalo milk currently on the market is very different, and there are more the phenomenon that pretending to be buffalo's milk with common milk cow's milk.And water
Cow's milk nutriment is abundant, studies have shown that in addition to lactose, total solid, fat, protein, ash content are above milk and people's milk
[1], especially protein, average content are about 4.5%, about the 1.5 of holstein cow times, 2.8 times of human breast milk, are praised
For " king in milk ", there is the title of " Chinese cheese ".Studies have shown that buffalo's milk has various active polypeptide, disappear with antibacterial
Effect that is scorching, enhancing immune, anti-oxidant, anti-aging, drop ester decompression and anticancer.In the market, buffalo's milk price is about He Sitan
3 times of ox cow's milk.This makes illegal businessman blend black-and-white flower cream in buffalo's milk, and milk quality is also resulted in while reaping staggering profits
The hidden danger of amount and safety.
Judging other detection method for buffalo's milk at present mainly has: LTQ-Orbitrap liquid-mass chromatography technical appraisement water
Milk casein, that is, using LTQ-Orbitrap liquid-mass chromatography technology respectively to 4 kinds of main components of newborn source casein into
Row analysis, search database obtain the overall amino acid sequence of 4 kinds of components, are compared with 4 kinds of Caseinum componemts of buffalo milk；Capillary
Blood vessel electrophoresis system analyzes buffalo milk casein and carries out quantitative detection；Based on mass spectrographic finger-print identification buffalo milk casein and
Soybean casein otherness；Utilize buffalo milk composition in real-time fluorescent PCR technology detection dairy products etc..The above several method is all
Carry out the identification of buffalo source by research object of casein in buffalo milk, and instrument equipment is expensive, conventional foundation laboratory without
Method matching.And Holstein cow quantity is more, and the output of milk of lactation period is big, thus black-and-white flower cream is at the first choice for blending buffalo's milk,
It is, thus, sought for a kind of solution solves the above problems.
Summary of the invention
It is an object of the invention to: in view of the above problems, provide whether a kind of quickly detect mixes in buffalo's milk
The detection method of miscellaneous black-and-white flower cow's milk, this method have good reproducibility and specificity, and can obtain sample room has representative
Property finger-print, can fast and stable analyze He Sitan milk supply whether is mixed in buffalo milk.
In order to achieve the above-mentioned object of the invention, The technical solution adopted by the invention is as follows:
Adulterated detection method in a kind of identification buffalo's milk, comprising the following steps:
(1) sample acquisition and instrument prepare: it is stored refrigerated after acquisition buffalo's milk and milk sample to be measured, while getting out height
It imitates liquid phase automatic sampling chromatograph and its matches chromatographic column and clean up spare；
(2) prepared by solution: preparing the mixed solution of BisTris, guanidine hydrochloride, sodium citrate and DTT as working solution I, matches
Guanidine hydrochloride solution processed is as working solution II；
(3) prepared by test sample: taking the buffalo's milk acquired in step (1) respectively and milk sample to be measured is appropriate and progress is following
Operation: isometric working solution I is added, forms mixed liquor after vortex mixing and stands, then mixed liquor is centrifuged and skims upper layer rouge
Fat takes bottom solution to be placed in new centrifuge tube, and working solution II is added and is uniformly mixed and obtains upper machine test product after membrane filtration；
(4) test sample is tested: machine test product on the buffalo's milk and cow's milk to be measured that prepare in step (3) is placed in efficient liquid phase certainly
In dynamic sample introduction chromatograph and test condition progress gradient elution test is set, obtains fingerprint chromatogram；(5) interpretation of result: by step
(4) buffalo's milk and the fingerprint chromatogram of cow's milk to be measured test are compared in, by finding to buffalo's milk Protein Separation situation
The characteristic peak not interfered identifies cow's milk to be measured.
Further, in step (1), the stored refrigerated temperature is 4 DEG C, and the chromatographic column is ZORBAX
300SB-C8 column.
Further, in step (2), the concentration of BisTris is 0.1-0.3mol/L, guanidine hydrochloride in the working solution I
Concentration be 3-8mol/L, the concentration of sodium citrate is 5-7mmol/L, and the concentration of DTT is 15-25mmol/L, the working solution
Guanidine hydrochloride 3-6mol/L in II.
Further, in step (3), the time of repose is 1-1.5h, and the temperature of the mixed liquor centrifugation is 3-10
DEG C, described to take the volume ratio of bottom solution and working solution II for (1-3): (3-10), the aperture of the diafiltration membrane are 0.2-
0.5um。
Further, in step (4), the efficient liquid phase automatic sampling chromatograph test condition: Detection wavelength
214nm, 35-45 DEG C of column temperature, sample volume 5-10ul, flow velocity 0.5-1.0mL/min, mobile phase includes mobile phase A and Mobile phase B.
Further, the mobility A is 0.1%TFA aqueous solution, and Mobile phase B is 0.1%TFA acetonitrile.
Further, the solvent of guanidine hydrochloride solution is mobility A0.1%TFA aqueous solution in the working solution II.
In conclusion by adopting the above-described technical solution, the beneficial effects of the present invention are:
The present invention is in precision, repeatability and stability experiment, the κ-CN/A, α s2-CN, κ-CN/B, the α s1- that isolate
CN, β-CN, β-LgB, α-La peak area relative standard deviation (RSD%) < 5%；Discovery characteristic peak is compared by finger-print,
As adulterated label, can be detected when non-buffalo cow's milk incorporation 2%, in the range of the incorporation non-buffalo cow's milk of 5%-40%
Interior, related coefficient is greater than 0.99.This method has good reproducibility and specificity, and it is representative can to obtain sample room
Finger-print can analyze non-aqueous source of milk whether is mixed in buffalo milk to fast and stable.
Lactoprotein is main ingredient in cow's milk, and the present invention is based on HPLC to carry out Protein Separation to cow's milk, establishes fingerprint image
Spectrum, carries out analysis comparison for 6 kinds of lactoproteins, and specificity is strong, easy to operate, favorable reproducibility, and detection lower bound meets actual demand,
Purity detecting quickly can be carried out to buffalo's milk sample.The otherness of protein expression, buffalo can also be analyzed from finger-print
In finger-print, similarity is related to Polymorphisms of Milk Protein, and the similarity between S9, S12 and S14 is higher than the phase with other samples
Like degree, illustrate that three's protein expression homology is higher.
Detailed description of the invention
Fig. 1 is respectively using chromatographic column ZORBAX 300SB-C8 (A) and Welch XB-C18 (B) to same sample albumen
Isolated HPLC chromatogram；
Fig. 2 is the finger-print of buffalo lactoprotein；
Fig. 3 is Holstein cow lactoprotein finger-print；
Fig. 4 is that 40% Holstein cow lactoprotein finger-print is mixed in buffalo's milk.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention more comprehensible, preferred embodiment is enumerated below, to this hair
Bright further description.However, it is necessary to illustrate, many details listed in specification are used for the purpose of making reader to this
The one or more aspects of invention have a thorough explanation, also may be implemented even without these specific details of the invention
These aspects.
Embodiment 1
Adulterated detection method in a kind of identification buffalo's milk, comprising the following steps:
(1) sample acquisition and instrument prepare: it is stored refrigerated after acquisition buffalo's milk and milk sample to be measured, while getting out height
It imitates liquid phase automatic sampling chromatograph and its matches chromatographic column and clean up spare；Cow's milk to be measured is the water for mixing Holstein cow cream
Cow's milk, stored refrigerated temperature are 4 DEG C, and the chromatographic column is ZORBAX 300SB-C8 column.
(2) prepared by solution: preparing the mixed solution of BisTris, guanidine hydrochloride, sodium citrate and DTT as working solution I, matches
Guanidine hydrochloride solution processed is as working solution II；The concentration of BisTris is 0.1 mol/L in working solution I, and the concentration of guanidine hydrochloride is
6mol/L, the concentration of sodium citrate are 5.37mmol/L, and the concentration of DTT is 19.5mmol/L, guanidine hydrochloride in the working solution II
4.5mol/L, the solvent of guanidine hydrochloride solution are mobility A0.1%TFA aqueous solution.
(3) prepared by test sample: taking the buffalo's milk acquired in step (1) respectively and milk sample to be measured is appropriate and progress is following
Operation: isometric working solution I is added, forms mixed liquor after vortex mixing and stands, then mixed liquor is centrifuged and skims upper layer rouge
Fat takes bottom solution to be placed in new centrifuge tube, and working solution II is added and is uniformly mixed and obtains upper machine test product after membrane filtration；
Time of repose is 1h, and the temperature of the mixed liquor centrifugation is 4 DEG C, described to take the volume ratio of bottom solution and working solution II for 1:3,
The aperture of the diafiltration membrane is 0.45um.
(4) test sample is tested: machine test product on the buffalo's milk and cow's milk to be measured that prepare in step (3) is placed in efficient liquid phase certainly
In dynamic sample introduction chromatograph and test condition progress gradient elution test is set, obtains fingerprint chromatogram, efficient liquid phase automatic sampling color
Spectrometer test condition: Detection wavelength 214nm, 45 DEG C of column temperature, sample volume 10ul, flow velocity 0.5mL/min, mobile phase is mobility A:
0.1%TFA aqueous solution, Mobile phase B: 0.1%TFA acetonitrile.
(5) interpretation of result: the fingerprint chromatogram that buffalo's milk in step (4) and cow's milk to be measured are tested is compared, is led to
It crosses to find and cow's milk to be measured is identified to the characteristic peak that buffalo's milk Protein Separation situation does not interfere.
Embodiment 2
Adulterated detection method in a kind of identification buffalo's milk, comprising the following steps:
(1) sample acquisition and instrument prepare: it is stored refrigerated after acquisition buffalo's milk and milk sample to be measured, while getting out height
It imitates liquid phase automatic sampling chromatograph and its matches chromatographic column and clean up spare；Cow's milk to be measured is the water for mixing Holstein cow cream
Cow's milk, stored refrigerated temperature are 4 DEG C, and the chromatographic column is ZORBAX 300SB-C8 column.
(2) prepared by solution: preparing the mixed solution of BisTris, guanidine hydrochloride, sodium citrate and DTT as working solution I, matches
Guanidine hydrochloride solution processed is as working solution II；The concentration of BisTris is 0.1 mol/L in working solution I, and the concentration of guanidine hydrochloride is
3mol/L, the concentration of sodium citrate are 5mmol/L, and the concentration of DTT is 15mmol/L, guanidine hydrochloride 3mol/ in the working solution II
L, the solvent of guanidine hydrochloride solution are mobility A0.1%TFA aqueous solution.
(3) prepared by test sample: taking the buffalo's milk acquired in step (1) respectively and milk sample to be measured is appropriate and progress is following
Operation: isometric working solution I is added, forms mixed liquor after vortex mixing and stands, then mixed liquor is centrifuged and skims upper layer rouge
Fat takes bottom solution to be placed in new centrifuge tube, and working solution II is added and is uniformly mixed and obtains upper machine test product after membrane filtration；
Time of repose is 1h, and the temperature of the mixed liquor centrifugation is 3 DEG C, described to take the volume ratio of bottom solution and working solution II for 1:3,
The aperture of the diafiltration membrane is 0.2um.
(4) test sample is tested: machine test product on the buffalo's milk and cow's milk to be measured that prepare in step (3) is placed in efficient liquid phase certainly
In dynamic sample introduction chromatograph and test condition progress gradient elution test is set, obtains fingerprint chromatogram, efficient liquid phase automatic sampling color
Spectrometer test condition: Detection wavelength 214nm, 35 DEG C of column temperature, sample volume 5ul, flow velocity 1mL/min, mobile phase is mobility A:
0.1%TFA aqueous solution, Mobile phase B: 0.1%TFA acetonitrile.
(5) interpretation of result: the fingerprint chromatogram that buffalo's milk in step (4) and cow's milk to be measured are tested is compared, is led to
It crosses to find and cow's milk to be measured is identified to the characteristic peak that buffalo's milk Protein Separation situation does not interfere.
Embodiment 3
Adulterated detection method in a kind of identification buffalo's milk, comprising the following steps:
(1) sample acquisition and instrument prepare: it is stored refrigerated after acquisition buffalo's milk and milk sample to be measured, while getting out height
It imitates liquid phase automatic sampling chromatograph and its matches chromatographic column and clean up spare；Cow's milk to be measured is the water for mixing Holstein cow cream
Cow's milk, stored refrigerated temperature are 4 DEG C, and the chromatographic column is ZORBAX 300SB-C8 column.
(2) prepared by solution: preparing the mixed solution of BisTris, guanidine hydrochloride, sodium citrate and DTT as working solution I, matches
Guanidine hydrochloride solution processed is as working solution II；The concentration of BisTris is 0.3mol/L in working solution I, and the concentration of guanidine hydrochloride is
8mol/L, the concentration of sodium citrate are 7mmol/L, and the concentration of DTT is 25mmol/L, guanidine hydrochloride 6mol/ in the working solution II
L, the solvent of guanidine hydrochloride solution are mobility A0.1%TFA aqueous solution.
(3) prepared by test sample: taking the buffalo's milk acquired in step (1) respectively and milk sample to be measured is appropriate and progress is following
Operation: isometric working solution I is added, forms mixed liquor after vortex mixing and stands, then mixed liquor is centrifuged and skims upper layer rouge
Fat takes bottom solution to be placed in new centrifuge tube, and working solution II is added and is uniformly mixed and obtains upper machine test product after membrane filtration；
Time of repose is 1.5h, and the temperature of the mixed liquor centrifugation is 10 DEG C, and the volume ratio for taking bottom solution and working solution II is
3:10, the aperture of the diafiltration membrane are 0.5um.
(4) test sample is tested: machine test product on the buffalo's milk and cow's milk to be measured that prepare in step (3) is placed in efficient liquid phase certainly
In dynamic sample introduction chromatograph and test condition progress gradient elution test is set, obtains fingerprint chromatogram, efficient liquid phase automatic sampling color
Spectrometer test condition: Detection wavelength 214nm, 40 DEG C of column temperature, sample volume 6ul, flow velocity 1mL/min, mobile phase is mobility A:
0.1%TFA aqueous solution, Mobile phase B: 0.1%TFA acetonitrile.
(5) interpretation of result: the fingerprint chromatogram that buffalo's milk in step (4) and cow's milk to be measured are tested is compared, is led to
It crosses to find and cow's milk to be measured is identified to the characteristic peak that buffalo's milk Protein Separation situation does not interfere.
Embodiment 4
1.1 materials and instrument
Buffalo's milk picks up from Guangxi buffalo research institute cattle farm and the Guangxi cattle farm Hua Xushui, and He Sitan buffalo's milk picks up from guest area
Pasture, raw milk refrigerate immediately after sampling to 4 DEG C, and transport to laboratory carries out analysis measurement.The source Bis Tris leaf biology；
Two citric acid monohydrate trisodiums Chengdu Kingsoft chemical reagent Co., Ltd；DTT, guanidine hydrochloride Solarbio；TFACNW chromatographically pure；Acetonitrile
Fisher Chemical chromatographically pure；Experimental water is pure water, other reagents used are that analysis is pure.
1260 efficient liquid phase automatic sampling chromatograph of Agilent；ZORBAX 300SB-C8 (4.6 × 150mm, 3.5 μm)
Agilent；XB-C18 column (4.6 × 250nm, 5 μm) Welch；Balance；Ultrasonic cleaner.
1.2 experimental method
1.2.1 instrument condition
Detection wavelength: 214nm；Column temperature: 45 DEG C；Sample volume: 10 μ L；Flow velocity: 0.5mL/min；Mobile phase: 0.1%TFA water
Solution (A) -0.1%TFA acetonitrile (B), gradient elution program is shown in Table 1
1.2.2 the preparation of solution
Working solution I is 0.1mol/L BisTris, 6mol/L guanidine hydrochloride, 5.37mmol/L sodium citrate, 19.5mmol/L
The mixed solution of DTT；Working solution II is using mobile phase A as the 4.5mol/L guanidine hydrochloride solution of solvent.
1.2.3 the preparation of test sample
Freezing 500 μ L of milk sample is taken, is added after isometric I vortex of working solution mixes and is stored at room temperature 1h.By mixed solution
12000g, 4 DEG C of centrifugation 5min, skims upper-layer fat, takes 300 μ L of bottom solution in new centrifuge tube, and 900 μ L working solutions are added
II, it mixes, crosses 0.45 μm of filter membrane, analyzed for HPLC.
1.2.4 it after the selection of chromatographic column prepares sample according to 1.2.3, is evaluated respectively using the instrument condition of 1.2.1
Welch XB-C18 column (4.6 × 250nm, 5 μm) and ZORBAX 300SB-C8 column (4.6 × 150nm, 3.5 μm) are to lactoprotein
Separation situation, select the preferable chromatographic column of separating degree as analysis tool.
1.2.5 Precision Experiment
D16 sample is chosen, after sample is handled according to 1.2.3, precision draws 10 μ L sample liquids, parallel under the conditions of 1.2.1
Measurement 6 times records κ-CN, α-CN, β-CN, β-LG peak area.
1.2.6 repeated experiment
6 parts of F98 sample are pipetted respectively, according to the preparation of 1.2.3 method for sample, 6 parts of operation repetitive.Precision draws 10 μ L
By 1.2.1 chromatographic condition into HPLC, κ-CN, α-CN, β-CN, β-LG, α-La peak area in each sample are measured, and is calculated each
The average mass fraction of component.
1.2.7 stability experiment
Sample is chosen, precision draws 10 μ L sample liquids, under the conditions of 1.2.1, measured respectively in 0,2,4,6,8,12h sample introduction,
Record each albumen calculated by peak area relative standard deviation.
1.2.8 buffalo's milk finger-print
Totally 50, sample of two cattle farms are taken, carry out test sample preparation according to 1.2.3, precision draws 10 μ L sample liquids and exists
1.2.1 it is measured under the conditions of, then carries out spectrogram integral and obtain the information such as retention time, peak area, record chromatogram.It will be newborn
Protein chromatography figure chromatographic work station exports AIA data file, uses " similarity evaluation 2012
Version " buffalo's milk finger-print is established, carry out similitude and diversity factor evaluation.
1.2.9 Holstein cow cream finger-print
Totally 50, sample for fetching the green strong pasture of guest carry out test sample preparation according to 1.2.3, and precision draws 10 μ L sample liquids
It is measured under the conditions of 1.2.1.Spectrogram integral obtains the information such as retention time, peak area, records chromatogram.By lactoprotein color
Spectrogram chromatographic work station exports AIA data file, and " similarity evaluation 2012 editions " is used to establish
Buffalo's milk finger-print carries out similitude and diversity factor evaluation.
1.2.10 the investigation of the Holstein cow cream range of linearity is mixed in buffalo's milk
Take number F8 as buffalo's milk sample, 322 black and white evaporated milk of number is as adulterated source.Respectively in F8 addition 2%,
5%, 10%, 15%, 20%, 40% black and white evaporated milk measures peak face after preparing sample according to 1.2.3 under conditions of 1.2.1
Product, with incorporation of concentration ratio as abscissa, peak area is ordinate, draws standard curve, calculates to obtain regression equation.
1.2.11 linear repeated experiment
6 parts of buffalo's milk samples are taken, the black and white evaporated milk of ratio in the range of linearity is mixed respectively, is carried out after being prepared according to 1.2.3
Analysis measurement, calculates each peak area RSD% value.
1.3 result judgements and data statistic analysis
Polymorphisms of Milk Protein is analyzed.Experimental data is respectively by Excel, origin7.5 and Chinese medicine chromatographic fingerprint figure
Compose the 2012 editions software analysis processing of similarity evaluation system.
2 results and analysis
The selection of 2.1 chromatographic columns
Investigated in test Welch XB-C18 column (4.6 × 250nm, 5 μm) and ZORBAX 300SB-C8 column (4.6 ×
150nm, 3.5 μm) to the separating effect of lactoprotein.The result shows that lactoprotein is under identical liquid-phase condition, in ZORBAX
Preferable separation and higher response are obtained in 300SB-C8 column.Each albumen appearance situation is as shown in Figure 1.Moreover, ZORBAX
300SB-C8 has superior stability and longer service life at a low ph.So this test select ZORBAX 300SB-C8 for
The separation chromatography column of this method.Chromatogram is shown in Fig. 1, wherein 1: κ-CN/A；2:αs2-CN；3: κ-CN/B；4: α s1-CN；5: β-
CN；6: β-LgB；7: α-La.
2.2 methodological study
2.2.1 precision experiment result
κ-CN, α s2-CN, α s1-CN, β-CN, β-LgB, α-La average peak area be respectively 8631,9462,
181827,355192,4075,5984, RSD be respectively 0.94%, 4.29%, 2.53%, 2.74%, 4.21%,
1.51%, respectively less than 5%.
2.2.2 stability experiment result
The RSD value of each lactoprotein peak area of test solution is respectively 1.86%, 1.31%, 2.18%, 3.71%,
2.74%, 1.59%.Show that various lactoproteins keep stablizing in 12h in test solution.
2.2.3 repeated experiment result
By being measured after carrying out 6 parallel processing to the milk sample randomly selected, κ-CN, α s2-CN, α s1-CN, β-CN, β-
The RSD value of LgB, α-La peak area is respectively 4.29%, 4.49%, 3.76%, 4.94%, 3.02%, 4.29%.
2.2.4 buffalo's milk finger-print
The chromatogram for randomly selecting 15 batches of buffalo's milk test samples of two cattle farms imports chromatographic fingerprints of Chinese materia medica similarity
Evaluation system is labeled as the peak Mark using κ-CN shown in Fig. 1, α s2-CN, α s1-CN, β-CN, α-La as major protein, carries out peak
After matching, the control of buffalo's milk finger-print is generated.Finger-print such as Fig. 2.And similarity calculation is carried out to resulting finger-print
[15], the similarity for calculating separately sample room the results are shown in Table 2, and wherein R is buffalo's milk protein control map, and S1-S15 is for examination
Product spectrum.
1 buffalo lactoprotein similarity mode result of table
From table 1 it follows that using the buffalo's milk protein fingerprint spectrum of generation as template, S9 is removed, outside S12, S14, sample
For similarity between product between 0.95~0.99, similarity is higher, and it is preferable to illustrate that the buffalo colpus albumen of separate sources has
Correlation.
2.2.4 Holstein cow cream finger-print
The chromatogram for randomly selecting 15 batches of Holstein cow milk test samples imports chromatographic fingerprints of Chinese materia medica similarity evaluation system
System.Consulting literatures determine Polymorphisms of Milk Protein, selection and the consistent κ-CN of buffalo's milk, α s2-CN, α s1-CN, β-CN,
The protein classes of α-La are labeled as the peak Mark, carry out peak match and generate finger-print control.Finger-print such as Fig. 3.With soft
Part to resulting finger-print carry out similarity calculation, calculate separately the similarity of sample room, the results are shown in Table 3, wherein R be lotus this
Smooth lactoprotein compares map, and S1-S15 is test sample spectrogram.
2 He Sitan lactoprotein similarity mode result of table
Using the He Sitan lactoprotein finger-print of generation as template, consider 15 batches of test sample similarities, S1~S7, S9,
S10, S12~S14 similarity are between 0.95~0.99；Tri- batches of samples of S8, S11, S15 with compare the similarity of map 0.82
Between~0.89, but three compares similarity and is all larger than 0.95, this is likely due to the influence that lactoprotein has polymorphism.
2.2.6 Holstein cow cream finger-print is mixed in buffalo's milk
S1~S6 sample in Fig. 2 is chosen, using S1 buffalo's milk chromatogram as template, selection and the consistent κ-CN of buffalo's milk, α
S2-CN, α s1-CN, β-CN, α-La protein classes be labeled as the peak Mark, carry out peak match simultaneously generate finger-print control.Refer to
Line map such as Fig. 4.It analyzes buffalo's milk and mixes the sample progress sample room similarity-rough set of 40% Holstein cow cream.It the results are shown in Table
3.S1~S6 buffalo's milk sample room similarity illustrates that sample room similarity is high, has between albumen higher between 0.98~0.99
Correlation.And it is added to sample S7~S12 of 40% Holstein cow cream, only have 0.32~0.55 with the similarity of S1~S6, with
Pure water cow's milk has significant difference.It can be used as the foundation for identifying purity.Comparison diagram 2, Fig. 3 checks in the visible lotus of document [16]
α s1-CN appearance time does not interfere buffalo's milk Protein Separation situation, can be used as and mix in 21min or so in this smooth cow's milk
False characteristic peak, as shown in figure 4, wherein S1-S6 is buffalo milk sample spectrogram, S7-S12 is 40% Holstein cow milk sample product of incorporation
Spectrogram.
3 buffalo's milk of table and incorporation Holstein cow milk sample product similarity matching result
2.2.7 Holstein cow milk-line is mixed in buffalo's milk to consider
Choose buffalo's milk F8 sample, Holstein cow No. 317 samples of cream, according to 2%, 5%, 10%, 15%, 20%,
Appearance situation and peak area, calculate correlation, find 5% when mixing sample post analysis record 21min is made in 40% ratio
In~40% range, the peak area linear equation of 21min institute appearance is y=13195x-406.4, r=0.9985.Explanation
The peak source is Holstein cow lactoprotein, has characteristic, can be used for identifying buffalo's milk adulterated.In terms of 3 signal-to-noise ratio of S/N >
It calculates, 2% incorporation can be used as detection lower bound.Whether there is generality to explore, has chosen 6 parts of buffalo's milks and 6 parts of He Sitan
Cow's milk is added according to 5%, 10%, 15%, 20%, 40%.Peak area and fit equation related coefficient are as follows.R value is big
In 0.99, illustrate that the peak is stabilized, there is generality, and it is linear good to add recycling.It can satisfy He Sitan in buffalo's milk
The testing requirements of Cow's Milk Adulteration.
Table 4 adds Holstein cow milk-line correlated results
3 conclusions
Lactoprotein is main ingredient in cow's milk, this experiment is based on HPLC and carries out Protein Separation to cow's milk, establishes Guangxi
Local buffalo's milk and Holstein cow cream finger-print.Analysis comparison is carried out for 6 kinds of lactoproteins, specificity is strong, and it is easy to operate,
Favorable reproducibility, detection lower bound meet actual demand, quickly can carry out purity detecting to buffalo's milk sample.May be used also from finger-print
To analyze the otherness of protein expression, in buffalo finger-print, similarity is related to Polymorphisms of Milk Protein, S9, S12 and S14
Between similarity be higher than similarity with other samples, illustrate that three's protein expression homology is higher.
In precision, repeatability and stability experiment, the κ-CN/A, α s2-CN, κ-CN/B, α s1-CN, β-that isolate
CN, β-LgB, α-La peak area relative standard deviation (RSD%) < 5%；According to the difference of buffalo and He Sitan protein polymorphism
It is different, finger-print is established after separating to lactoprotein, passes through the phase of Similarity measures buffalo's milk and Holstein cow cream sample room
It is all larger than 0.95 like property, correlation is high.Discovery characteristic peak is compared by finger-print, as adulterated label, when Holstein cow cream
It can be detected when mixing 2%, in the range of incorporation 5%~40% Holstein cow cream, related coefficient is greater than 0.99.This method
With good reproducibility and specificity, the representative finger-print of sample room can be obtained, can be analyzed to fast and stable
Whether He Sitan milk supply is mixed in buffalo milk.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the principle of the present invention, it can also make several improvements and retouch, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (7)
1. adulterated detection method in a kind of identification buffalo's milk, it is characterised in that: the following steps are included:
(1) sample acquisition and instrument prepare: it is stored refrigerated after acquisition buffalo's milk and milk sample to be measured, while getting out efficient liquid
It phase automatic sampling chromatograph and its matches chromatographic column and cleans up spare；
(2) prepared by solution: preparing the mixed solution of BisTris, guanidine hydrochloride, sodium citrate and DTT as working solution I, matches salt manufacturing
Sour guanidine solution is as working solution II；
(3) prepared by test sample: taking the buffalo's milk acquired in step (1) and milk sample to be measured in right amount respectively and carries out following behaviour
Make: isometric working solution I be added, forms mixed liquor after vortex mixing and stands, then mixed liquor is centrifuged to and is skimmed upper-layer fat,
It takes bottom solution to be placed in new centrifuge tube, working solution II is added and is uniformly mixed and obtains upper machine test product after membrane filtration；
(4) test sample is tested: by machine test product on the buffalo's milk and cow's milk to be measured that prepare in step (3) be placed in efficient liquid phase automatically into
In sample chromatograph and test condition progress gradient elution test is set, obtains fingerprint chromatogram；
(5) interpretation of result: the fingerprint chromatogram that buffalo's milk in step (4) and cow's milk to be measured are tested is compared, by seeking
It looks for and cow's milk to be measured is identified to the characteristic peak that buffalo's milk Protein Separation situation does not interfere.
2. adulterated detection method in a kind of identification buffalo's milk according to claim 2, it is characterised in that: in step (1)
In, the stored refrigerated temperature is 4 DEG C, and the chromatographic column is ZORBAX 300SB-C8 column.
3. adulterated detection method in a kind of identification buffalo's milk according to claim 3, it is characterised in that: in step (2)
In, the concentration of BisTris is 0.1-0.3mol/L in the working solution I, and the concentration of guanidine hydrochloride is 3-8mol/L, sodium citrate
Concentration is 5-7mmol/L, and the concentration of DTT is 15-25mmol/L, guanidine hydrochloride 3-6mol/L in the working solution II.
4. adulterated detection method in a kind of identification buffalo's milk according to claim 4, it is characterised in that: in step (3)
In, the time of repose is 1-1.5h, and the temperature of the mixed liquor centrifugation is 3-10 DEG C, described to take bottom solution and working solution II
Volume ratio be (1-3): (3-10), the aperture of the diafiltration membrane is 0.2-0.5um.
5. adulterated detection method in a kind of identification buffalo's milk according to claim 5, it is characterised in that: in step (4)
In, the efficient liquid phase automatic sampling chromatograph test condition: Detection wavelength 214nm, 35-45 DEG C of column temperature, sample volume 5-10ul,
Flow velocity 0.5-1.0mL/min, mobile phase include mobile phase A and Mobile phase B.
6. adulterated detection method in a kind of identification buffalo's milk according to claim 6, it is characterised in that: the mobility
A is 0.1%TFA aqueous solution, and Mobile phase B is 0.1%TFA acetonitrile.
7. adulterated detection method in a kind of identification buffalo's milk according to claim 7, it is characterised in that: the working solution
The solvent of guanidine hydrochloride solution is mobility A0.1%TFA aqueous solution in II.
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