CN102331471A - Method for determining cattle lactalbumin content in milk powder and milk by ultra performance liquid chromatography - Google Patents
Method for determining cattle lactalbumin content in milk powder and milk by ultra performance liquid chromatography Download PDFInfo
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Abstract
The invention discloses a method for determining cattle lactalbumin content in milk powder and milk by an ultra performance liquid chromatography. The method is characterized by comprising the following steps of: adding a hydrochloric acid extracting solution containing TritonX-100 into the milk powder and milk to be detected and dissolving by using ultrasound, adjusting pH to be 4.4 to 4.6 by using a sodium acetate aqueous solution, metering volume and homogenizing by using water, standing at room temperature and then filtering, allowing filtrate to pass through a filtration membrane, and putting on an ACQUITYUPLCBEH300C18 chromatographic column, and determining the content of three kinds of cattle lactalbumin, namely Ba-Lalpha, Bbetab-Lg and Bbetaa-Lg in the milk powder and the milk by an ACQUITYUPLC-TUVCQUITYUPLCBEH300C18 chromatographic column. Relatively simple and efficient purification steps are applied to operation, so that the separation of Ba-Lalpha, Bbetab-Lg and Bbetaa-Lg is realized, quantification accuracy is guaranteed, the interference of impurities such as casein, lipid and the like can be effectively removed, and the final detection flexibility is improved. The detection limit of the method is 1 mu g/g.
Description
Technical field
The invention belongs to a kind of Ultra Performance Liquid Chromatography and measure the method for milk albumin content in milk powder and the milk.
Background technology
Generally speaking, essential amino acid kind and content protein complete and that can provide human body to need can be called good protein, also is complete protein.Essential amino acid is meant needed by human but self can not synthesize, the amino acid that must from food, absorb.In phytoprotein, have only soybean protein to belong to complete protein, but the animal protein of soybean protein too late high-quality on absorbing.Lactalbumin belongs to the complete protein of high-quality, also is animal protein.It contains 8 seed amino acids of needed by human, and reasonable mixture ratio, near the demand percentage of human body, is people's bulk-growth, growth, the indispensable elite material of the anti-ageing vital movement of waiting for a long time.In range protein, the nutritive value of lactalbumin is the highest, and its is prone to digested and assimilated, and lactalbumin contains 60% in the breast milk, and casein contains 40%, so it is softer to drink the infant faeces of breast milk, measures also less.In addition, be rich in halfcystine and methionine in the whey, they can keep the level of anti-oxidant in the human body.Also have many experimental studies all to prove, take whey protein concentrate and can promote humoral immunity and cellular immunity, stimulate the human immune system, stop the generation of chemically induced property cancer.So lactalbumin is again a kind of albumen of extraordinary enhance immunity power.
From the angle of nutrition, in the food in animal protein source, contain objectionable impuritiess such as harmful excessive saturated fat, cholesterol, excessive eating is prone to cause body fat and cholesterol to raise, thereby causes the generation of angiocardiopathy.Fat in the lactalbumin, lactose content is low, but it contains beta lactoglobulin, ALA, immunoglobulin (Ig), also has other various active compositions.These active components have possessed lactalbumin just is of value to many health cares of human body, so it is considered to one of good protein source of needed by human body.Lactalbumin is one type of protein that is of high nutritive value, is prone to digest and assimilate, contain the various active composition, especially is fit to infant, the elderly, sport people and preoperative and postoperative patient.In food industry, used more and more widely, like child nutrition albumen powder, babies ' formula milk powder etc.Yet, to the detection method of lactalbumin suitable limitation also, the demand in a kind of easy, quick, the effective and detection cost is cheap relatively detection method seasonable generation and arising at the historic moment.
Lactalbumin is the main protein in the milk; Lactalbumin mainly comprises α-cow's milk albumin (B α-Lactalbumin; (the above two are principal ingredients of lactalbumin for B β-Lactoglbulin, B β-Lg), proteinase-peptone and bovine serum albumin(BSA) etc. for B α-La), β-cow's milk globulin; Account for the 10-20% and the 40-50% of its total amount respectively, β-cow's milk globulin comprises B β b-Lg and two genetic variants of B β a-Lg.
In " People's Republic of China's food security national standard " " mensuration of lactalbumin (exposure draft) in infant food and the dairy products " improvement " GB/T 5413.2-1997 " mensuration of dispensed food for baby and milk powder lactalbumin " "; Discussed: first method high performance liquid chromatography-mass spectrometry method, adopt 1.7 μ m siloyl group C
18Post, mass-to-charge ratio (m/z) 1-3000 mass-to-charge ratio (m/z); Resolution is the liquid chromatograph-mass spectrometer system of 0.1 atomic mass unit, makes detecting of ALA, β b-lactoglobulin and β a-lactoglobulin be limited to 5 mg/100g, and the quantitative measurement scope is 5-50 mg/100g; The second method gel permeation chromatography is applicable to detect the mensuration of ALA in infant food and the dairy products and be limited to 50 mg/100g.Because the limitation that the mass spectrometer of 3000 used karyoplasmic ratios costs an arm and a leg and gel permeation chromatography detects in the National Standard Method.
Summary of the invention
The purpose of this invention is to provide a kind of can fast detecting milk powder and milk in the Ultra Performance Liquid Chromatography method of milk albumin content.
The objective of the invention is to realize
The instituteThat states utilizes Ultra Performance Liquid Chromatography, measures the method for milk albumin content in milk powder and the milk, and concrete steps are:
In milk powder to be measured or milk, add hydrochloric acid extraction liquid, the ultrasonic dissolution that contains Triton X-100; Adjust pH between 4.4-4.6 with aqueous sodium acetate solution; Water constant volume, homogeneous; Room temperature leaves standstill the back and filters, last ACQUITY UPLC BEH300 C18 chromatographic column, the content of B α-La, B β b-Lg and three kinds of milk albumins of B β a-Lg in employing ACQUITY UPLC-TUV Ultra Performance Liquid Chromatography system measurement milk powder or the milk.
The 0.1mol/L hydrochloric acid extraction liquid that contains the preferred 0.2% Triton X-100 of hydrochloric acid extraction liquid of Triton X-100.
Described chromatographic column is ACQUITY UPLC BEH300 C18 2.1mm * 100mm, 1.7 μ m
Liquid phase systems is that water generation ACQUITY UPLC joins ACQUITY TUV UV-detector; The column temperature of chromatographic column: 35 ℃, mobile phase A: 0.1% trifluoroacetic acid acetonitrile solution; Mobile phase B: 0.1% trifluoroacetic acid aqueous solution; Adopt gradient elution, flow velocity is 0.2 mL/min; Detect wavelength: 280 nm; Analysis time: 15 min; Content through external standard method B α-La, B β b-Lg and three kinds of milk albumins of B β a-Lg.Above-mentioned % is weight percentage.
It is 300 ACQUITY UPLC BEH300 C that described chromatographic column preferably adopts the Waters aperture
18Chromatographic column is separated.
The principal feature of detection method of the present invention:
The present invention adopts more easy pre-treating method with reference to national standard method, and improved moving phase and detection mode, with " ACQUITY UPLC BEH300 C
18Chromatographic column; ACQUITY UPLC-TUV Ultra Performance Liquid Chromatography system " measure in milk powder and the milk three kinds of milk albumins (α-cow's milk albumin (B α-Lactalbumin; the content of B α-La), β-cow's milk globulin (B β-Lactoglbulin, B β-Lg)).Samples such as satisfying cow's milk, milk powder detect be limited to make every effort under the prerequisite that 1 mg/kg requires simple, fast, cost is low and can handle sample by high flux; Through method optimization; Make the detection limit of three kinds of milk albumins reach 0.1 mg/100g; Quantitative measurement scope 0.1-10 mg/100g; More help the quantitative measurement of three kinds of milk albumins in the sample of low concentration content, handled sample is at the perfluorooctane sulfonate buffer salt: also can measure the detection of vitamin B6, nicotinic acid in the moving phase system of methyl alcohol, help the extensive utilization in the production testing.
Advantage of the present invention is: the present invention has set up the quantitative analysis method that detects B α-La, B β b-Lg and three kinds of milk albumins of B β a-Lg in milk powder and the milk actual sample with ACQUITY UPLC-TUV system; Reduced the experiment initial stage in the fund input of aspects such as experiment reagent, instrument and equipment; Use relative advantages of simplicity and high efficiency purification step to operate; Realize B α-La, B β b-Lg and B β a-Lg, separation, guaranteed quantitative accuracy; And effectively remove impurity such as casein protein and lipid and disturb, improved the sensitivity of final detection.Detecting of method is limited to 1 μ g/g, compares similar high performance liquid chromatography-mass spectrometry method, on detection limit, increases, and more effectively guarantees the accuracy of experimental data, helps the relatively low sample detection of protein content of whey.Hydrochloric acid mixes generation sodium chloride with NaOH, help the extraction of lactalbumin, removes in the detection method of the same type the interpolation of sodium chloride medicine from.
Description of drawings
Fig. 1 is α of the present invention-La cow's milk albumin ultraviolet spectrogram.
Fig. 2 is a β b-Lg cow's milk albumin ultraviolet spectrogram of the present invention.
Fig. 3 is a β a-Lg cow's milk albumin ultraviolet spectrogram of the present invention.
Fig. 4 is that three kinds of lactalbumin 280nm wavelength detect chromatogram.
Four chromatograms of Fig. 5 for selecting to obtain under four kinds of eluent gradient methods of 1-4.
Fig. 6 is that the pH of pre-treating method of the present invention selects lab diagram.
Fig. 7 is α of the present invention-La canonical plotting.
Fig. 8 is a β b-Lg canonical plotting of the present invention.
Fig. 9 is a β a-Lg canonical plotting of the present invention.
Figure 10 is three kinds of lactalbumin standard colorss of 1.000 mg/L spectrogram.
Figure 11 is a powdered milk sample A chromatogram.
Figure 12 is a powdered milk sample B chromatogram.
Figure 13 is liquid milk sample chromatogram figure.
Figure 14 is the blank overlapping chromatogram of standard items, actual sample and sample extracting solution.
Figure 15 adds the chromatogram of standard items at last for milk sample and milk sample.
Figure 16 is two kinds of overlapping chromatograms of deciding the solution standard items.
Figure 17 is B
6, the nicotinic acid sample adds the overlapping chromatogram of target.
Embodiment
Below in conjunction with embodiment the present invention is elaborated
Experimental technique
Material, reagent and instrument
Acetonitrile (chromatographically pure), trifluoroacetic acid (top grade is pure), Triton X-100; Sodium chloride (top grade is pure); Hydrochloric acid (top grade is pure), NaOH (analyzing pure), experimental water are ultrapure water (18 M Ω cm; TOC 3 ppb), milk albumin standard items B α-La, B β b-Lg and B β a-Lg; Acquity TUV detecting device, FILTER MIXER (425 μ L, P/N 205000403) join in ACQUITY UPLC Ultra Performance Liquid Chromatography system.
Reagent is used in test
1. 0.1% trifluoroacetic acid acetonitrile solution: draw 1 mL trifluoroacetic acid with dilution in acetonitrile to 1000 mL;
2. 0.1% trifluoroacetic acid aqueous solution: draw 1 mL trifluoroacetic acid and be diluted with water to 1000 mL;
3. 0.1 mol/L hydrochloric acid: draw 9 mL hydrochloric acid, water is diluted with water to 1000 mL, adds 2 g Triton X-100, ultrasonic dissolution;
4. sodium hydroxide solution: take by weighing 50 g NaOH, be dissolved in the 1000 mL water.
5. three kinds of lactalbumin standards are all with the ultrapure water dilution, and the standard specimen that is mixed with variable concentrations detects, and can obtain better chromatographic peak profile, and preparation is simple.
6. standard substance:
1) cow's milk ALA, purity is not less than 85%;
2) cow's milk β-lactoglobulin, purity is not less than 90%.
β b-lactoglobulin wherein should separate through liquid chromatography with β a-lactoglobulin, detects absorption intensity at wavelength 280 nm places.According to the area of chromatographic peak, confirm content ratio separately respectively by area normalization method.
Pre-treating method
Take by weighing 1.00 g powdered milk samples or 10 mL milk samples, add the dissolve with hydrochloric acid solution of 30 mL (adding 20 mL), 0.1 mol/L, ultrasonic 15 min for milk; Regulate pH to 4.5 ± 0.1 with aqueous sodium acetate solution after being cooled to room temperature; Water is settled to 50 mL, leaves standstill 5 min, and room temperature leaves standstill the back and filters; Filtrating is through 0.2 μ m filter membrane, last machine testing.
Find in the test that hydrochloric acid solution adopts the hydrochloric acid extraction liquid that contains Triton X-100, it is better to the extraction and the stablizing effect of lactalbumin to contain Triton X-100, the 0.1mol/L hydrochloric acid extraction liquid of preferred 0.2% Triton X-100.
Chromatographic condition
Liquid phase systems: water generation ACQUITY UPLC joins ACQUITY TUV UV-detector
Chromatographic column: ACQUITY UPLC BEH300 C18 2.1mm * 100mm, 1.7 μ m
Column temperature: 35 ℃
Mobile phase A: 0.1% trifluoroacetic acid acetonitrile solution
Mobile phase B: 0.1% trifluoroacetic acid aqueous solution; Gradient elution
Detect wavelength: 280nm
Analysis time: 15 min
Sample size: 10 μ L (sample feeding 2 μ L)
Gradient method sees the following form 1:
Table 1 detects gradient table
| Time/(min) | Flow/(mL/min) | A% | | Curve | |
| 0 | 0.20 | 39 | 61 | Initially | |
| 3 | 0.20 | 41 | 59 | 6 | |
| 4 | 0.20 | 44 | 56 | 6 | |
| 8 | 0.20 | 46 | 54 | 6 | |
| 10 | 0.20 | 70 | 30 | 1 | |
| 15 | 0.20 | 39 | 61 | 1 |
(on show % refer to percent by volume).
Result and discussion
Detect the selection of wavelength
Scan the uv absorption spectra that obtains three kinds of lactalbumins with Waters PDA detecting device, extremely shown in Figure 3 like Fig. 1.
By visible among the figure; Lactalbumin all has uv absorption under 220 nm and 280 nm; This paper selects two kinds of wavelength that three kinds of lactalbumins (concentration is 5 ppm) are detected; Though result's discovery analyte has maximum uv absorption under the ultraviolet wavelength of 220 nm, it also has higher baseline noise and interference under this wavelength simultaneously, and the present invention still selects ultraviolet wavelength 280 nm as detecting wavelength; Reduce the baseline interference in the moving phase, the standard colors spectrogram of 5 ppm as shown in Figure 4.
Flow velocity, moving phase optimization
Like Fig. 4 is the chromatogram of three kinds of milk albumin hybrid standards, is B α-La, B β b-Lg and B β a-Lg successively according to retention time, and for the analysis of protein of HMW, employing Waters aperture is 300 ACQUITY UPLC BEH300 C
18Chromatographic column is separated, and in addition, considers the mass transfer characteristic of high molecular weight protein, needs to use less flow velocity, can realize separating preferably.This paper has tested 0.1 mL/min, 0.2 mL/min and three kinds of flow velocitys of 0.3 mL/min; 0.1 the mL/min dead time reaches 2.5 min, whole appearance time is later, and peak width strengthens; Final comprehensive peak shape, quick and degree of separation consideration selection 0.2 mL/min flow velocity, as shown in Figure 4.
B α-La has reservation preferably under current analysis condition; Change proportion of mobile phase then; Obtain four kinds of proportioning chromatograms (like Fig. 5) of standard items; Can find out that by figure two kinds of genetic variants of B β b-Lg and B β a-Lg reach perfect and separate (degree of separation=2.79) among No. 1 chromatographic peak figure; Along with the variation of gradient, keeping accelerating the appearance time of B β b-Lg and B β a-Lg gradually under the constant situation of B α-La retention time, the degree of separation of two kinds of compounds is gradually varied to 1.97 (No. 4 chromatograms), and sensitivity also has increase slightly.But consider that the impurity that minimizing possibly exist is to the interference of analyte when analyzing actual sample, the present invention adopts chromatogram gradient method (seeing table 1) to detect.
Actual sample detects to be found; B α-La the front of sample has more impurity peaks to exist; Therefore for preventing the low interference that keeps impurity to B α-La in the actual sample; Liquid phase process B α-La retention time to Fig. 5 has carried out adjusting (as shown in Figure 4), and B β b-Lg and B β a-Lg still have 2.22 degree of separation.
Pre-treating method is discussed
Several lime lights are arranged in the pre-treatment, at first just be to use the 0.1mol/L hydrochloric acid extraction liquid that contains 0.2% Triton X-100, try not to surpass 3 days, can descend to the extraction and the stablizing effect of lactalbumin because this solution after 3 days is found in experiment.Secondly sample adds extract, needs to filter behind the adjustment pH, because unfiltered solution need be placed centrifuging and taking supernatant behind 50 min, filters and can save more experimental period.
Its key point also is the grasp of extracting liquid pH value; This paper compares the extraction situation of same milk powder product under different pH, and is as shown in Figure 6, and pH does not have too big difference between 4.4-4.6; But pH reaches 4.8 and at 5.0 o'clock; The peak shape variation of B β b-Lg and B β a-Lg, especially B α-La the peak position that goes out flooded by impurity fully, therefore need control pH value in the experiment above 4.6.
The linear dependence of method and detection limit
Three kinds of lactalbumin standards that concentration is respectively 1.000,40.000,60.000,80.000,100.000 mg/L are sample introduction successively, and sample size is 10 μ L, and external standard method is quantitative.With peak area A is ordinate, and concentration C is that horizontal ordinate carries out linear regression, obtains B α-La linear equation A=2.34 * 10 respectively
3C-2.36 * 10
3, coefficient R
2=0.9997; B β b-Lg linear equation A=1.32 * 10
3C-1.30 * 10
3, coefficient R
2=0.9995; B β a-Lg linear equation A=1.24 * 10
3C-1.36 * 10
3, coefficient R
2=0.9987.Three kinds of lactalbumins have good linear relationship (like Fig. 7-shown in Figure 9) in 1.000-100.000 mg/L scope.Through measuring, method detects and is limited to 1.000 mg/kg, and Figure 10 is three kinds of lactalbumin standard colorss of 1.000 mg/L spectrogram, and B α-La, B β b-Lg, B β a-Lg signal to noise ratio (S/N ratio) reach 8,3 and 3 respectively under this concentration, and wherein B α-La can realize quantitative requirement.
The actual sample testing result
Figure 11-Figure 13 is respectively two kinds of milk powder and a kind of milk actual sample is analyzed chromatogram and result.Table 2, table 3 and table 4 are seen in the quantitative result report respectively.
The quantitative result of table 2 powdered milk sample A
The quantitative result of table 3 powdered milk sample B
The quantitative result of table 4 liquid milk sample
By Figure 14, the overlapping chromatogram of the blank solution of the standard items of three kinds of lactalbumins, actual sample and sample extracting solution can be seen, in the extraction solvent of milk powder and milk, does not have the interference to lactalbumin analyte in the sample.Referring to following Figure 15, be the chromatogram that milk sample and milk sample add standard items at last in addition, can further qualitative milk extraction sample in the existence of three kinds of lactalbumins.
Find simultaneously, lactalbumin standard items water or sample extracting solution (the NaCl solution that contains Triton X-100) constant volume, sample detection does not have influence basically to the peak area of three kinds of lactalbumins, referring to Figure 16.
Adopt above-mentioned identical pre-treating method of the present invention, the sample of gained+mark article are at the perfluoroetane sulfonic acid sodium water solution: chromatogram such as Figure 17 of gained in methyl alcohol (90:10) the moving phase system.
Conclusion
This paper has set up the quantitative analysis method that detects B α-La, B β b-Lg and three kinds of milk albumins of B β a-Lg in milk powder and the milk actual sample with ACQUITY UPLC-TUV system; Reduced the experiment initial stage in the fund input of aspects such as experiment reagent, instrument and equipment; Use relative advantages of simplicity and high efficiency purification step to operate; Realize B α-La, B β b-Lg and B β a-Lg, separation, guaranteed quantitative accuracy; And effectively remove impurity such as casein protein and lipid and disturb, improved the sensitivity of final detection.Detecting of method is limited to 1 μ g/g, compares similar high performance liquid chromatography-mass spectrometry method, on detection limit, increases, and more effectively guarantees the accuracy of experimental data, helps the relatively low sample detection of protein content of whey.Hydrochloric acid mixes generation sodium chloride with NaOH, help the extraction of lactalbumin, removes in the detection method of the same type the interpolation of sodium chloride medicine from.
Use ACQUITY UPLC BEH300 C
18Chromatographic column flows mutually with the trifluoroacetic acid adjuvant, makes B β b-Lg and two kinds of genetic variants of B β a-Lg can reach baseline separation; Adopt 280nm to detect wavelength, the moving phase that effectively reduces under the low wavelength detection is disturbed, and can be used for the assay of milk powder and three kinds of milk albumins of milk.
List of references
[1]?Kinghorn?N?M,?Norris?C?S,?Paterson?G?R.?Chromatogr?A,?1995，700:111。
[2] Cai Zengxuan, Chu Xiaojun etc. the 17 national chromatogram symposium and instrument exhibition proceeding, KB02:23-24.
[3] Li Hui, Chen Min etc. chromatogram .2007.1,25:116-117
[4]?Ena?J?M,?van?Beresteijn?E?C?H,?Robben?A?J?P?M,?Schmidt?D?G.?J?Food?Sci,?1995，60(1):104。
Claims (3)
1. one kind
Ultra Performance Liquid Chromatography is measured the method for milk albumin content in milk powder and the milkIt is characterized in that: in milk powder to be measured or milk, add hydrochloric acid extraction liquid, the ultrasonic dissolution that contains Triton X-100, adjust pH between 4.4-4.6 with aqueous sodium acetate solution, water constant volume, homogeneous; Room temperature leaves standstill the back and filters; Filtrating is passed through filter membrane, last ACQUITY UPLC BEH300 C18 chromatographic column, the content of B α-La, B β b-Lg and three kinds of milk albumins of B β a-Lg in employing ACQUITY UPLC-TUV Ultra Performance Liquid Chromatography system measurement milk powder or the milk.
2. according to claim 1
Ultra Performance Liquid Chromatography is measured the method for milk albumin content in milk powder and the milk, it is characterized in that:
Said filtrating is through 0.2 μ m filter membrane, and described chromatographic column is ACQUITY UPLC BEH300 C18 2.1mm * 100mm, 1.7 μ m
ACQUITY UPLC-TUV Ultra Performance Liquid Chromatography system is that water generation ACQUITY UPLC joins ACQUITY TUV UV-detector; The column temperature of chromatographic column: 35 ℃, mobile phase A: 0.1% trifluoroacetic acid acetonitrile solution; Mobile phase B: 0.1% trifluoroacetic acid aqueous solution, adopt gradient elution, flow velocity is 0.2 mL/min; Detect wavelength: 280 nm; Analysis time: 15 min; Content through external standard method B α-La, B β b-Lg and three kinds of milk albumins of B β a-Lg.
3. according to claim 1 and 2
Ultra Performance Liquid Chromatography is measured the method for milk albumin content in milk powder and the milk, it is characterized in that: described chromatographic column adopting Waters aperture is that the ACQUITY UPLC BEH300 C18 chromatographic column of 300 à is separated.
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| CN115480002A (en) * | 2022-08-12 | 2022-12-16 | 卡士乳业(深圳)有限公司 | Method for analyzing content of active whey protein based on dynamic high-pressure micro-jet combined high performance liquid chromatography technology |
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