CN113063860A - Method for determining lactoferrin content in infant milk powder - Google Patents
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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
- G01N30/06—Preparation
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- Physics & Mathematics (AREA)
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- Chemical & Material Sciences (AREA)
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Abstract
The invention discloses a method for determining the content of lactoferrin in infant milk powder, which comprises the following steps: adding infant milk powder to be measured into a phosphate buffer solution A to completely dissolve the infant milk powder, and adjusting the pH value to be acidic to form casein precipitate to obtain a mixed solution; after the mixed solution is centrifugally separated, taking supernatant and adding the phosphate buffer solution A to obtain a sample injection solution; and (3) taking the sample injection solution to detect in a high performance liquid chromatography, wherein a chromatographic column of the high performance liquid chromatography is a heparin affinity column. The invention precipitates impurity protein in infant milk powder by adjusting pH, then directly uses a high performance liquid chromatograph (a chromatographic column is a HiTratpTMHeparin HP (1mL) heparin affinity column) to determine, and extracts a target object by buffer salt. The method optimizes the pretreatment step and uses the heparin affinity column for determination, so that the method is simple and has high detection efficiency.
Description
Technical Field
The invention belongs to the field of lactoferrin determination, and particularly relates to a method for determining lactoferrin content in infant milk powder.
Background
Lactoferrin is a natural, immunologically functional, globular monomeric glycoprotein belonging to a member of the transferrin family. Sorensen et al were found in animal milks in 1939; in 1959, Groves was purified by chromatography; montreuij & Johansonb was found in human milk in 1960, and Blanc and isiker were isolated from human milk in 1961 and formally named lactoferrin. Lactoferrin has the functions of sterilization, bacteriostasis, antivirus, antioxidation, immunoregulation, adjustment of intestinal flora, promotion of iron absorption and the like, is a novel functional food nutrition enhancer and is commonly used in infant formula food. Research shows that human and bovine colostrum has high content of lactoferrin, the content of lactoferrin in human milk is about 22% of total protein content, and the content of lactoferrin in cow milk is less than 1%, and the content of lactoferrin in human milk is much higher than that in cow milk. With the development of economy, people have higher and higher requirements on food nutrition, particularly infant food, and infant formula food tends to be emulsified by adding separated and purified bovine colostrum lactoferrin to the infant formula food. In recent years, lactoferrin has become a hot spot, and in order to better study lactoferrin and its functions, it is necessary to establish an effective detection method.
At present, a method for detecting the total protein content of lactoferrin as a food nutrition enhancer is simply introduced in GB 1903.17-2016, but the method is not suitable for detecting the lactoferrin in milk powder; in CN201210030215.9, "a method for determining lactoferrin content in dairy products", pretreatment is performed by purifying with a heparin affinity column, and then target substances are separated with a C18 chromatographic column, which has many steps.
Therefore, it is highly desirable to provide a new method for determining the lactoferrin content in infant milk powder.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method for measuring the lactoferrin content in infant milk powder.
The invention adopts the following specific technical scheme:
the invention provides a method for measuring the lactoferrin content in infant milk powder, which comprises the following steps:
s1: adding infant milk powder to be measured into a phosphate buffer solution A to completely dissolve the infant milk powder, and adjusting the pH value to be acidic to form casein precipitate to obtain a mixed solution;
s2: after the mixed solution is centrifugally separated, taking supernatant and adding the phosphate buffer solution A to obtain a sample injection solution;
s3: and (3) taking the sample injection solution to detect in a high performance liquid chromatography, wherein a chromatographic column of the high performance liquid chromatography is a heparin affinity column.
Preferably, in S1, 5.0g of the infant milk powder to be measured is weighed, 30mL of phosphate buffer a is added, and the infant milk powder is completely dissolved in the phosphate buffer a by shaking.
Preferably, in S1, the pH is adjusted to 4.5 with a phosphoric acid solution to obtain a mixed solution.
Preferably, the phosphate buffer solution A is 0.1mol/L NaH2PO4-Na2HPO4Buffered saline solution.
Preferably, in S2, the centrifugation speed is 10000r/min, and the centrifugation time is 10 min.
Further, in S2, the supernatant after centrifugation was transferred to a 50mL volumetric flask, and the volume was fixed to a scale with phosphate buffer a and mixed.
Furthermore, after the sample injection solution is filtered, 2mL of the sample injection solution is detected in a high performance liquid chromatography.
Further, the column temperature of the chromatographic column is 35 ℃, the sample injection amount is 100 mu L, the detection wavelength is 280nm, and the flow rate is 0.5 mL/min.
Further, when the mobile phase gradient elution in the high performance liquid chromatography is carried out, phosphate buffer salt A for 0-3 min, phosphate buffer salt B for 3-7 min, phosphate buffer salt C for 7-22 min and phosphate buffer salt A for 22-30 min are sequentially adopted.
Further, the phosphate buffer salt A is 0.1mol/L NaH2PO4-Na2HPO4Buffering the salt solution; the phosphate buffer salt B contains 0.1mol/L NaCl and 0.05mol/L NaH2PO4-Na2HPO4Buffer salt; the phosphate buffer salt C contains 1.0mol/L NaCl and 0.05mol/L NaH2PO4-Na2HPO4And (4) buffer salt.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention precipitates impurity protein in infant milk powder by adjusting pH, then directly uses a high performance liquid chromatograph (a chromatographic column is a HiTratpTMHeparin HP (1mL) heparin affinity column) to determine, and extracts a target object by buffer salt. The method optimizes the pretreatment step and uses the heparin affinity column for determination, so that the method is simple and has high detection efficiency.
(2) The invention adopts high performance liquid chromatography to serially connect a heparin affinity column to detect the lactoferrin content in the infant milk powder, firstly proposes to apply the heparin affinity column to the lactoferrin on-machine analysis, and optimizes the application of the lactoferrin in the sample by phosphate buffer salt A (0.1mol/L NaH)2PO4-Na2HPO4 buffer salt), processing on membrane, and HiTrap processingTMSeparating by using an affinity column with a Heparin HP 1mL specification, detecting by using a diode array detector, and quantifying by using an external standard method. Compared with the method reported in the literature, the method has the advantages of simple and rapid pretreatment operation, low detection cost, mass on-machine detection, good repeatability, high sensitivity and the like, can meet the detection of the lactoferrin content in the milk powder at present, and provides technical support for the national detection standards and the requirement of addition limit.
Drawings
FIG. 1 is a high performance liquid chromatogram obtained in example 1, measured using the method of the present invention;
FIG. 2 is a high performance liquid chromatogram measured using a C18 column as a chromatographic column in comparative example 1;
figure 3 is a standard curve for lactoferrin in example 2.
Detailed Description
The invention will be further elucidated and described with reference to the drawings and the detailed description. The technical features of the embodiments of the present invention can be combined correspondingly without mutual conflict.
Example 1
S1: weighing 5.0g of infant milk powder to be measured in a 50mL centrifuge tube, adding 30mL of phosphate buffer solution A into the centrifuge tube, and performing vortex oscillation to completely dissolve the infant milk powder in the phosphate buffer solution A. And then adjusting the pH value to 4.5 by using a phosphoric acid solution to precipitate impurity proteins in the infant milk powder to obtain a mixed solution. The phosphate buffer solution A can adopt 0.1mol/L NaH2PO4-Na2HPO4Buffered saline solution.
S2: and (4) centrifuging the mixed solution obtained in the step (S1) for 10min at the centrifugation speed of 10000r/min to completely separate a solid phase from a liquid phase in the mixed solution. Transferring the supernatant obtained after centrifugal separation into a 50mL volumetric flask, fixing the volume of the supernatant to a scale position by using a phosphate buffer solution A, and mixing and shaking up. Filtering the solution by a filter membrane for later use to obtain a sample injection solution.
S3: and filtering the sample solution, and detecting 2mL of the sample solution in a high performance liquid chromatography to obtain the content of lactoferrin in the sample solution. The column temperature of the chromatographic column was 35 ℃, the sample injection amount was 100. mu.L, the detection wavelength was 280nm, and the flow rate was 0.5 mL/min. And when the mobile phase in the high performance liquid chromatography is subjected to gradient elution, sequentially adopting 0-3 min of phosphate buffer salt A, 3-7 min of phosphate buffer salt B, 7-22 min of phosphate buffer salt C and 22-30 min of phosphate buffer salt A.
Wherein, the phosphate buffer salt A is NaH with 0.1mol/L2PO4-Na2HPO4The buffer salt solution has the specific configuration method that: weighing 1.92g of sodium dihydrogen phosphate and 11.93g of disodium hydrogen phosphate, adding a proper amount of water to dissolve the two, and fixing the volume to 1L to obtain the phosphoric acid bufferSalt A. The phosphate buffer salt B contains 0.1mol/L NaCl and 0.05mol/L NaH2PO4-Na2HPO4The buffer salt is prepared by the following specific preparation method: 0.96g of sodium dihydrogen phosphate, 5.96g of disodium hydrogen phosphate and 5.84g of sodium chloride are weighed, and a proper amount of water is added for dissolution, so that the volume is determined to be 1L, and the phosphate buffer salt B is obtained. The phosphate buffer salt C contains 1.0mol/L NaCl and 0.05mol/L NaH2PO4-Na2HPO4The buffer salt is prepared by the following specific preparation method: 0.96g of sodium dihydrogen phosphate, 5.96g of disodium hydrogen phosphate and 58.4g of sodium chloride are weighed, and a proper amount of water is added for dissolution, so that the volume is determined to be 1L, and the phosphate buffer salt C is obtained.
The spectrogram obtained by the method is shown in figure 1, and as can be seen from the figure, the peak shape of the spectrogram in figure 1 is neat and clear and is easy to read.
Comparative example 1
The same test method as in example 1 was used, with the only difference that in this comparative example, a C18 column (instead of a heparin affinity column) was used as the chromatographic column, and the spectrum is shown in FIG. 2. As can be seen from the figure, when a C18 column was used as the column, the peak shape was not good, and the effect was far inferior to that obtained in example 1.
Example 2
1. Taking the peak area y of the map in example 1 as the ordinate and the mass concentration x (mg/mL) of the lactoferrin standard solution as the abscissa, a standard curve was plotted and a linear regression was performed. The standard curve is shown in figure 3, and it can be seen from the figure that the linear relation between the peak area and the concentration is good within the concentration range of 0.01-1.0 mg/mL. The linear regression equation for lactoferrin is 2546380x-640.579 with correlation coefficient 0.99998.
The lactoferrin peak area was determined by repeating the injection of 0.5mg/mL standard lactoferrin solution 6 times as in example 1, as shown in the following table. The calculated Relative Standard Deviation (RSD) is 1.46 percent, the RSD is lower than 5.00 percent, and the result shows that the precision is good and the stability of the instrument is good.
2. The same commercially available infant milk powder is taken, analyzed and detected by the method in the embodiment 1, 6 times of parallel tests are carried out, the average value of 6 times of tests is 44.9mg/100g, the range is 42.4-47.2 mg/100g, the RSD is 3.85%, and specific data are shown in the following table.
Therefore, the precision and the stability of the results obtained by the measurement method are very excellent.
Example 3
1. Lactoferrin with different concentrations is added into the blank milk powder for a labeling recovery test. Using the procedure of example 1, each set was subjected to 6 replicates to obtain the results shown in the following table. As can be seen from the table, the recovery rate of the normalized product is 89.9 to 101%, and the relative standard deviation is 1.61 to 4.00%.
Therefore, the relative standard deviation of the measuring method is small, and the standard adding recovery rate is high.
2. By adopting the method in the embodiment 1, the infant milk powder of different brands in the market is detected, and the detection results are shown in the following table.
The results show that the milk powder of infants of different brands contains different lactoferrin contents, the milk powder added with exogenous lactoferrin is detected in the nutrient composition table, and the milk powder not added with exogenous lactoferrin is not detected in the nutrient composition table. Therefore, the detection method has good accuracy in the practical application process.
The above-described embodiments are merely preferred embodiments of the present invention, which should not be construed as limiting the invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, the technical scheme obtained by adopting the mode of equivalent replacement or equivalent transformation is within the protection scope of the invention.
Claims (10)
1. A method for measuring the lactoferrin content in infant milk powder is characterized by comprising the following steps:
s1: adding infant milk powder to be measured into a phosphate buffer solution A to completely dissolve the infant milk powder, and adjusting the pH value to be acidic to form casein precipitate to obtain a mixed solution;
s2: after the mixed solution is centrifugally separated, taking supernatant and adding the phosphate buffer solution A to obtain a sample injection solution;
s3: and (3) taking the sample injection solution to detect in a high performance liquid chromatography, wherein a chromatographic column of the high performance liquid chromatography is a heparin affinity column.
2. The method according to claim 1, wherein in S1, 5.0g of the infant milk powder to be measured is weighed, 30mL of phosphate buffer solution A is added, and the infant milk powder is completely dissolved in the phosphate buffer solution A by shaking.
3. The method according to claim 1, wherein a mixed solution is obtained by adjusting the pH of the solution to 4.5 with a phosphoric acid solution in S1.
4. The method according to claim 1, wherein the phosphate buffer A is 0.1mol/L NaH2PO4-Na2HPO4Buffered saline solution.
5. The method according to claim 1, wherein in S2, the centrifugation rate is 10000r/min, and the centrifugation time is 10 min.
6. The method according to claim 2, wherein in S2, the supernatant after centrifugation is transferred to a 50mL volumetric flask, and the volume is fixed to a scale with phosphate buffer A and mixed.
7. The assay method according to claim 6, wherein 2mL of the sample solution is filtered and detected by high performance liquid chromatography.
8. The method according to claim 7, wherein the column temperature of the column is 35 ℃, the amount of the sample is 100. mu.L, the detection wavelength is 280nm, and the flow rate is 0.5 mL/min.
9. The method according to claim 8, wherein the mobile phase gradient elution in the high performance liquid chromatography comprises phosphate buffer A of 0 to 3min, phosphate buffer B of 3 to 7min, phosphate buffer C of 7 to 22min, and phosphate buffer A of 22 to 30min in this order.
10. The method according to claim 9, wherein the phosphate buffer salt A is NaH at 0.1mol/L2PO4-Na2HPO4Buffering the salt solution; the phosphate buffer salt B contains 0.1mol/L NaCl and 0.05mol/L NaH2PO4-Na2HPO4Buffer salt; the phosphate buffer salt C contains 1.0mol/L NaCl and 0.05mol/L NaH2PO4-Na2HPO4And (4) buffer salt.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115598278A (en) * | 2022-12-14 | 2023-01-13 | 黑龙江飞鹤乳业有限公司（Cn） | Quantitative detection method for lactoferrin |
| NL2031571B1 (en) * | 2022-01-13 | 2023-07-25 | Beijing Meizheng Biotechnology Co Ltd | Binding buffer for lactoferrin extraction, preparation method and application thereof, and method for detecting bovine lactoferrin |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102854262A (en) * | 2012-08-24 | 2013-01-02 | 青岛谱尼测试有限公司 | High-performance affinity chromatography for determining lactoferrin in dairy product |
| CN104280498A (en) * | 2014-09-20 | 2015-01-14 | 中山鼎晟生物科技有限公司 | Detection kit and detection method for lactoferrin in milk product |
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2021
- 2021-02-04 CN CN202110158656.6A patent/CN113063860A/en active Pending
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| CN102854262A (en) * | 2012-08-24 | 2013-01-02 | 青岛谱尼测试有限公司 | High-performance affinity chromatography for determining lactoferrin in dairy product |
| CN104280498A (en) * | 2014-09-20 | 2015-01-14 | 中山鼎晟生物科技有限公司 | Detection kit and detection method for lactoferrin in milk product |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| NL2031571B1 (en) * | 2022-01-13 | 2023-07-25 | Beijing Meizheng Biotechnology Co Ltd | Binding buffer for lactoferrin extraction, preparation method and application thereof, and method for detecting bovine lactoferrin |
| CN115598278A (en) * | 2022-12-14 | 2023-01-13 | 黑龙江飞鹤乳业有限公司（Cn） | Quantitative detection method for lactoferrin |
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