CN112557483A

CN112557483A - Method for analyzing osteopontin in dairy product

Info

Publication number: CN112557483A
Application number: CN202011315213.5A
Authority: CN
Inventors: 王象欣; 解庆刚; 崔东影; 蒋士龙; 梁爱梅
Original assignee: Heilongjiang Feihe Dairy Co Ltd
Current assignee: Heilongjiang Feihe Dairy Co Ltd
Priority date: 2020-11-20
Filing date: 2020-11-20
Publication date: 2021-03-26
Anticipated expiration: 2040-11-20
Also published as: CN112557483B

Abstract

The invention provides an analysis method of osteopontin in a dairy product. The analysis method comprises the following steps: step S1, removing casein in the aqueous solution of the dairy product to obtain a treatment solution; step S2, adjusting the pH value of the treatment solution to 6.5-7.5 to obtain a solution to be detected; and step S3, analyzing osteopontin in the solution to be detected by capillary electrophoresis with a neutral coating capillary. The inventor screens out a neutral coating capillary tube to detect OPN in the dairy product. In view of the serious interference effect of casein at the peak position of OPN, the method firstly removes the casein from the aqueous solution of the dairy product and then adjusts the pH value to obtain the solution to be detected, and then utilizes the neutral coating capillary to reduce the hydrophobicity and the interaction between the electrostatic solute and the tube wall to the maximum extent under the condition of the pH value, thereby eliminating the electroosmotic flow to the maximum extent and further accurately detecting the osteopontin in the dairy product.

Description

Method for analyzing osteopontin in dairy product

Technical Field

The invention relates to the technical field of dairy products, in particular to an analysis method of osteopontin in a dairy product.

Background

Osteopontin (OPN) is an important protein present in breast milk and is classified as an extracellular matrix protein. In view of the structure, OPN is a highly phosphorylated acidic glycoprotein consisting of about 300 amino acid residues, and OPN in cow's milk and human milk is very similar in amino acid sequence and phosphorylation pattern, and contains a specific RGD (arginine-glycine-aspartic acid, Arg-Glu-Asp) sequence, which plays an important role in the adhesion function of OPN, and both cow's milk and human milk OPN are O-glycosylated in the same region. Human milk has an OPN content of about 138mg/L, which is much higher than in cow's milk (about 18 mg/L). The aim of the infant formula milk powder is to realize the breast milk, so that the biological influence of OPN on the infant can be clearly understood to have profound significance. The infant formula milk powder on the market at present hardly has reinforced OPN, so that the OPN is more and more concerned by manufacturers of the infant formula milk powder as a potential candidate protein for enhancing the similarity of the infant formula milk powder and breast milk, and the quantitative detection of the OPN in milk products is also more and more concerned.

At present, the detection research of OPN in infant formula milk powder is less, wherein the mass spectrometry developed by AOAC (Analytical method to quantitative osteopontin in dairy powder and inhibitor for mula by signature peptide quantification with UHPLC-MS/MS) is based on the principle of proteomics, and is characterized in that the osteopontin is subjected to enzymolysis by trypsin, and the specific peptide segment generated by enzymolysis is detected by mass spectrometry to identify and indirectly quantify the osteopontin in the milk product. However, the above method is limited to quantitative determination of osteopontin in a dairy product in which no maillard reaction product is produced, and the detection accuracy is low, and thus the content of osteopontin cannot be effectively quantified.

In addition, since OPN is a secreted, acidic phosphorylated glycoprotein, which is negatively charged in the milk system, it can form a protein complex with positively charged proteins by electrostatic binding, and the protein complex cannot be dissociated by chromatographic methods; therefore, an efficient and convenient pretreatment means is needed, and the content of OPN in the milk matrix is detected by combining an accurate quantitative method so as to ensure that the content of the raw materials and the content of the finished product meet the requirements.

Disclosure of Invention

The invention mainly aims to provide an analytical method of osteopontin in a dairy product, so as to solve the problem that the analytical method of osteopontin in the dairy product in the prior art is difficult to accurately quantify OPN.

In order to achieve the above object, according to an aspect of the present invention, there is provided an analysis method of osteopontin in a milk product, the analysis method comprising: step S1, removing casein in the aqueous solution of the dairy product to obtain a treatment solution; step S2, adjusting the pH value of the treatment solution to 6.5-7.5 to obtain a solution to be detected; and step S3, analyzing osteopontin in the solution to be detected by capillary electrophoresis with a neutral coating capillary.

Further, the buffer solution used in the capillary electrophoresis method includes any one or a combination of more of a phosphate buffer solution, a borate buffer solution, an acetate buffer solution, a borax buffer solution, and a citric acid buffer solution, preferably the pH value of the buffer solution is 6.5 to 7.5, further preferably the pH value of the buffer solution is 6.8 to 7.2, and preferably the phosphate buffer solution includes a sodium dihydrogen phosphate solution and a disodium hydrogen phosphate solution.

Further, the pH value of the solution to be detected is the same as that of the buffer solution.

Further, when the capillary electrophoresis method is used for analysis, the working voltage is-5 to-30 kv, and more preferably-8 to-12 kv; the preferable detection wavelength is 200-450 nm, and further preferable is 210-218 nm; the detection frequency is preferably 2-18 Hz, and more preferably 2-4 Hz.

Further, the neutral coating capillary tube is a quartz capillary tube, preferably the length of the quartz capillary tube is 20-100 cm, and further preferably 25-40 cm; the inner diameter of the quartz capillary is preferably 25 to 75 μm, and more preferably 50 to 75 μm.

Further, the temperature of the quartz capillary tube is 0 to 50 ℃, preferably 20 to 30 ℃.

Further, the inner wall coating layer of the quartz capillary tube is polyacrylamide or polyethylene glycol.

Furthermore, the outer wall of the quartz capillary tube is coated with an elastic coating.

Further, the elastic coating is polyimide.

Further, the step S1 includes: step S11, adjusting the pH value of the aqueous solution of the dairy product to 4.0-4.6 to obtain an acidic system; in step S12, the acidic system is subjected to solid-liquid separation to obtain a treated liquid, and the preferred mode of solid-liquid separation is centrifugal separation.

By applying the technical scheme of the invention, the inventor finds that the OPN cannot be effectively separated by adopting a capillary electrophoresis method because the glycosylation degree of Osteopontin (OPN) in the dairy product is higher and the adsorption of the uncoated capillary to the OPN is more serious through a large amount of experiments, and finally screens out the neutral coated capillary to detect the OPN in the dairy product through a large amount of tests. In view of the serious interference effect of casein at the peak position of OPN, the method firstly removes the casein from the aqueous solution of the dairy product and then adjusts the pH value to obtain the solution to be detected, and then utilizes the neutral coating capillary to reduce the hydrophobicity and the interaction between the electrostatic solute and the tube wall to the maximum extent under the condition of the pH value, thereby eliminating the electroosmotic flow to the maximum extent and further accurately detecting the osteopontin in the dairy product.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows a capillary electrophoresis diagram of an OPN standard sample provided in accordance with example 1 of the present invention;

fig. 2 shows a capillary electrophoresis chart of a test solution of infant formula provided in example 1 of the present application.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

As analyzed by the background technology, the method for analyzing the osteopontin in the dairy product in the prior art has the problem that the OPN is difficult to accurately quantify, and the invention provides the method for analyzing the osteopontin in the dairy product to solve the problem.

In an exemplary embodiment of the present application, there is provided a method for analyzing osteopontin in a milk product, the method comprising: step S1, removing casein in the aqueous solution of the dairy product to obtain a treatment solution; step S2, adjusting the pH value of the treatment solution to 6.5-7.5 to obtain a solution to be detected; and step S3, analyzing osteopontin in the solution to be detected by capillary electrophoresis with a neutral coating capillary.

The inventor finds that the OPN is seriously adsorbed by the uncoated capillary tube due to the higher glycosylation degree of the Osteopontin (OPN) in the dairy product, so that the OPN cannot be effectively separated by adopting a capillary electrophoresis method, and finally the neutral coated capillary tube is screened out through a large amount of tests to detect the OPN in the dairy product. In view of the serious interference effect of casein at the peak position of OPN, the method firstly removes the casein from the aqueous solution of the dairy product and then adjusts the pH value to obtain the solution to be detected, and then utilizes the neutral coating capillary to reduce the hydrophobicity and the interaction between the electrostatic solute and the tube wall to the maximum extent under the condition of the pH value, thereby eliminating the electroosmotic flow to the maximum extent and further accurately detecting the osteopontin in the dairy product.

The dairy products include but not limited to cow milk, goat milk and other animal milks and products produced by taking the animal milks as main raw materials and carrying out industrial processing.

Under the condition that the pH value of the treatment liquid is 6.5-7.5, the method is favorable for accurately detecting osteopontin in the dairy product. In order to reduce the influence of the buffer solution on the pH value of the solution to be tested, the buffer solution used in the capillary electrophoresis method preferably includes any one or a combination of more of a phosphate buffer solution, a borate buffer solution, an acetate buffer solution, a borax buffer solution, and a citric acid buffer solution, the pH value of the buffer solution is preferably 6.5 to 7.5, the pH value of the buffer solution is further preferably 6.8 to 7.2, and the phosphate buffer solution preferably includes a sodium dihydrogen phosphate solution and a disodium hydrogen phosphate solution.

The pH affects electroosmosis and the adsorption level of components in the solution to be measured on the tube wall, and further preferably, the pH of the solution to be measured is the same as the pH of the buffer solution in order to reduce the influence of the buffer solution on the pH of the solution to be measured to the greatest extent and thereby eliminate electroosmotic flow.

In one embodiment of the present application, the capillary electrophoresis method is used for analyzing, wherein the working voltage is-5 to-30 kv, and more preferably-8 to-12 kv; the preferable detection wavelength is 200-450 nm, and further preferable is 210-218 nm; the detection frequency is preferably 2-18 Hz, and more preferably 2-4 Hz.

The conditions during the analysis by the capillary electrophoresis method are favorable for improving the detection accuracy of the osteopontin and improving the analysis efficiency of the capillary electrophoresis method.

In an embodiment of the present application, the neutral coating capillary is a quartz capillary, and preferably the length of the quartz capillary is 20 to 100cm, and more preferably 25 to 40 cm; the inner diameter of the quartz capillary is preferably 25 to 75 μm, and more preferably 50 to 75 μm.

The preferred quartz capillary tube of the above length and inner diameter dimensions can improve the efficiency of osteopontin separation.

The temperature can affect the pH value of the liquid to be detected and the separation effect of each component in the liquid to be detected, and in order to better separate osteopontin from other components in the liquid to be detected, the temperature of the quartz capillary is preferably 0-50 ℃, and is preferably 20-30 ℃.

The neutral coating of the quartz capillary may be a coating formed of a conventional neutral material currently used for the quartz capillary, and in one embodiment of the present application, the inner wall coating layer of the quartz capillary is polyacrylamide or polyethylene glycol. The combination of the inner wall coating layer of the quartz capillary tube and the quartz is stable, and a long-term stable neutral coating environment can be provided for the inner wall of the quartz capillary tube, so that the electroosmotic flow can be eliminated to the maximum extent.

In order to improve the elasticity of the quartz capillary tube and thus the durability thereof, it is preferable that the outer wall of the quartz capillary tube is coated with an elastic coating.

Those skilled in the art can coat the outer wall of the quartz capillary tube with a substance having a certain flexibility in the prior art to enhance the durability thereof, and in order to further reduce the production cost, the above polyimide is preferably used as the elastic coating.

In order to more thoroughly remove casein from the aqueous milk solution, it is preferable that the above step S1 includes: step S11, adjusting the pH value of the aqueous solution of the dairy product to 4.0-4.6 to obtain an acidic system; in step S12, the acidic system is subjected to solid-liquid separation to obtain a treated liquid, and the preferred mode of solid-liquid separation is centrifugal separation. The acidic environment with the pH value of 4.0-4.6 is beneficial to ensure that casein molecules are gathered into larger precipitate floccules to be removed.

In addition, during analysis by the capillary electrophoresis method, in order to reduce the interference effect of the environmental temperature on the test conditions as much as possible and control the sample introduction condition of the liquid to be tested within a reasonable range, thereby being beneficial to the separation of the osteopontin, the temperature of the sample chamber is preferably 0-30 ℃, and is preferably 10-15 ℃; the preferable operation time is 10-60 min, and the preferable operation time is 20-40 min; the sample injection mode is pressure sample injection or vacuum sample injection, and the pressure of the pressure sample injection is preferably 0.1-10 psi, preferably 2-5 psi; the sample introduction time is 1-20 s, preferably 10-20 s.

The advantageous effects of the present application will be described below with reference to specific examples and comparative examples.

Example 1

Instruments and conditions:

capillary electrophoresis apparatus: SCIEX PA800 Plus; a detector: UV (ultraviolet detector); detection wavelength: 210nm, 2 Hz; quartz capillary (SCIEX, 477441): the inner diameter is 50 μm; effective length/total length: 25cm/30.2 cm; window slit: 200 μm × 100 μm; temperature of the quartz capillary: 20 ℃; temperature of the sample chamber: 10 ℃.

Preparation of phosphate buffer solution (BGE) of 40mmol and pH 7:

after 6.1mL of a 40mmol disodium hydrogenphosphate solution was aspirated and 3.9mL of a 40mmol sodium dihydrogenphosphate solution was further aspirated, the two were mixed uniformly to prepare a 40mmol phosphate buffer solution having a pH of 7.

Thirdly, an instrument operation method comprises the following steps:

the sample introduction condition is that the sample introduction lasts for 10s under the pressure of 2 psi; the separation condition was-8 kV for 20 min.

Fourthly, capillary tube washing conditions:

the capillary was pre-rinsed before each sample analysis, with ultra pure water at 20psi for 5min, and then with the phosphate buffer solution at 20psi for 5 min. The capillary also needs to be rinsed before each sample run, with ultra pure water at 20psi for 2min, and then with phosphate buffer at 20psi for 2 min.

Preparing an OPN standard sample:

0.10526g (accurate to 0.01mg) of OPN standard substance (purity is 95%) is accurately weighed and dissolved in water, the volume is determined to be 10mL, stock solution with the concentration of 10mg/mL is prepared, and the stock solution is stored in a refrigerator at-20 ℃.

Accurately transferring 1mL of OPN standard stock solution, diluting to 10mL with water, mixing, and making into OPN standard intermediate solution (prepared at present) with concentration of 1 mg/mL.

Accurately sucking 5 muL, 10 muL, 20 muL, 50 muL, 100 muL and 200 muL to 1mL of sample injection vials from the OPN standard intermediate solution respectively, and adding corresponding volumes of water respectively to make the total volume be 1mL, thus obtaining standard samples, wherein the concentrations of the standard samples are respectively and sequentially: 5mg/L, 10mg/L, 20mg/L, 50mg/L, 100mg/L, 200mg/L (prepared immediately before use).

Preparing a solution to be tested:

accurately weighing 2.5g (accurate to 0.001g) of infant formula milk powder, and adding 30mL of ultrapure water for dissolving; adjusting the pH value to 4.6 by using 8mol/L acetic acid and fixing the volume to 50mL, centrifuging at 10000rpm for 15min, taking 5mL supernatant, adjusting the pH value to 6.8 by using 3mol/L NaOH and fixing the volume to 25mL to obtain the solution to be detected.

Making a standard curve:

and respectively injecting the OPN standard samples into capillary electrophoresis to obtain corresponding peak areas, and preparing a standard curve by taking the concentration of the OPN in the OPN standard samples as an abscissa and the corresponding peak area (or peak height) as an ordinate.

FIG. 1 is a capillary electrophoresis chart of an OPN standard sample, and the peak position of OPN can be seen from FIG. 1. The upper curve in fig. 2 is the response of OPN in the infant formula base and the separation from other major proteins in the infant formula after the infant formula has been fortified with OPN; the lower curve shows the separation of the major proteins in the infant formula, and it can be seen that there is no interference of other proteins in the peak position of OPN.

Measurement of a solution to be measured:

and respectively injecting the solution to be detected into a capillary electrophoresis apparatus to obtain peak areas, and obtaining the concentration c of the OPN in the sample to be detected by referring to the standard curve.

Ninthly, data processing:

the content of OPN in the sample to be detected is measured by concentration X, the unit is mg/100g, and the content is calculated according to the following formula:

in the formula: c-OPN concentration in milligrams per liter (mg/L) of the solution to be tested;

v is the constant volume of the sample to be detected, mL;

m is the mass of the sample to be measured, g;

n is the dilution multiple of the sample to be detected;

the result of the calculation retains two significant digits.

The capillary electrophoresis method is confirmed by reference to GB/T27417-.

Example 2

Example 2 differs from example 1 in that the pH values of the test solution and the buffer solution were both 7.2.

Example 3

Example 3 differs from example 1 in that the pH values of the test solution and the buffer solution were both 6.5.

Example 4

Example 4 differs from example 1 in that the pH values of the test solution and the buffer solution were both 7.5.

Example 5

Example 5 differs from example 1 in that the neutral coating of the quartz capillary (SCIEX, 477601) is polyethylene glycol.

Example 6

Example 6 differs from example 1 in that the operating voltage conditions were-12 kV, the detection wavelength was 218nm, the detection frequency was 4Hz, the length of the quartz capillary was 40cm, the inner diameter of the quartz capillary was 75 μm, and the temperature of the quartz capillary was 30 ℃.

Example 7

Example 7 is different from example 1 in that the operating voltage condition was-5 kV, the detection wavelength was 200nm, the detection frequency was 2Hz, the length of the quartz capillary was 20cm, the inner diameter of the quartz capillary was 25 μm, and the temperature of the quartz capillary was 0 ℃.

Example 8

Example 8 differs from example 1 in that the operating voltage conditions were-30 kV, the detection wavelength was 450nm, the detection frequency was 18Hz, the length of the quartz capillary was 100cm, the inner diameter of the quartz capillary was 75 μm, and the temperature of the quartz capillary was 50 ℃.

Example 9

Example 9 differs from example 1 in that the pH was adjusted to 4.0 with 8mol/L acetic acid and the volume was adjusted to 50mL to obtain an acidic system.

Example 10

Example 10 differs from example 1 in that the solid dairy product is whey protein powder.

Example 11

Example 11 differs from example 1 in that the buffer solution is a citric acid buffer solution.

Comparative example 1

The difference between comparative example 1 and example 1 is that the pH values of the test solution and the buffer solution were both 6.0.

Comparative example 2

Comparative example 2 is different from example 1 in that the pH values of the test solution and the buffer solution are both 8.0.

The concentrations X of OPN in the above examples 1 to 11, comparative examples 1 and 2 were tested, and the test results are shown in Table 1.

TABLE 1

Examples/comparative examples	X/mg/100g
		Example 1	529.21
Example 2	522.37
		Example 3	518.41
Example 4	521.74
		Example 5	522.11
Example 6	528.13
		Example 7	525.71
Example 8	523.32
		Example 9	521.31
Example 10	687.32
		Example 11	525.32
Comparative example 1	Not detected out
		Comparative example 2	Not detected out

As can be seen from table 1 above, the pH value has a significant influence on the separation of OPN in the dairy product, and if the pH value is too large or too small, the OPN cannot be separated from other proteins in the dairy product, so that the OPN in the dairy product cannot be qualitatively and quantitatively analyzed.

From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects:

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An analytical method for osteopontin in a dairy product, comprising:

step S1, removing casein in the aqueous solution of the dairy product to obtain a treatment solution;

step S2, adjusting the pH value of the treatment liquid to 6.5-7.5 to obtain a liquid to be detected; and

and step S3, analyzing the osteopontin in the solution to be detected by a capillary electrophoresis method by using a neutral coating capillary.

2. The analysis method according to claim 1, wherein the buffer solution used in the capillary electrophoresis method comprises any one or more of a phosphate buffer solution, a borate buffer solution, an acetate buffer solution, a borax buffer solution, and a citric acid buffer solution, preferably the pH value of the buffer solution is 6.5 to 7.5, further preferably the pH value of the buffer solution is 6.8 to 7.2, and preferably the phosphate buffer solution comprises a sodium dihydrogen phosphate solution and a disodium hydrogen phosphate solution.

3. The analytical method according to claim 2, wherein the pH of the test solution is the same as the pH of the buffer solution.

4. The method according to any one of claims 1 to 3, wherein the capillary electrophoresis method is used for analysis at an operating voltage of-5 to-30 kv, preferably-8 to-12 kv; the preferable detection wavelength is 200-450 nm, and further preferable is 210-218 nm; the detection frequency is preferably 2-18 Hz, and more preferably 2-4 Hz.

5. The analytical method according to any one of claims 1 to 4, wherein the neutral coated capillary is a quartz capillary, preferably the quartz capillary has a length of 20 to 100cm, further preferably 25 to 40 cm; the inner diameter of the quartz capillary tube is preferably 25-75 μm, and more preferably 50-75 μm.

6. The analytical method according to claim 5, wherein the temperature of the quartz capillary is 0 to 50 ℃, preferably 20 to 30 ℃.

7. The analytical method of claim 5, wherein the inner wall coating layer of the quartz capillary is polyacrylamide or polyethylene glycol.

8. The analytical method of claim 5, wherein the outer wall of the quartz capillary is coated with an elastomeric coating.

9. The assay of claim 8, wherein the elastic coating is polyimide.

10. The analysis method according to any one of claims 1 to 4, wherein the step S1 includes:

step S11, adjusting the pH value of the aqueous solution of the dairy product to 4.0-4.6 to obtain an acidic system;

and step S12, performing solid-liquid separation on the acidic system to obtain the treatment liquid, wherein the solid-liquid separation mode is preferably centrifugal separation.