CN112557483B

CN112557483B - Analysis method of osteopontin in dairy product

Info

Publication number: CN112557483B
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: 2023-08-11
Anticipated expiration: 2040-11-20
Also published as: CN112557483A

Abstract

The application provides an analysis method of osteopontin in dairy products. The analysis method comprises the following steps: step S1, casein in the aqueous solution of the dairy product is removed, and a treatment solution is obtained; s2, regulating the pH value of the treatment liquid to 6.5-7.5 to obtain a liquid to be detected; and step S3, adopting a neutral coating capillary to analyze the osteopontin in the liquid to be tested by a capillary electrophoresis method. The inventors screened out neutral coated capillaries to detect OPN in dairy products. In view of the serious interference effect of casein at the peak position of OPN, the method firstly carries out casein removal treatment on the dairy product aqueous solution, then adjusts the pH value of the dairy product aqueous solution, and then utilizes the neutral coating capillary to furthest reduce hydrophobicity and interaction between electrostatic solute and the pipe wall under the condition of the pH value, thereby furthest eliminating electroosmotic flow, further accurately detecting the osteopontin in the dairy product, and the method is simple, convenient and quick to operate.

Description

Analysis method of osteopontin in dairy product

Technical Field

The application 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. Structurally, OPN is a highly phosphorylated acidic glycoprotein consisting of about 300 amino acid residues, and OPN in bovine and human milk is very similar in amino acid sequence and phosphorylation pattern, 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 bovine and human milk OPN are O-glycosylated in the same region. The OPN content in human milk was about 138mg/L, which is much higher than that in cow's milk (about 18 mg/L). The infant formula milk powder aims at realizing mother emulsification, so that the biological influence of OPN on infants can be clear. The infant formula powder on the current market has almost no reinforced OPN, so OPN is used as a potential candidate protein for enhancing the similarity between the infant formula powder and breast milk, and is increasingly paid attention to by infant formula powder manufacturers, and quantitative detection of the OPN in dairy products is also gradually paid attention to.

Currently, less research is done on the detection of OPN in infant formula milk powder, wherein mass spectrometry (Analytical method to quantify osteopontin in dairy powders and infant formulas by signature peptide quantification with UHPLC-MS/MS) developed by AOAC is based on proteomics principle, and is characterized by using trypsin to carry out enzymolysis on osteopontin, and a method for identifying and indirectly quantifying the osteopontin in milk products by detecting specific peptide fragments generated by enzymolysis through mass spectrometry. However, the above method is limited to quantitative determination of osteopontin in dairy products which do not produce maillard reaction products, and the detection accuracy is low, so that the content of osteopontin cannot be effectively quantified.

In addition, since OPN is a secreted, acidic phosphorylated glycoprotein, negatively charged in milk systems can form a protein complex by electrostatic binding with positively charged proteins, which 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 that the content of raw materials and finished products can be ensured to meet the requirements.

Disclosure of Invention

The application mainly aims to provide an analysis method of osteopontin in dairy products, which aims to solve the problem that the analysis method of the osteopontin in dairy products in the prior art is difficult to accurately quantify OPN.

In order to achieve the above object, according to one aspect of the present application, there is provided an analysis method of osteopontin in a dairy product, the analysis method including: step S1, casein in the aqueous solution of the dairy product is removed, and a treatment solution is obtained; s2, regulating the pH value of the treatment liquid to 6.5-7.5 to obtain a liquid to be detected; and step S3, adopting a neutral coating capillary to analyze the osteopontin in the liquid to be tested by a capillary electrophoresis method.

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

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

Further, in the analysis by the capillary electrophoresis method, the operating voltage is from-5 to-30 kv, and more preferably from-8 to-12 kv; preferably, the detection wavelength is 200 to 450nm, and more preferably 210 to 218nm; the detection frequency is preferably 2 to 18Hz, more preferably 2 to 4Hz.

Further, the neutral coating capillary is a quartz capillary, preferably the length of the quartz capillary is 20-100 cm, and more preferably 25-40 cm; the quartz capillary preferably has an inner diameter of 25 to 75. Mu.m, more preferably 50 to 75. Mu.m.

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

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

Further, an elastic coating is coated on the outer wall of the quartz capillary tube.

Further, the elastic coating is polyimide.

Further, the step S1 includes: step S11, regulating the pH value of the dairy product aqueous solution 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 treatment liquid, and the solid-liquid separation is preferably performed by centrifugation.

By applying the technical scheme of the application, the inventor finds that, due to higher glycosylation degree of Osteopontin (OPN) in the dairy product, the adsorption of the OPN by the uncoated capillary is serious, so that the OPN can not be effectively separated by adopting a capillary electrophoresis method, and finally, the OPN in the dairy product is detected by screening out the neutral coating capillary through a large number of tests. In view of the serious interference effect of casein at the peak position of OPN, the method firstly carries out casein removal treatment on the dairy product aqueous solution, then adjusts the pH value of the dairy product aqueous solution, and then utilizes the neutral coating capillary to furthest reduce hydrophobicity and interaction between electrostatic solute and the pipe wall under the condition of the pH value, thereby furthest eliminating electroosmotic flow, further accurately detecting the osteopontin in the dairy product, and the method is simple, convenient and quick to operate.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

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

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

Detailed Description

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

As analyzed by the background art, the problem that the analysis method of the osteopontin in the dairy product is difficult to accurately quantify the OPN exists in the prior art, and in order to solve the problem, the application provides the analysis method of the osteopontin in the dairy product.

In an exemplary embodiment of the present application, there is provided a method for analyzing osteopontin in a dairy product, the method comprising: step S1, casein in the aqueous solution of the dairy product is removed, and a treatment solution is obtained; s2, regulating the pH value of the treatment liquid to 6.5-7.5 to obtain a liquid to be detected; and step S3, adopting a neutral coating capillary to analyze the osteopontin in the liquid to be tested by a capillary electrophoresis method.

The inventor finds through a large number of experiments that, as the glycosylation degree of Osteopontin (OPN) in the dairy product is higher, the adsorption of the OPN by the uncoated capillary is serious, so that the OPN can not be effectively separated by adopting a capillary electrophoresis method, and finally, the OPN in the dairy product is detected by screening out the neutral coating capillary through a large number of tests. In view of the serious interference effect of casein at the peak position of OPN, the method firstly carries out casein removal treatment on the dairy product aqueous solution, then adjusts the pH value of the dairy product aqueous solution, and then utilizes the neutral coating capillary to furthest reduce hydrophobicity and interaction between electrostatic solute and the pipe wall under the condition of the pH value, thereby furthest eliminating electroosmotic flow, further accurately detecting the osteopontin in the dairy product, and the method is simple, convenient and quick to operate.

The dairy products include, but are not limited to, cow's milk, sheep's milk and other animal milks, and products produced by industrial processing of such animal milks as main raw materials.

The method is favorable for accurately detecting the osteopontin in the dairy product under the condition that the pH value of the treatment liquid is 6.5-7.5. In order to reduce the influence of the buffer solution on the pH value of the solution to be measured, it is preferable that the buffer solution used in the above-mentioned capillary electrophoresis method includes any one or a combination 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.

The pH influences electroosmosis and the adsorption level of components in the liquid to be tested on the pipe wall, so that the influence of the buffer solution on the pH value of the liquid to be tested is reduced to the greatest extent, and electroosmosis flow is eliminated, and further, the pH value of the liquid to be tested is preferably the same as the pH value of the buffer solution.

In one embodiment of the present application, the operating voltage is-5 to-30 kv, and more preferably-8 to-12 kv, when analyzed by the capillary electrophoresis method; preferably, the detection wavelength is 200 to 450nm, and more preferably 210 to 218nm; the detection frequency is preferably 2 to 18Hz, more preferably 2 to 4Hz.

The conditions in 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 one embodiment of the present application, the neutral coating capillary is a quartz capillary, preferably the length of the quartz capillary is 20-100 cm, and more preferably 25-40 cm; the quartz capillary preferably has an inner diameter of 25 to 75. Mu.m, more preferably 50 to 75. Mu.m.

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

The temperature affects the pH value of the solution to be measured and the separation effect of each component in the solution to be measured, and in order to separate osteopontin from other components in the solution to be measured better, the temperature of the quartz capillary is preferably 0 to 50 ℃, and more preferably 20 to 30 ℃.

Neutral coating of quartz capillary the neutral coating of quartz capillary can be considered to be a coating formed of conventional neutral materials currently used for quartz capillary, and in one embodiment of the present application, the inner wall coating of the quartz capillary is polyacrylamide or polyethylene glycol. The combination of the inner wall coating layer of the quartz capillary tube and 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 greatest extent.

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

The material having certain flexibility in the prior art can be coated on the outer wall of the quartz capillary tube by a person skilled in the art to enhance the durability, and the polyimide is preferably used as the elastic coating layer in order to further reduce the production cost.

In order to more thoroughly remove casein from the aqueous dairy solution, it is preferred that the step S1 comprises: step S11, regulating the pH value of the dairy product aqueous solution 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 treatment liquid, and the solid-liquid separation is preferably performed by centrifugation. An acidic environment at a pH of 4.0 to 4.6 helps to aggregate casein molecules into larger precipitated flocs for removal.

In addition, in the analysis of 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 injection condition of the liquid to be tested within a reasonable range, thereby being beneficial to separating the osteopontin, the temperature of a sample chamber is preferably 0-30 ℃, and the temperature of a sample chamber is preferably 10-15 ℃; preferably, the operation time is 10-60 min, preferably 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, and the pressure of the pressure sample injection is preferably 2-5 psi; the sample injection 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

(1) Instrument and conditions:

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

(2) Preparation of 40mmol phosphate buffer solution (BGE) at ph=7:

6.1mL of a 40mmol sodium phosphate dibasic solution is absorbed, 3.9mL of a 40mmol sodium phosphate monobasic solution is absorbed, and the two solutions are uniformly mixed to prepare a phosphate buffer solution with the pH value of 40mmol and 7.

(3) The operation method of the instrument comprises the following steps:

the sample injection condition is that the pressure is 2psi for 10s; the separation conditions were-8 kV and 20min.

(4) Capillary wash conditions:

the capillary tube was pre-rinsed with ultrapure water at 20psi for 5min and then with the phosphate buffer solution at 20psi for 5min before each analysis of the sample. The capillary tube was also rinsed with ultrapure water at 20psi for 2min and then with phosphate buffer at 20psi for 2min before each sample run.

(5) Preparing an OPN standard sample:

0.10526g (accurate to 0.01 mg) of OPN standard (purity: 95%) was accurately weighed, dissolved in water, and fixed to 10mL to prepare a stock solution having a concentration of 10mg/mL, and the stock solution was stored in a refrigerator at-20 ℃.

Accurately transferring 1mL of OPN standard stock solution, fixing the volume to 10mL by water, and uniformly mixing to prepare OPN standard intermediate solution (in-situ preparation) with the concentration of 1 mg/mL.

Accurately sucking 5 mu L, 10 mu L, 20 mu L, 50 mu L, 100 mu L and 200 mu L to 1mL sample injection vials from OPN standard intermediate liquid respectively, and adding water with corresponding volumes respectively to make the total volume be 1mL to obtain standard samples, wherein the concentrations of the standard samples are as follows: 5mg/L, 10mg/L, 20mg/L, 50mg/L, 100mg/L, 200mg/L (formulated immediately before use).

(6) Preparation of the solution to be tested:

accurately weighing 2.5g (accurate to 0.001 g) of infant formula milk powder, and adding 30mL of ultrapure water for dissolution; after adjusting the pH value to 4.6 with 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 with 3mol/L NaOH and fixing the volume to 25mL to obtain the solution to be measured.

(7) And (3) manufacturing 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 OPN in the OPN standard samples as an abscissa and the corresponding peak areas (or peak heights) as an ordinate.

Wherein, FIG. 1 is a capillary electrophoresis chart of OPN standard sample, and the peak position of OPN can be seen from FIG. 1. The upper graph in fig. 2 shows the response of OPN in the infant formula matrix and 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 main proteins in infant formula, and the peak position of OPN is free from the interference of other proteins.

(8) Measurement of the 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 OPN in the sample to be detected by referring to the standard curve.

(9) And (3) data processing:

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

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

v-the constant volume, mL, of the sample to be tested;

m-mass of the sample to be measured g;

n-dilution of the sample to be measured;

the result of the calculation retains two significant digits.

The capillary electrophoresis method is confirmed by referring to GB/T27417-2017 'inspection and verification guidelines for qualification analysis methods', and all standard requirements are met.

Example 2

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

Example 3

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

Example 4

Example 4 differs from example 1 in that the pH of the test solution and the buffer solution are 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 differs 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 set to 50mL to give 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

Comparative example 1 differs from example 1 in that the pH of the test solution and the buffer solution are both 6.0.

Comparative example 2

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

The concentration X of OPN in the above examples 1 to 11, comparative examples 1 and 2 was 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
		Comparative example 2	Not detected

As can be seen from table 1 above, the pH has a significant effect on the separation of OPN from the dairy product, and too large or too small a pH cannot separate OPN from other proteins in the dairy product, and thus the qualitative and quantitative analysis of OPN in the dairy product cannot be performed.

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

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method of analyzing osteopontin in a dairy product, the method comprising:

step S1, casein in the aqueous solution of the dairy product is removed, and a treatment solution is obtained;

s2, adjusting the pH value of the treatment liquid to 6.5-7.5 to obtain a liquid to be detected; and

s3, adopting a neutral coating capillary to analyze the osteopontin in the liquid to be tested by a capillary electrophoresis method;

the pH value of the buffer solution used in the capillary electrophoresis method is 6.5-7.5;

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

2. The method according to claim 1, wherein the buffer solution used in the capillary electrophoresis method comprises any one or more of phosphate buffer, borate buffer, acetate buffer, borax buffer, and citric acid buffer.

3. The method according to claim 2, wherein the buffer solution has a pH of 6.8-7.2.

4. The method of claim 2, wherein the phosphate buffer solution comprises a sodium dihydrogen phosphate solution and a disodium hydrogen phosphate solution.

5. The method according to any one of claims 1 to 4, wherein the operating voltage is-5 to-30 kv when the capillary electrophoresis is used for analysis.

6. The method according to claim 5, wherein the operating voltage is-8 to-12 kv.

7. The method according to claim 5, wherein the detection wavelength is 200 to 450nm.

8. The method according to claim 7, wherein the detection wavelength is 210 to 218nm.

9. The method according to claim 5, wherein the detection frequency is 2 to 18Hz.

10. The method according to claim 9, wherein the detection frequency is 2-4 hz.

11. The method of any one of claims 1 to 4, wherein the neutral coated capillary is a quartz capillary.

12. The method according to claim 11, wherein the length of the quartz capillary tube is 20-100 cm.

13. The method of claim 12, wherein the quartz capillary has a length of 25-40 cm.

14. The method according to claim 11, wherein the quartz capillary has an inner diameter of 25 to 75 μm.

15. The method according to claim 14, wherein the quartz capillary has an inner diameter of 50 to 75 μm.

16. The method according to claim 11, wherein the quartz capillary tube has a temperature of 0-50 ℃.

17. The method according to claim 16, wherein the temperature of the quartz capillary tube is 20-30 ℃.

18. The method of claim 11, wherein the inner wall coating of the quartz capillary is polyacrylamide or polyethylene glycol.

19. The method of claim 11, wherein the outer wall of the quartz capillary tube is coated with an elastic coating.

20. The method of claim 19, wherein the elastic coating is polyimide.

21. The method according to any one of claims 1 to 4, wherein the step S1 comprises:

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

and step S12, carrying out solid-liquid separation on the acid system to obtain the treatment liquid.

22. The method according to claim 21, wherein the solid-liquid separation is performed by centrifugation.