Quantitative method for whey protein in milk and dairy products

Abstract

The invention relates to a quantitative method for whey protein in milk and dairy products, which comprises the following steps: preparing samples including liquid dairy products and solid dairy products; quantitative detection is carried out, and analysis is carried out by combining a capillary electrophoresis system CEInfinite with an acrylamide derivative AD coating capillary column; according to the method verification, reproducibility on the same batch of AD and different batches of AD coating capillaries is compared, and then repeatability summary of isoelectric points and peak areas is obtained; and obtaining a result. According to the method disclosed by the invention, high-efficiency separation and simultaneous quantitative analysis of alpha-lactalbumin, beta-lactoglobulin A and beta-lactoglobulin B in whey protein in dairy products are rapidly realized by using full-column imaging capillary isoelectric focusing electrophoresis (icIEF) and acrylamide derivative AD coating capillary; the method has important significance in promoting scientific research, new product development and the like while guaranteeing the product quality, protecting the health of consumers and optimizing the production process.

Description

Technical Field

[0001] The invention relates to the field of analytical chemistry and food science and technology, and in particular to a quantitative method for whey protein in milk and dairy products. Background Art

[0002] Whey protein is an important nutrient in milk and dairy products. Its quantitative detection is of great significance for ensuring the safety and nutritional value of dairy products, protecting product quality and consumer rights, guiding production and processing and optimizing product formulas, adapting to market demand and regulatory requirements, and promoting technological innovation and industry development.

[0003] Traditional whey protein analysis methods include immunological techniques, infrared spectroscopy, slab-gel isoelectric focusing (IEF), capillary electrophoresis (CE) and high performance liquid chromatography (HPLC), etc. However, these methods generally have the problems of low throughput, cumbersome operation, poor reproducibility and insufficient quantitative ability. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a method for quantifying whey protein in milk and dairy products to solve one or more problems in the prior art.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] A method for quantifying whey protein in milk and dairy products comprises the following steps:

[0007] Sample preparation, including liquid and solid dairy products;

[0008] Quantitative detection was performed using the capillary electrophoresis system CEInfinite combined with an acrylamide derivative AD-coated capillary column;

[0009] Method validation: Compare the reproducibility of the coated capillaries of the same batch of AD and different batches of AD to obtain the reproducibility summary of isoelectric point and peak area;

[0010] Get the result.

[0011] Furthermore, the sample preparation comprises the following steps:

[0012] Weigh the sample and add solvent to make up to volume;

[0013] After mixing, a solvent is added to acidify to a specified pH value, thereby separating casein and whey protein;

[0014] The acidified sample was centrifuged to separate the whey and obtain the supernatant;

[0015] The supernatant is mixed with an amphoteric electrolyte, a pI marker, and a denaturant.

[0016] Furthermore, the sample is weighed and volume is determined, and the ratio of the sample to the solvent for liquid dairy products is 1:2 to 1:8; the ratio of the sample to the solvent for solid dairy products is 1:4 to 1:20.

[0017] Furthermore, the acidification reaches a specified pH value in the range of 4.6.

[0018] Furthermore, the speed range of the acidified sample rotation centrifugation separation is 11000-15000 r / min, and the centrifugation time is 2-8 min.

[0019] Furthermore, the amphoteric electrolyte is 6% (v / v) 3-7, the markers are 1% (v / v) pI Marker 4.65 and 1% (v / v) pI Marker 6.14, and the denaturant is 5 M urea.

[0020] Furthermore, the capillary electrophoresis system CEInfinite performs detection at a wavelength of 280 nm.

[0021] Furthermore, during the quantitative detection, the focusing conditions are 1000-2000V for 1-5min and 2000-4000V for 5-15min.

[0022] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0023] The present invention uses whole-column imaging capillary isoelectric focusing electrophoresis icIEF and acrylamide derivative AD coated capillaries to quickly achieve efficient separation and simultaneous quantitative analysis of α-lactalbumin, β-lactoglobulin A and β-lactoglobulin B in whey protein in dairy products, which is of great significance in promoting scientific research and new product development while ensuring product quality, protecting consumer health, and optimizing production processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A schematic flow chart of a method for quantifying whey protein in milk and dairy products according to an embodiment of the present invention is shown.

[0025] Figure 2 A schematic diagram of the sample preparation process of a quantitative method for whey protein in milk and dairy products according to an embodiment of the present invention is shown.

[0026] Figure 3 A data graph showing a method for quantifying whey protein in milk and dairy products according to an embodiment of the present invention, wherein the whey protein in three milk products is analyzed by icIEF.

[0027] Figure 4The graph shows the test data of the reproducibility of a method for quantifying whey protein in milk and dairy products using the same batch of AD coated capillaries according to an embodiment of the present invention.

[0028] Figure 5 A graph showing the reproducibility of test data of a method for quantifying whey protein in milk and dairy products using different batches of AD-coated capillaries according to an embodiment of the present invention.

[0029] Figure 6 The standard curve diagram of α-lactalbumin, β-lactoglobulin A and β-lactoglobulin B in a method for quantifying whey protein in milk and dairy products according to an embodiment of the present invention is shown. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present invention clearer, the following is a further detailed description of a quantitative method for whey protein in milk and dairy products proposed by the present invention in conjunction with the accompanying drawings and specific embodiments. According to the following description, the advantages and features of the present invention will be clearer. It should be noted that the accompanying drawings adopt a very simplified form and all use non-precise proportions, which are only used to conveniently and clearly assist in explaining the purpose of the embodiments of the present invention. In order to make the purpose, features and advantages of the present invention more obvious and easy to understand, please refer to the accompanying drawings. It should be noted that the structure, proportion, size, etc. illustrated by the accompanying drawings of this specification are only used to match the content disclosed in the specification, so that people familiar with this technology can understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no technical substantive significance. Any modification of the structure, change of the proportional relationship or adjustment of the size, without affecting the effect that the present invention can produce and the purpose that can be achieved, should still fall within the scope of the technical content disclosed by the present invention.

[0031] See also Figure 1 The quantitative method for whey protein in milk and dairy products of this embodiment comprises the following steps:

[0032] Step 1: Sample preparation, including liquid dairy products and solid dairy products.

[0033] Step 1.1: Weigh the sample and add solvent to make up to volume.

[0034] For liquid dairy samples: the ratio of the sample to the solvent for the liquid dairy product is 1:2-1:8, 1:4. For example, taking the ratio of the sample to the solvent as 1:4, 5 g of the sample is weighed, and 20 ml of the solvent is added to make the volume to 25 ml. The solvent in this step in this embodiment is ionized water.

[0035] For solid dairy products: the ratio of sample to solvent for solid dairy products is 1:4 to 1:20. For example, taking the ratio of sample to solvent as 1:11.5, 2 g of sample is weighed and 20 ml of deionized water is added to make the volume to 25 ml.

[0036] Step 1.2: After mixing, add solvent to acidify to a specified pH value to separate casein and whey protein.

[0037] The solution fixed to volume in step 1 above was shaken and mixed, and then acetic acid was added until the pH value reached 4.6 to precipitate casein, while the whey protein remained in the solution.

[0038] Step 1.3: The acidified sample was centrifuged to separate the whey and obtain the supernatant.

[0039] The sample acidified in step 1.2 is centrifuged at 11000-15000 r / min for 2-8 min to separate the whey portion and collect the supernatant.

[0040] Step 1.4: Mix the supernatant with the zwitterionic electrolyte, pI marker and denaturant.

[0041] The supernatant collected in step 1.3 was mixed with 6% (v / v) UR 3-7. 1% (v / v) pI Marker 4.10, 1% (v / v) pI Marker 7.05 and 5M urea were mixed and ready for detection.

[0042] The amphoteric electrolyte is 6% (v / v) 3-7, suitable for experiments such as isoelectric focusing. The markers are 1% (v / v) pI Marker 4.65 and 1% (v / v) pI Marker 6.14, which are isoelectric point (pI) markers used to mark specific pH values ​​or isoelectric point positions during electrophoresis to help determine the pI value of proteins in the sample. The denaturant is 5M urea, which is a commonly used denaturant used in electrophoresis to destroy hydrogen bonds between proteins and help proteins migrate better in the electric field.

[0043] Step 2: Quantitative detection was performed using the capillary electrophoresis system CE Infinite combined with an acrylamide derivative AD coated capillary column.

[0044] Since biological molecules in the sample, such as proteins, have a strong UV absorption peak at 280nm, the capillary electrophoresis system CEInfinite is performed at a wavelength of 280nm, and the detection achieves efficient and high-resolution detection of biological macromolecules such as proteins in the entire micro-separation channel. This technology can directly perform "imaging" detection without moving the focused and fixed protein zone, greatly improving the accuracy and throughput of the analysis.

[0045] Furthermore, the acrylamide derivative coated capillary column forms a coating composed of acrylamide or its derivatives on the inner wall of the capillary to achieve the purpose of reducing or eliminating electroosmotic flow, preventing the adsorption of biomacromolecules on the tube wall, etc. This coating can effectively improve the separation efficiency and peak shape quality, while increasing the stability and service life of the capillary.

[0046] Furthermore, during quantitative detection, the focusing conditions are 1000-2000V for 1-5min and 2000-4000V for 5-15min. That is, the initial focusing is performed at a voltage of 1000-2000V for 1-5min, which can quickly place the sample or observation object within the approximate focusing range, laying the foundation for subsequent fine adjustment. Subsequently, the fine focusing is performed at a voltage of 2000-4000V for 5-15min. At higher voltages and longer times, the focusing effect can be further adjusted and optimized to ensure the best observation or separation effect.

[0047] Step 3: Method validation, compare the reproducibility of the coated capillaries of the same batch of AD and different batches of AD to obtain the reproducibility summary of isoelectric point and peak area.

[0048] Whey proteins in three milk products were analyzed by capillary isoelectric focusing (icIEF). Figure 3 As shown: the horizontal axis represents the pI point, the vertical axis represents the absorption intensity detected at a wavelength of 280nm UV, which is used for quantitative analysis. In addition, A is a blank sample, B is brand 1 milk, C is brand 2 milk, and D is brand 3 milk. 1 represents α-lactalbumin, 2 represents β-lactoglobulin A, and 3 represents β-lactoglobulin B. The blank sample here refers to a sample without milk, which is a sample that replaces the milk sample with a solvent (water). Compared with the actual sample, except for the sample without milk, the types and concentrations of other components including pI marker, amphoteric electrolyte, denaturant, etc. are exactly the same, which can be examined when no milk is added, the three target whey proteins should not be detected.

[0049] Furthermore, by comparing the reproducibility of the same batch of AD-coated capillaries, Figure 4 As shown in Figure 2, by comparing the reproducibility of different batches of AD-coated capillaries, Figure 5As shown, the reproducibility of the isoelectric point and the reproducibility of the peak area are obtained, and the summary data are shown in the following Table 1:

[0050] Table 1

[0051]

[0052] According to the results of the relative standard method RSD in Table 1 above, the RSD value is low, which means that the preparation process of this batch of AD is stable, the product quality is uniform, and the analytical system of the coated capillary runs stably on this batch of samples, which can produce reliable and consistent results. The RSD values ​​of different batches of AD in isoelectric point and peak area are also low, indicating that the entire production process is properly controlled, the difference between batches is small, and the product quality is stable. The reliability of the analytical system of this embodiment and the feasibility of the experimental method are effectively proved, providing strong data support for production and quality control.

[0053] Step: 4 Get the result.

[0054] Under the verification of step 3, the standard curves of α-lactalbumin, β-lactoglobulin A and β-lactoglobulin B were obtained as follows: Figure 6 As shown, the quantitative results of different actual samples can be obtained as shown in Table 2 below:

[0055]

[0056] It achieves rapid, accurate and efficient analysis of whey protein in milk and dairy products, providing an important tool for quality control in the dairy industry.

[0057] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0058] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for quantifying whey protein in milk and dairy products, characterized in that: The steps include: Sample preparation, including liquid and solid dairy products; Quantitative detection was performed using the capillary electrophoresis system CEInfinite combined with an acrylamide derivative AD-coated capillary column; Method validation: Compare the reproducibility of the coated capillaries of the same batch of AD and different batches of AD to obtain the reproducibility summary of isoelectric point and peak area; Get the result.

2. A method for quantifying whey protein in milk and dairy products as claimed in claim 1, characterized in that: The sample preparation comprises the following steps: Weigh the sample and add solvent to make up to volume; After mixing, a solvent is added to acidify to a specified pH value, thereby separating casein and whey protein; The acidified sample was centrifuged to separate the whey and obtain the supernatant; The supernatant is mixed with an amphoteric electrolyte, a pI marker, and a denaturant.

3. A method for quantifying whey protein in milk and dairy products as claimed in claim 2, characterized in that: The sample is weighed and volume is determined. The ratio of the sample to the solvent for liquid dairy products is 1:2 to 1:8; the ratio of the sample to the solvent for solid dairy products is 1:4 to 1:

20.

4. A method for quantifying whey protein in milk and dairy products as claimed in claim 2, characterized in that: The acidification was performed to achieve a specified pH value in the range of 4.

6.

5. A method for quantifying whey protein in milk and dairy products as claimed in claim 2, characterized in that: The rotation speed range of the acidified sample rotation centrifugation separation is 11000-15000 r / min, and the centrifugation time is 2-8 min.

6. A method for quantifying whey protein in milk and dairy products as claimed in claim 2, characterized in that: The amphoteric electrolyte is 6% (v / v) The pI markers were 1% (v / v) pI Marker 4.65 and 1% (v / v) pI Marker 6.14, and the denaturant was 5 M urea.

7. A method for quantifying whey protein in milk and dairy products according to claim 1, characterized in that: The capillary electrophoresis system CE Infinite performs detection at a wavelength of 280 nm.

8. A method for quantifying whey protein in milk and dairy products according to claim 1, characterized in that: During the quantitative detection, the focusing conditions are 1000-2000V for 1-5min, and 2000-4000V for 5-15min.

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