Method for detecting the content and proportion of whey protein and casein in milk and dairy products

Abstract

The application belongs to the technical field of food detection, and discloses a method for detecting the contents of beta-lactoglobulin, alpha-lactalbumin, bovine serum albumin, immunoglobulin, lactoferrin and casein, and determining the content and proportion of whey protein and casein. The method is based on the covalent combination of whey protein and fluorescent dye to generate a fluorescent signal, the separation of proteins through capillary gel electrophoresis, the fluorescence detection of protein signals, and the quantitative determination of five kinds of whey protein and casein according to the differences in peak areas. The method has the advantages of simple operation, no complex pretreatment steps, short analysis time (only 500s for sample injection and detection), high throughput, low cost, and is suitable for the rapid quantitative analysis of five kinds of whey protein and casein in milk and dairy products. In addition, the characteristic peak area of whey protein and casein within a certain range is used to calculate the proportion of whey protein in milk and dairy products. The method can accurately and effectively determine the proportion of whey protein and casein without absolute quantification of individual proteins.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, and specifically relates to a method for detecting the content of five whey proteins and casein in milk and dairy products, as well as the ratio of whey protein to casein, using capillary gel electrophoresis. Background Technology

[0002] Whey protein contains a variety of essential amino acids, possessing rich nutritional value and unique physiological functions. Its types and content are important indicators for evaluating the nutritional and commercial value of milk and dairy products. Whey protein mainly includes β-lactoglobulin, α-lactalbumin, immunoglobulins, lactoferrin, and bovine serum albumin. Determining the content and / or ratio of whey protein and casein in milk and dairy products is beneficial not only for evaluating the nutritional value of dairy products, improving industry standards, and protecting consumer rights, but also for providing a reference for product process control and monitoring. Furthermore, it plays a crucial role in the development of dairy products and meeting market demands.

[0003] GB10765-2021, "Infant Formula," stipulates that whey protein should account for ≥60% of the total protein in milk-based infant formula. GB10766-2021, "Follow-up Formula," also stipulates that whey protein content in infant formula should be ≥40%. The latest national standard does not specify a testing method; the testing still follows GB / T 5413.2-1997. Currently, the standard methods for whey protein content detection mainly include sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance liquid chromatography, liquid chromatography-mass spectrometry, and capillary gel electrophoresis (as shown in Table 1). However, all of these methods generally suffer from cumbersome and time-consuming sample pretreatment steps, long testing times, and high costs. In addition, Chen Lijun (ZL 202110904028) proposed a method using liquid chromatography-mass spectrometry (LC-MS) to detect the characteristic peptide signals of whey protein and casein, thereby achieving the detection of whey protein content, casein content, and / or the ratio of the two in milk powder. This method improves the accuracy and stability of peptide detection using external standard mass spectrometry. However, this method is time-consuming (protein sample digestion requires 26-30 hours, and instrument detection requires about 75 minutes) and requires screening for characteristic peptides of different proteins.

[0004] Table 1 Comparison of various standard detection methods for whey protein

[0005] Summary of the Invention

[0006] Addressing the numerous shortcomings of existing technologies, this invention utilizes fluorescent dyes to label proteins, followed by capillary gel electrophoresis for protein separation. Fluorescence detection of protein signals is then employed to establish a linear equation between protein electrophoretic peak area and concentration. This invention proposes a fully automated capillary gel electrophoresis method for the quantitative detection of five whey proteins and casein in milk and dairy products, as well as for determining the ratio of whey protein to casein. This method requires only simple centrifugation to remove fat and labeling of the actual sample before loading for detection. The detection time is only approximately 500 seconds, and the detection cost is low.

[0007] The technical solution of the present invention is as follows:

[0008] A method for detecting the content and ratio of whey protein and casein in milk and dairy products, comprising the following steps:

[0009] 1) Sample preparation: Dissolve the target proteins β-lactoglobulin, α-lactalbumin, bovine serum albumin, immunoglobulin, lactoferrin, and casein in 1×PBS to a concentration of 10 mg / mL to prepare a standard protein stock solution. Label the protein with a certain concentration of fluorescent dye, add buffer to prepare a 5 μL system, centrifuge, and incubate in a thermal cycler. Then dilute the protein sample, add reducing agent, and finally denature in a thermal cycler.

[0010] 2) Sample loading and detection: The above samples are placed into a capillary gel electrophoresis instrument for analysis to obtain the electrophoretic peak diagram of the protein sample to be tested;

[0011] 3) Standard curve plotting: Using protein standards, set different concentration gradients and perform capillary gel electrophoresis. Use Origin 9.0 software or a software data processing system to collect peak area data as the ordinate and protein concentration as the abscissa to plot the standard curve.

[0012] 4) Actual sample determination: After centrifuging the actual sample liquid milk or formula milk powder to remove fat, the proteins were labeled, diluted and denatured according to the above steps. Then, the samples were placed in a capillary gel electrophoresis instrument to obtain the electrophoretic peak diagram. The corresponding electrophoretic peak area was substituted into the standard curve to determine the specific content of the six proteins.

[0013] 5) Determination of whey protein ratio: Whey protein and casein are treated as a family, and the characteristic peak area within a certain range is measured. The proportion of whey protein in the total protein is obtained by the following calculation formula.

[0014]

[0015] Where X is the proportion of whey protein in the total protein, M1 is the peak area of ​​whey protein in the actual sample, and M2 is the peak area of ​​the casein family in the actual sample.

[0016] Further, the fluorescent dye mentioned in step 1) is one of Chromeo P503, FQ, AF488, Cy5, FITC, OPA, NDA, FC, CBQCA, 6-AQC, NBD, ICG, NanoOrange, Sypro Red, ANS, and Dylight 488, and the pH range of the solution during labeling is 8-9.

[0017] Furthermore, the incubation time and temperature marked on the thermal cycler in step 1) are 10 min, 50℃~60℃.

[0018] Further, the reducing agent mentioned in step 1) is one of DTT, β-mercaptoethanol, and TCEP.

[0019] Furthermore, the denaturation time and temperature of the thermal cycler described in step 1) are 5 min, 90℃~100℃.

[0020] Furthermore, the centrifugation conditions used for degreasing in step 4) are 12,000 rpm, 4°C, 10 min, and repeated twice.

[0021] The beneficial technical effects of the present invention are as follows:

[0022] Compared with existing technologies, the method of this invention for detecting the content of five whey proteins and casein in milk and dairy products, as well as determining the ratio of whey protein to casein, has high sensitivity and separation efficiency. It is simple to operate, fast (sample injection and detection only requires 500 s), high-throughput, and low-cost, suitable for the quantitative analysis of whey proteins in milk and dairy products, and can provide quantitative data for protein characterization. Furthermore, by treating whey protein and casein as separate families and measuring their characteristic peak areas within a certain range, this method ensures that no individual protein is missed, and accurately determines the ratio of whey protein to casein in the total protein of dairy products without absolute quantification of individual proteins. Attached Figure Description

[0023] Figure 1 Capillary electrophoresis images of α-lactalbumin, β-lactoglobulin, casein, bovine serum albumin, immunoglobulins, lactoferrin standards, and mixed standards. A) Capillary electrophoresis images of the six protein standards and mixed standards without reducing agent; B) Capillary electrophoresis images of the six protein standards and mixed standards with reducing agent.

[0024] In the diagram: α-Lac represents α-lactalbumin; β-Lg represents β-lactoglobulin; Casein represents casein; IgG represents immunoglobulin; BSA represents bovine serum albumin; and LF represents lactoferrin.

[0025] Figure 2 Capillary electrophoresis images of six protein standards at different concentration ranges. A) Mixed standard of α-lactalbumin and β-lactoglobulin; B) Casein; C) Bovine serum albumin; D) Reduced immunoglobulins; E) Unreduced immunoglobulins; F) Lactoferrin.

[0026] In the diagram: α-Lac represents α-lactalbumin; β-Lg represents β-lactoglobulin; Casein represents casein; IgG represents immunoglobulin; BSA represents bovine serum albumin; and LF represents lactoferrin.

[0027] Figure 3 Standard curves of six protein concentrations versus electrophoretic peak areas. A) α-lactalbumin; B) β-lactoglobulin; C) casein; D) bovine serum albumin; E) reduced and unreduced immunoglobulins; F) lactoferrin.

[0028] In the diagram: α-Lac represents α-lactalbumin; β-Lg represents β-lactoglobulin; Casein represents casein; IgG represents immunoglobulin; BSA represents bovine serum albumin; and LF represents lactoferrin.

[0029] Figure 4 Confirmation of the types of whey protein and casein electrophoresis peaks in actual sample electrophoresis images. A) Electrophoresis images of liquid milk with α-lactalbumin, β-lactoglobulin, lactoferrin, immunoglobulin, and casein standards added respectively; B) Electrophoresis images of milk powder with α-lactalbumin, β-lactoglobulin, lactoferrin, immunoglobulin, and casein standards added.

[0030] In the diagram: α-Lac represents α-lactalbumin; β-Lg represents β-lactoglobulin; Casein represents casein; LF represents lactoferrin; and IgG represents immunoglobulin.

[0031] Figure 5 : Schematic diagram of the peak ranges of whey protein and casein in the electrophoresis images of actual samples. A) Liquid milk; B) Milk powder.

[0032] In the diagram: Casein represents casein; whey protein represents whey protein.

[0033] Figure 6 Capillary gel electrophoresis images of liquid milk treated under four different heat processing conditions. A) Infrared ultra-brief sterilization; B) No heat processing; C) Pasteurization; D) Ultra-high temperature sterilization.

[0034] In the diagram: α-Lac represents α-lactalbumin; β-Lg represents β-lactoglobulin; Casein represents casein; IgG represents immunoglobulin; BSA represents bovine serum albumin; and LF represents lactoferrin.

[0035] Figure 7 Capillary gel electrophoresis images of six common commercially available milk powders. A) Infant formula; B) Follow-up formula; C) Toddler formula; D) Whole milk powder; E) Orange juice-based milk powder; F) Energy-structured maternal milk powder.

[0036] In the diagram: α-Lac represents α-lactalbumin; β-Lg represents β-lactoglobulin; Casein represents casein; IgG represents immunoglobulin; and LF represents lactoferrin.

[0037] Figure 8 Capillary gel electrophoresis images of different milk sources. A) Human milk; B) Goat milk; C) Cow milk; D) Camel milk.

[0038] In the diagram: α-Lac represents α-lactalbumin; β-Lg represents β-lactoglobulin; Casein represents casein; IgG represents immunoglobulin; BSA represents bovine serum albumin; and LF represents lactoferrin. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention. The described embodiments are some, but not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0040] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0041] Example 1: Capillary gel electrophoresis detection of α-lactalbumin

[0042] 1.1 Solution Preparation

[0043] 1.1.1 PBS Buffer (20×): Dissolve one packet of contents in 50 mL of distilled or deionized water to prepare a 20× buffer solution. The pH is 7.4 at 25°C. Store at -20°C. Dilute to 1× as needed before use.

[0044] 1.1.2 Fluorescent dye stock solution: Weigh 1 g of powder and dissolve it in 1 mL of dimethyl sulfoxide. Mix thoroughly to obtain a 20 mg / mL solution (dilution to 0.2 mg / mL or 0.5 mg / mL during the experiment). Store in a -20°C refrigerator.

[0045] 1.1.3 NaHCO3 buffer (pH 9): Weigh 0.0840 g sodium bicarbonate powder, dilute to 10 mL with ultrapure water, and adjust the pH to 9 with NaOH powder.

[0046] 1.1.4 α-Lactalbumin Standard Stock Solution: Weigh 0.010 g of α-lactalbumin standard protein powder and dilute to 1 mL with 1×PBS to prepare a 10 mg / mL protein standard stock solution. Dilute to 2 mg / mL with ultrapure water before use.

[0047] 1.2 Sample Preparation

[0048] 1.2.1 Labeling and Denaturation of Protein Standards: Take the α-lactalbumin standard stock solution and dilute it to 2 mg / mL with ultrapure water. In a 200 μL centrifuge tube, add 2.4 μL of ultrapure water, 1.25 μL of 2 mg / mL α-lactalbumin, 0.6 μL of labeling buffer, 0.5 μL of denature buffer, and 0.25 μL of 0.2 mg / mL fluorescent dye sequentially. Mix thoroughly to prepare a 5 μL system. At this point, the protein concentration is 0.5 mg / mL. Centrifuge for 30 s and incubate at 50℃~60℃ for 10 min in a thermal cycler. Take the labeled sample according to the required experimental concentration, add 0.5 μL of 0.1 mol / L reducing agent, and dilute with dilution buffer to a 15 μL system. Mix thoroughly with a pipette. Centrifuge the diluted sample for 30 s and denature at 90℃~100℃ for 5 min in a thermal cycler.

[0049] 1.2.2 Determination of standard curve: Take the standard of α-lactalbumin, after labeling and denaturation as described in 1.2.1, and dilute it to different concentrations (4.17 μg / mL, 8.33 μg / mL, 16.67 μg / mL, 33.33 μg / mL, 66.67 μg / mL, 100.00 μg / mL) according to experimental requirements for later use.

[0050] 1.3 Sample Testing

[0051] Determination of the standard curve: Denatured α-lactalbumin standards diluted to different concentrations were added to 200 μL centrifuge tubes, with binding buffer used as a blank control. The standard clamp (P2, 4 kV) was used for detection in a capillary gel electrophoresis apparatus. Instrument parameters were set as follows: injection voltage 4 kV, injection time 10 s; separation voltage 4 kV; separation time 500 s; effective capillary separation length 13 cm; baseline set to 1000.

[0052] 1.4 Data Analysis

[0053] After capillary electrophoresis, the raw electrophoresis data was imported into Origin 9.0 software for data analysis. The peak areas of α-lactalbumin at different concentrations were collected to establish a linear relationship between protein concentration and peak area, and to determine the detection limit. Results are as follows: Figure 1 , Figure 2 A, Figure 3 As shown in A).

[0054] The linear equation between the peak area and concentration of α-lactalbumin was obtained as y = 1011.07995x - 3058.02205, with R² = 0.95167. The calculated limit of detection for α-lactalbumin was 3.185 μg / mL. This demonstrates a good linear relationship and correlation coefficient between the peak area and protein concentration of α-lactalbumin.

[0055] Example 2: Capillary gel electrophoresis detection of β-lactoglobulin

[0056] 2.1 Solution Preparation

[0057] β-lactoglobulin standard stock solution: Weigh 0.010 g of β-lactoglobulin standard powder and dilute to 1 mL with 1×PBS to prepare a 10 mg / mL protein standard stock solution. Dilute to 2 mg / mL with ultrapure water before use.

[0058] Other solutions are prepared in the same way as in 1.1.

[0059] 2.2 Sample Preparation

[0060] Sample preparation is the same as in 1.2, except that the protein standard is replaced with the β-lactoglobulin standard.

[0061] 2.3 Sample Testing

[0062] The sample testing procedure is the same as in 1.3, except that the protein standard is replaced with the β-lactoglobulin standard.

[0063] 2.4 Data Analysis

[0064] The data collection and analysis process is the same as in 1.4. The results are as follows: Figure 1 , Figure 2A, Figure 3 As shown in B).

[0065] The linear equation between the peak area and concentration of β-lactoglobulin was obtained as y = 742.17612x - 1452.96376, with R² = 0.96431. The calculated limit of detection for β-lactoglobulin was 2.215 μg / mL. This indicates that within the protein concentration range of 4.17–100 μg / mL, there is a good linear relationship and correlation coefficient between the peak area and protein concentration of β-lactoglobulin.

[0066] Example 3: Capillary gel electrophoresis detection of casein

[0067] 3.1 Solution Preparation

[0068] Casein standard stock solution: Weigh 0.010 g of casein standard powder and dilute to 1 mL with 1×PBS to prepare a 10 mg / mL protein standard stock solution. Dilute to 2 mg / mL with ultrapure water before use.

[0069] Other solutions are prepared in the same way as in 1.1.

[0070] 3.2 Sample Preparation

[0071] Sample preparation is the same as in 1.2, except that the protein standard is replaced with the casein standard.

[0072] 3.3 Sample Testing

[0073] The sample testing procedure is the same as in 1.3, except that the protein standard is replaced with the casein standard.

[0074] 3.4 Data Analysis

[0075] The data collection and analysis process is the same as in 1.4. The results are as follows: Figure 1 , Figure 2 B) Figure 3 As shown in C).

[0076] The linear equation between casein peak area and concentration is y = 963.67691x + 7116.06373, R² = 0.99874. The calculated limit of detection for casein is 1.043 μg / mL. This indicates that within the casein concentration range of 4.17–100 μg / mL, there is a good linear relationship and correlation coefficient between casein peak area and protein concentration.

[0077] Example 4: Capillary gel electrophoresis detection of bovine serum albumin

[0078] 4.1 Solution Preparation

[0079] Bovine serum albumin standard stock solution: Weigh 0.010 g of bovine serum albumin standard powder and dilute to 1 mL with 1×PBS to prepare a 10 mg / mL protein standard stock solution. Dilute to 2 mg / mL with ultrapure water before use.

[0080] Other solutions are prepared in the same way as in 1.1.

[0081] 4.2 Sample Preparation

[0082] Sample preparation is the same as in 1.2, except that the protein standard is replaced with bovine serum albumin standard.

[0083] 4.3 Sample Testing

[0084] The sample testing steps are the same as in 1.3, except that the protein standard is replaced with bovine serum albumin standard.

[0085] 4.4 Data Analysis

[0086] The data collection and analysis process is the same as in 1.4. The results are as follows: Figure 1 , Figure 2 C Figure 3 As shown in D).

[0087] The linear equation between the peak area and concentration of bovine serum albumin is y = 1284.43307x + 15713.29575, with R² = 0.98282. This indicates that within the protein concentration range of 4.17–100 μg / mL, there is a good linear relationship and correlation coefficient between the peak area and protein concentration of bovine serum albumin.

[0088] Example 5: Capillary gel electrophoresis detection of immunoglobulins

[0089] 5.1 Solution Preparation

[0090] Immunoglobulin standard stock solution: Weigh 0.010 g of immunoglobulin standard powder and dilute to 1 mL with 1×PBS to prepare a 10 mg / mL protein standard stock solution. Dilute to 2 mg / mL with ultrapure water before use.

[0091] Other solutions are prepared in the same way as in 1.1.

[0092] 5.2 Sample Preparation

[0093] The sample was prepared in two steps. The first preparation was the same as in 1.2, except that the protein standard was replaced with the immunoglobulin standard. The second preparation did not add a reducing agent, and the excess volume was made up with dilution buffer. The protein standard was replaced with the immunoglobulin standard, and the other preparation steps were the same as in 1.2.

[0094] 5.3 Sample Testing

[0095] The sample testing steps are the same as in 1.3, except that the protein standard is replaced with an immunoglobulin standard.

[0096] 5.4 Data Analysis

[0097] The data collection and analysis process is the same as in 1.4. The results are as follows: Figure 1 , Figure 2 D) Figure 2 E) Figure 3 As shown in E).

[0098] The linear equation between the peak area and concentration of unreduced immunoglobulins is y = 1650.445x + 8332.696, R² = 0.96373; the linear equation between the peak area and concentration of reduced immunoglobulins is y = 1806.94245x + 4959.18604, R² = 0.98967. It can be seen that within the protein concentration range of 4.17–100 μg / mL, there is a good linear relationship and correlation coefficient between the peak area and protein concentration of immunoglobulins before and after reduction.

[0099] Example 6: Capillary gel electrophoresis detection of lactoferrin

[0100] 6.1 Solution Preparation

[0101] Lactoferrin Standard Stock Solution: Weigh 0.010 g of lactoferrin standard powder and dilute to 1 mL with 1×PBS to prepare a 10 mg / mL protein standard stock solution. Dilute to 2 mg / mL with ultrapure water before use.

[0102] Other solutions are prepared in the same way as in 1.1.

[0103] 6.2 Sample Preparation

[0104] Sample preparation is the same as in 1.2, except that the protein standard is replaced with the lactoferrin standard.

[0105] 6.3 Sample Testing

[0106] The sample testing steps are the same as in 1.3, except that the protein standard is replaced with the lactoferrin standard.

[0107] 6.4 Data Analysis

[0108] The data collection and analysis process is the same as in 1.4. The results are as follows: Figure 1 , Figure 2 F) Figure 3 As shown in F).

[0109] The linear equation between the peak area and concentration of lactoferrin is y = 173.7197x + 30884.99716, with R² = 0.99054. This indicates that within the protein concentration range of 10–150 μg / mL, there is a good linear relationship and correlation coefficient between the peak area and protein concentration of lactoferrin.

[0110] Example 7: Capillary gel electrophoresis detection of actual samples

[0111] 7.1 Sample Preparation

[0112] 7.1.1 Liquid milk samples: Four common commercially available liquid milk samples were placed in 2 mL centrifuge tubes and degreased using a high-speed centrifuge. The centrifugation conditions were set to 12000 rpm, 4℃, and 10 min. The middle layer was collected, and the process was repeated twice before storing the samples at -20℃ for later use. The degreased liquid milk was diluted to a protein concentration of 2 mg / mL before protein labeling, dilution, and denaturation.

[0113] 7.1.2 Solid Milk Powder Samples: Weigh milk powder into 2 mL centrifuge tubes and bring the volume to 2 mL with 1×PBS to achieve a final protein concentration of 6 mg / mL. After mixing the sample thoroughly, remove fat using a high-speed centrifuge. Set the centrifuge to 12000 rpm, 4℃, and 10 min. Collect the middle layer, repeat the process twice, and then store at -20℃ for later use. The six defatted milk powder samples were labeled with a fluorescent dye, denatured, and then used for further processing.

[0114] 7.1.3 Identification of protein signal peak types in electrophoresis images

[0115] In six defatted milk powder samples, 0 μL, 0.3 μL, and 0.5 μL of labeled α-lactalbumin and β-lactoglobulin standards at 2 mg / mL were added respectively. The samples were then labeled with fluorescent dyes, diluted, and denatured for later use.

[0116] 7.1.4 Detection of liquid milk from different sources

[0117] Liquid milk from four different common sources was collected in 2 mL centrifuge tubes and degreased using a high-speed centrifuge. Centrifugation conditions were set at 12000 rpm, 4℃, and 10 min. The intermediate layer was collected, and the process was repeated twice before the tubes were stored at -20℃ for later use. The degreased liquid milk was then diluted and subjected to protein labeling, dilution, and denaturation.

[0118] 7.2 Sample Testing

[0119] Labeled liquid milk and solid milk powder samples were added to 200 μL centrifuge tubes, with buffer as a blank control. Detection was performed using a standard clamp (P2, 4 kV) in a capillary gel electrophoresis apparatus. Instrument parameters were set as follows: injection voltage 4 kV, injection time 10 s; separation voltage 4 kV; separation time 500 s; effective capillary separation length 13 cm; baseline set to 1000.

[0120] 7.3 Data Analysis

[0121] 7.3.1 Identification of protein signal peak types in electrophoresis images

[0122] After capillary electrophoresis of the actual samples, the raw electrophoretic peak data was imported into Origin 9.0 software for data analysis. The signal peak components were determined based on the signal changes in the electrophoretic graph after the addition of protein standards, thus identifying the positions of α-lactalbumin, β-lactoglobulin, and casein in the electrophoretic graph. The results are as follows: Figure 4 As shown.

[0123] Different proteins have different elution times and positions in electrophoresis due to their different molecular weights. The larger the molecular weight, the longer the elution time. In capillary gel electrophoresis, α-lactalbumin has the shortest elution time, while casein has the longest.

[0124] 7.3.2 Determination of Protein Content

[0125] After capillary electrophoresis of the actual sample, the raw electrophoresis data was imported into Origin 9.0 software for data analysis. The peak areas of whey proteins (α-lactalbumin, β-lactoglobulin) and casein were obtained. The peak areas of α-lactalbumin, β-lactoglobulin, and casein were substituted into their respective standard curve equations to calculate the content of the three proteins in the actual sample. The results are as follows: Figure 6 , Figure 7 As shown in Tables 2 and 3.

[0126] 7.3.3 Determination of whey protein ratio

[0127] After capillary electrophoresis of the actual samples, Origin 9.0 was used to group the five whey proteins and casein into separate families, and then... Figure 5 The electrophoretic peak areas of whey protein and casein were obtained as shown, and the proportion of whey protein in the actual sample was calculated using the formula. The results are as follows: Figure 6 , Figure 7 As shown in Tables 2 and 3.

[0128] Where X is the proportion of whey protein in the total protein, M1 is the peak area obtained by treating β-lactoglobulin and α-lactalbumin as a family in the actual sample, and M2 is the peak area of ​​the casein family in the actual sample.

[0129] Table 2. Detection data of four common commercially available liquid milk samples.

[0130]

[0131] Table 3. Test data of six common commercially available milk powder samples

[0132]

[0133] 7.3.4 Detection of liquid milk from different sources

[0134] After capillary electrophoresis of the actual samples, the raw electrophoresis data was imported into Origin 9.0 software for data analysis. Electrophoresis peak diagrams were obtained for four different milk sources (human milk, goat milk, cow milk, and camel milk). The results are as follows: Figure 8 As shown.

[0135] Example 8: Reagent Kit

[0136] A quantitative detection kit for whey protein and casein is prepared by packaging whey protein standards, casein standards, fluorescent dyes, and sample buffers (e.g., PBS, labeling buffer).

Claims

1. A method for detecting the content and ratio of whey protein and casein in milk and dairy products, characterized in that: Includes the following steps: 1) Sample preparation: Dissolve the target proteins β-lactoglobulin, α-lactalbumin, bovine serum albumin, immunoglobulin, lactoferrin, and casein in 1×PBS to a concentration of 10 mg / mL to prepare a standard protein stock solution. Label the protein with a certain concentration of fluorescent dye, add buffer to prepare a 5 μL system, centrifuge, and incubate in a thermal cycler. Then dilute the protein sample, add reducing agent, and finally denature in a thermal cycler. 2) Sample loading and detection: The above samples are placed into a capillary gel electrophoresis instrument for analysis to obtain the electrophoretic peak diagram of the protein sample to be tested; 3) Standard curve plotting: Using protein standards, set different concentration gradients and perform capillary gel electrophoresis. Use Origin 9.0 software or a software data processing system to collect peak area data as the ordinate and protein concentration as the abscissa to plot the standard curve. 4) Actual sample determination: After centrifuging the actual sample liquid milk or formula milk powder to remove fat, the proteins were labeled, diluted and denatured according to the above steps. Then, the samples were placed in a capillary gel electrophoresis instrument to obtain the electrophoretic peak diagram. The corresponding electrophoretic peak area was substituted into the standard curve to determine the specific content of the six proteins. 5) Determination of whey protein ratio: Whey protein and casein are treated as a family, and the characteristic peak area within a certain range is measured. The proportion of whey protein in the total protein is obtained by the following calculation formula. Where X is the proportion of whey protein in the total protein, M1 is the peak area of ​​whey protein in the actual sample, and M2 is the peak area of ​​the casein family in the actual sample.

2. The method for detecting the content and ratio of whey protein and casein in milk and dairy products according to claim 1, characterized in that: The thermal cycler described in step 1) is marked with an incubation time and temperature of 10 min, 50℃~60℃.

3. The method for detecting the content and ratio of whey protein and casein in milk and dairy products according to claim 1, characterized in that: The reducing agent mentioned in step 1) is one of DTT, β-mercaptoethanol, and TCEP.

4. The method for detecting the content and ratio of whey protein and casein in milk and dairy products according to claim 1, characterized in that: The denaturation time and temperature of the thermal cycler mentioned in step 1) are 5 min, 90℃~100℃.

5. The method for detecting the content and ratio of whey protein and casein in milk and dairy products according to claim 1, characterized in that: The centrifugation conditions used for degreasing in step 4) are 12,000 rpm, 4°C, 10 min, and repeated twice.

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