Method for detecting IgG in fat-containing formula food

Abstract

The invention relates to the technical field of inspection and detection, in particular to a method for detecting IgG in fat-containing formula food. The invention discloses a method for measuring the content of IgG in fat-containing formula food. According to the method, after a sample to be measured is subjected to enzymolysis, ultrasonic extraction is adopted, the extraction rate of IgG is increased, an extracting solution is filtered after being subjected to high-speed centrifugation, a sample solution is obtained, the sample is analyzed through high performance liquid chromatography, and repeatability and accuracy are high.

Description

Technical Field

[0001] This invention relates to the field of testing and detection technology, and in particular to a method for detecting IgG in fat-containing formula foods. Background Technology

[0002] Immunoglobulins (Ig) are essential components of the body's immune system, widely found in the blood, milk, colostrum, and lymph of mammals. They are important immune molecules that specifically recognize and bind to invading pathogens such as bacteria and viruses, and mediate immune responses including complement activation and opsonization. They play a crucial role in enhancing the body's immunity and preventing bacterial and viral invasion. With continuous advancements in immunoglobulin isolation technology, our understanding of their biological functions and types has deepened. Immunoglobulins mainly include five classes: IgG, IgA, IgM, IgD, and IgE. In bovine colostrum, the predominant Ig is isotype IgG (80%). In recent years, an increasing number of foods and health products have added immunoglobulins, primarily IgG, to enhance their functions, such as regulating human metabolism, strengthening immunity, and fighting infection.

[0003] Immunoglobulin IgG isolated from cow's milk has become an important additive in infant formula and various dairy products and health supplements. Since the content of immunoglobulin IgG directly determines the quality and price of various formula products, its detection is extremely important. Immunoglobulin IgG has a unique molecular structure and biological activity, making detection methods relatively complex. Detection methods for immunoglobulin IgG mainly include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), immunogold assay, and capillary electrophoresis. Among these, HPLC has the advantages of high sensitivity, fast analysis speed, and low detection limit. The current national standard GB / T 5009.194-2003, "Determination of Immunoglobulin IgG in Health Foods," adopts HPLC. A commonly used method in HPLC for determining immunoglobulin content is online analysis using an online chromatographic column. However, high-performance liquid chromatography (HPLC) has some drawbacks in detecting immunoglobulin IgG in lipid-containing formula foods: 1. The complex matrix of lipid-containing formula foods can contaminate and damage instruments and columns, increasing detection costs; 2. The extraction efficiency of immunoglobulin IgG is low, and it cannot be completely extracted, affecting the accuracy and repeatability of the detection; 3. There are many immunoglobulin subtypes, and other proteins may co-elut with IgG, leading to overestimation of the measured results. Therefore, improving the accuracy and repeatability of detection is crucial for the quality control of lipid-containing formula foods. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for detecting IgG in fat-containing formula foods, in order to improve the repeatability and / or accuracy of the detection.

[0005] The present invention provides a method for detecting IgG in fat-containing formulated foods, comprising:

[0006] The sample to be tested was diluted with phosphate buffer, then subjected to lipase enzymatic hydrolysis and ultrasonic extraction, and the supernatant was collected by centrifugation for HPLC detection.

[0007] The enzymatic hydrolysis conditions include: enzymatic hydrolysis at 35-40℃ for 30-120 min, and a lipase to sample mass ratio of 2:1-20:1.

[0008] The ultrasound has a power of 300–500W, a frequency of 30–50kHz, a temperature of 10–40℃, and a duration of 30–90min.

[0009] The method provided by this invention involves enzymatic hydrolysis and ultrasonic extraction of the sample before injection, which not only improves the extraction efficiency but also eliminates fats, avoiding contamination and clogging of the HPLC chromatograph and column by impurities such as fats, thereby improving the accuracy and repeatability of detection.

[0010] In this invention, the sample to be tested is at least one of liquid milk, milk powder, milk tablets, milk-containing beverages, or milk-containing candies. In a specific embodiment, the sample to be tested is a fat-containing formula food such as bovine colostrum calcium-fortified tablets, lactoferrin-yeast β-glucan-modified milk powder, or lactoferrin-bovine colostrum-modified milk powder.

[0011] In this invention, the buffer solution not only maintains the acid-base balance in the enzymatic hydrolysis system, protecting enzyme activity and improving reaction efficiency, but also resists interference from acidic and alkaline substances. Furthermore, it serves as a solvent for HPLC sample injection. The solvent's solubility, flow properties, and interaction with the sample directly affect sample separation and detection. This invention has conducted experiments and screening of buffer solutions. The results show that sodium phosphate buffer solution is the most effective. Preferably, the phosphate buffer solution is a 0.05 mol / L sodium phosphate buffer solution with a pH of 7.4.

[0012] The degree of dissolution of the test sample is related to the degree of enzymatic hydrolysis. The mass-to-volume ratio of the test sample to the phosphate buffer is 1 g:(50-400) mL. Preferably, the mass-to-volume ratio of the test sample to the phosphate buffer is 1 g:(50-500) mL. More preferably, the mass-to-volume ratio of the test sample to the phosphate buffer is 1 g:50 mL, 1 g:400 mL, or 1 g:500 mL.

[0013] The temperature, time, and amount of enzyme used in enzymatic hydrolysis significantly affect the degree of hydrolysis. To improve the effectiveness of enzymatic hydrolysis, this invention optimizes and screens the hydrolysis conditions, thereby further improving the degree of fat removal and the accuracy of detection. Experiments show that the hydrolysis effect is superior to that under other parameters under the following conditions: hydrolysis at 37℃ for 60 min, and a lipase-to-sample mass ratio of 2:1.

[0014] The power, frequency, temperature, and duration of ultrasound significantly affect the extraction effect. Sufficient extraction is more conducive to improving the accuracy and repeatability of detection. This invention optimizes the ultrasound parameters to better apply them to the enzymatically hydrolyzed products. Compared to other parameters, the following conditions are more conducive to improving the extraction effect: ultrasound power of 400W, frequency of 40kHz, temperature of 37℃, and duration of 60min.

[0015] Centrifugation is used to separate the extracted sample from impurities. In this invention, the centrifugation conditions include 5000–10000 r / min for 1–10 min. Preferably, the centrifugation conditions include 5000–8000 r / min for 1–10 min, or 8000–10000 r / min for 1–10 min, 5000–10000 r / min for 5–10 min, or 5000–10000 r / min for 1–5 min. In a specific embodiment, the centrifugation parameters include 10000 r / min for 10 min.

[0016] This invention utilizes a Bio-Monolith Protein G column, which can withstand pressures up to 140 MPa, exhibiting excellent pressure resistance and applicability to most common high-performance liquid chromatography (HPLC) methods. By optimizing the mobile phase elution program, the separation of the target IgG peak is improved, thereby enhancing detection accuracy.

[0017] In some embodiments, the chromatographic conditions for HPLC detection include:

[0018] Column: Bio-Monolith Protein G;

[0019] Mobile phase:

[0020] Phase A is a 0.05 mol / L phosphate buffer solution with a pH of 7.4;

[0021] Phase B is a 0.5 mol / L aqueous solution of acetic acid, pH 2.0;

[0022] The elution gradient is:

[0023] 0–0.50 min, 100%–100% mobile phase A;

[0024] 0.51–6.00 min, 0%–0% mobile phase A;

[0025] 6.01–12.00 min, 100%–100% mobile phase A.

[0026] In some embodiments, the chromatographic conditions for HPLC detection further include: flow rate: 0.1–2.0 mL / min, column pressure: 40 bar, column temperature: 25 °C, detection wavelength: 280 nm, and injection volume: 10–50 μL.

[0027] The method of the present invention further includes the preparation of a reference solution: IgG is dissolved in 0.05 mol / L phosphate buffer at pH 7.4 to obtain the reference solution; the concentration of the reference solution is 0.2 to 1 mg / mL.

[0028] The detection method of the present invention uses the peak area percentage method to qualitatively and / or quantitatively determine the components of the test solution based on the chromatogram of the reference solution.

[0029] This invention discloses a method for determining the IgG content in fat-containing formula foods. The method involves enzymatically hydrolyzing the sample to be tested, followed by ultrasonic extraction to improve the IgG extraction rate. The extract is then centrifuged at high speed and filtered to obtain a sample solution. The sample is analyzed by high-performance liquid chromatography, which has high repeatability and accuracy. Attached Figure Description

[0030] Figure 1 The chromatogram of the reference standard in Comparative Example 1 is shown.

[0031] Figure 2 The chromatogram of the test sample in Comparative Example 1 is shown.

[0032] Figure 3 The chromatogram of the reference standard in Comparative Example 2 is shown.

[0033] Figure 4 The chromatogram of the test sample in Comparative Example 2 is shown.

[0034] Figure 5 Using the sample preparation conditions of Group 1 and the chromatographic conditions of Group 2, the chromatogram of the reference standard was detected.

[0035] Figure 6 The chromatogram of the test sample was detected using the sample preparation conditions of Group 1 and the chromatographic conditions of Group 2. Detailed Implementation

[0036] This invention provides a method for detecting IgG in fat-containing formula foods. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0037] The complex matrix of lipid-containing food products causes significant interference and increases instrument maintenance costs for the detection of immunoglobulin IgG. Furthermore, the complex matrix can affect the extraction rate, and conventional shaking extraction cannot guarantee complete extraction.

[0038] This invention utilizes lipase to degrade fat in fat-containing food formulations, reducing the interference of food fat on the detection of immunoglobulin IgG, and also reducing the contamination and damage of food fat to high-performance liquid chromatography instruments and columns, thereby improving experimental repeatability and reducing instrument maintenance costs.

[0039] This invention employs ultrasonic extraction to extract IgG from lipid-containing food formulations, thereby improving the extraction efficiency of immunoglobulin IgG, lowering the limit of detection and limit of quantitation, and enhancing the precision and accuracy of detection. Specifically:

[0040] The method for determining the IgG content in fat-containing formula foods provided by this invention includes the following steps:

[0041] (1) Accurately weigh the sample, dilute it with sodium phosphate buffer, and then add lipase for enzymatic hydrolysis;

[0042] (2) The enzymatically hydrolyzed sample solution was extracted by ultrasonication, diluted to a certain volume, shaken, separated by centrifugation and allowed to stand. Finally, the supernatant after standing was taken and filtered through an organic membrane to obtain the sample to be tested for later use.

[0043] (3) The sample to be tested was detected using a Bio-Monolith Protein G column. The HPLC detection conditions included using sodium phosphate buffer (A) and acetic acid solution (B) as mobile phases, and the gradient elution program was as follows: 0–0.50 min, 100%–100% mobile phase A; 0.51–6.00 min, 0%–0% mobile phase A; 6.01–12.00 min, 100%–100% mobile phase A.

[0044] In some embodiments, the ratio of sample to sodium phosphate buffer used is 1:50 to 1:500.

[0045] In some embodiments, the amount of lipase added is between 100 and 1000 μL, and the enzymatic hydrolysis time is between 30 and 120 min.

[0046] In some embodiments, the ultrasound temperature is between 10 and 40°C, and the duration is 60 minutes.

[0047] In some embodiments, the ultrasonic power is 400W and the frequency is 40kHz.

[0048] In some embodiments, the centrifugation speed is 5000–10000 r / min, and the centrifugation time is 1–10 min.

[0049] In some embodiments, the detection wavelength is 280 nm; the flow rate is 0.1–2.0 mL / min; and the injection volume is 5–50 μL. Preferably, the flow rate is 0.5–1.2 mL / min; more preferably, the flow rate is 0.8–1.2 mL / min. Even more preferably, the flow rate is 1 mL / min. Preferably, the injection volume is 5 μL–20 μL; more preferably, the injection volume is 10 μL–20 μL; and most preferably, the injection volume is 10 μL or 20 μL.

[0050] In some embodiments, the molar concentration of phosphate buffer is 0.05–0.5 mol / L, and the mass percentage concentration of acetate buffer is 2%–8%.

[0051] The method for detecting immunoglobulin IgG content in samples provided by this invention uses all reference standards, reagents, and instruments purchased from the market. Among them, bovine colostrum calcium-fortified tablets, lactoferrin yeast β-glucan-modified milk powder, and lactoferrin bovine colostrum-modified milk powder are from Hubei Angel Yeast Co., Ltd., and their IgG contents are 4g / 100g, 10g / 100g, and 10g / 100g, respectively.

[0052] Instruments and equipment: Agilent 1260 high-performance liquid chromatograph; Mettler Toledo electronic analytical balance; ultrasonic extractor; centrifuge.

[0053] Reagents: Purified water, sodium dihydrogen phosphate, sodium dihydrogen phosphate, glacial acetic acid, sodium chloride, sodium hydroxide, phosphate, immunoglobulin IgG control (source: Sigma)

[0054] The present invention will be further illustrated below with reference to the embodiments:

[0055] Example 1: The Influence of Pretreatment Conditions on Detection Results

[0056] I. Sample Preparation

[0057] 1. Preparation of the reference stock solution: Accurately weigh 0.010 g of immunoglobulin IgG standard, dissolve it in 0.05 mol / L pH 7.4 phosphate buffer, and bring the volume to 10 mL. Shake well. This prepares a reference stock solution with an immunoglobulin IgG concentration of 1 mg / mL.

[0058] 2. Preparation of Standard Curves: Take appropriate amounts of the stock solution of the reference standard and dilute it with 0.05 mol / L pH 7.4 phosphate buffer to prepare a series of standard solutions containing IgG of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL. Prepare immediately before use.

[0059] 3. Sample preparation: Bovine colostrum calcium-compressed candy was used as the test sample. 0.2g of the test sample (approximately equivalent to 8mg of IgG) was accurately weighed and placed in a 10mL volumetric flask. An appropriate amount of 0.05mol / L pH7.4 phosphate buffer was added (the ratio of test sample to buffer is shown in Table 1). An appropriate amount of lipase was added (as shown in Table 1). After enzymatic hydrolysis, the sample was extracted by sonication (as shown in Table 1). The sample was shaken well, centrifuged, and allowed to stand. Finally, the supernatant was collected and filtered to obtain the finished product for later use.

[0060] The preparation conditions for each of the above samples are shown in Table 1.

[0061] Table 1. Sample preparation conditions for each group (1–11)

[0062]

[0063] Result calculation:

[0064] Immunoglobulin IgG in the sample is calculated using the following formula:

[0065] The analytical results are presented as follows: The IgG content in the sample is calculated according to formula (1):

[0066]

[0067] In the formula:

[0068] X — The content of immunoglobulin IgG in the sample, in grams per 100 grams (g / 100g);

[0069] c—The concentration of immunoglobulin IgG calculated from the standard curve, in grams per milliliter (g / mL);

[0070] V—The final volume of the sample, in milliliters (mL);

[0071] m — the mass of the sample, in grams (g).

[0072] 4. The test results under the sample preparation conditions of each group are shown in Table 2.

[0073] Table 2. Measurement results under the sample preparation conditions for each group (1–11)

[0074]

[0075] The data in the table above shows that:

[0076] When the sample-to-liquid ratio was 1:50, the enzymatic hydrolysis time was 30 min at 37℃, and the ultrasonic extraction time was 60 min, the IgG content in the bovine colostrum calcium-fortified compressed candy was close to the theoretical content, indicating that these conditions were suitable. Comparing the whole milk powder content and IgG content in different products, the optimal sample-to-liquid ratios for lactoferrin-yeast β-glucan-modified milk powder and lactoferrin-bovine colostrum-modified milk powder were 1:400 and 1:500, respectively, with enzymatic hydrolysis time of 30 min at 37℃ and ultrasonic extraction time of 60 min.

[0077] II. The samples prepared under the above conditions were analyzed using the following chromatographic conditions:

[0078] Chromatographic conditions include:

[0079] Column: Bio-Monolith Protein G;

[0080] Mobile phase: Phase A: 0.05 mol / L phosphate buffer, pH 7.4

[0081] Phase B: 0.5 mol / L acetic acid aqueous solution, pH 2.0

[0082] Gradient elution:

[0083] 0–0.50 min, 100%–100% mobile phase A;

[0084] 0.51–6.00 min, 0%–0% mobile phase A;

[0085] 6.01–12.00 min, 100%–100% mobile phase A;

[0086] Flow rate: 1 mL / min, column pressure: 40 bar, column temperature: 25 °C;

[0087] Detection wavelength: 280nm, injection volume: 10~50μL.

[0088] Table 3 Chromatographic conditions for Group 1, Group 2, and Group 3

[0089]

[0090] III. Detection Results

[0091] 1. Linearity evaluation:

[0092] The immunoglobulin IgG standard solutions were detected using the methods described in Part I, employing the sample preparation conditions of Group 1 and the chromatographic conditions of Group 1. The chromatograms are shown below. Figures 5-6 The test results are shown in Table 4:

[0093] Table 4. HPLC Detection Results of IgG Standards

[0094]

[0095] Linearity evaluation: Correlation coefficient R 2 The correlation coefficient (R) was 0.9995, indicating that this method showed good linearity in determining the content of immunoglobulin IgG in samples within a concentration range of 0.2 to 1.0 mg / mL, meeting the requirements of GB / T 27404-2008 "Laboratory Quality Control Standard for Physicochemical Testing of Food". GB / T 27404-2008 requires a correlation coefficient R... 2 ≥0.99.

[0096] 2. Repeatability test

[0097] Experimental method: Six samples were weighed and prepared according to the method in Part I. The content of immunoglobulin IgG in the samples was detected, and the RSD% was calculated. The experimental data of the scheme using the sample preparation conditions of Group 1 and the chromatographic conditions of Group 1 are shown in Table 5.

[0098] Table 5 Results of repeatable immunoglobulin IgG testing

[0099]

[0100] The RSD% of immunoglobulin IgG content in the 6 samples was 2.5%, indicating that the method has good repeatability and meets the requirements of GB / T 27404-2008 "Laboratory Quality Control Standard for Physicochemical Testing of Food". GB / T 27404-2008 requires a correlation coefficient RSD ≤ 2.7%.

[0101] 3. Recovery rate test

[0102] The experimental method involved accurately weighing an appropriate amount of sample powder (approximately equivalent to 4 mg of IgG reference standard) into a 10 mL volumetric flask, and then weighing six portions. For each portion, 4.5 mg of immunoglobulin IgG reference standard was added, along with an appropriate amount of diluent and 100 μL of lipase. After enzymatic hydrolysis, the sample was extracted using ultrasound (400 W) for 60 min. The extract was then cooled to room temperature, and the volume was adjusted to the mark and shaken well. The solution was transferred to a 2 mL centrifuge tube, centrifuged at 10000 rpm for 10 min, and the supernatant was filtered through a 0.45 μm filter membrane to obtain the sample spiked solution, which was then analyzed using an instrument. The recovery results for the tests using the sample preparation conditions of Group 1 and the chromatographic conditions of Group 1 are shown in Table 6.

[0103] Table 6 Results of Immunoglobulin IgG Recovery Rate Detection

[0104]

[0105] The average recovery rate of immunoglobulin IgG spiked in the 6 samples was 103.50%, and the relative standard deviation (RSD) was 1.5%. The recovery rate and RSD should meet the requirements of GB / T 27404-2008 "Laboratory Quality Control Standard for Physicochemical Testing of Food". When the content of the tested component is less than 10 / 100, the recovery rate is between 95% and 105%.

[0106] 4. Limit of detection and limit of quantitation

[0107] The experimental method involved accurately weighing an appropriate amount of IgG reference standard, making up to the required volume, serially diluting, and then performing the analysis. The first set of chromatographic conditions was used, and the limits of detection and quantitation were calculated based on the concentrations corresponding to 3 times and 10 times the signal-to-noise ratio of the IgG peak. The experimental results are shown in Table 7.

[0108] Table 7. Measurement results under the sample preparation conditions for each group of samples.

[0109]

[0110] 5. Other:

[0111] The immunoglobulin IgG standard solutions were analyzed using the methods described in Part I. Specifically, the solutions were analyzed six times (Group II 1–6) using the sample preparation conditions of Group 2 and the chromatographic conditions of Group 2; and six times (Group III 1–6) were analyzed using the sample preparation conditions of Group 2 and the chromatographic conditions of Group 3. The recovery, repeatability, and linearity results are shown in the table below.

[0112] Table 8. Results of IgG recovery rate under the preparation conditions of Groups 2 and 3

[0113]

[0114]

[0115] Table 9. IgG repeatability under preparation conditions in Groups 2-6 and 3-6

[0116]

[0117] Table 10 Linearity results under the second group of preparation conditions

[0118]

[0119] Table 11 Linearity results under the preparation conditions of the third group

[0120]

[0121] Comparative Example 1: Effect of Chromatographic Conditions on Detection Results

[0122] After processing the bovine colostrum calcium-fortified compressed candy using the sample preparation conditions described in Example 1, HPLC analysis was performed. The HPLC conditions were in accordance with GB / T5009.194-2003, "Determination of Immunoglobulin IgG in Health Foods".

[0123] The national standard uses Hi-Traprotein G columns, which can only withstand 3 bar. While suitable for rapid protein determination in liquid chromatography systems, the high back pressure can easily cause the packing material to detach from the column wall, leading to leakage, decreased column efficiency, and a short lifespan. Furthermore, it places stringent requirements on the entire liquid chromatography system's piping and interfaces. Our laboratory's liquid chromatography system cannot achieve the column pressure required by the national standard. Therefore, we can only explore solutions by reducing the flow rate and extending the elution time.

[0124] Chromatographic column: Pharmacia HI-Trap Protein G;

[0125] Mobile phase: Phase A: 0.05 mol / L phosphate buffer, pH 6.5

[0126] Phase B: 0.05 mol / L phosphate buffer, pH 2.5

[0127] Gradient elution:

[0128] 0~18min(100%A~100%A),

[0129] 18.01~22.00min (0%B~100%B),

[0130] 22.01~68.00min (100%B~100%B),

[0131] 68.01~70.00min (100%B~0%B),

[0132] 70.01~90.00min (100%A~100%A);

[0133] Flow rate: 0.1 mL / min;

[0134] Detection wavelength: 280nm, injection volume: 20μL.

[0135] Chromatograms during detection, such as Figures 1-2 Because the column pressure requirements in the national standard cannot be met, and the peak shape and resolution of the samples cannot meet the requirements, it is necessary to develop a method suitable for detecting immunoglobulin IgG.

[0136] Comparative Example 2

[0137] The Bio-Monolith Protein G column was selected, with a maximum pressure tolerance of 150 bar, and it also exhibits good adsorption properties for immunoglobulin IgG.

[0138] Column: Bio-Monolith Protein G;

[0139] Mobile phase: Phase A: 0.05 mol / L phosphate buffer, pH 7.4

[0140] Phase B: 0.5 mol / L acetic acid aqueous solution, pH 2.0

[0141] Gradient elution:

[0142] 0–1 min, 100%–100% mobile phase A;

[0143] 1.01–3.00 min, 0%–0% mobile phase A;

[0144] 3.01–7.00 min, 100%–100% mobile phase A;

[0145] Flow rate: 1 mL / min, column pressure: 40 bar, column temperature: 25 °C;

[0146] Detection wavelength: 280nm, injection volume: 10~50μL.

[0147] Chromatogram as shown Figures 3-4 The chromatographic peaks of the reference standard and the test sample showed some tailing, so the elution process needs to be further optimized to improve the peak shape of the reference standard and the test sample.

[0148] Determination of immunoglobulin IgG content in Comparative Example 3 samples

[0149] The content of immunoglobulin IgG in the same batch of samples in Example 1 was determined using the method for determining immunoglobulin IgG in health foods according to GB / T 5009.194-2003. The specific method is as follows:

[0150] Chromatographic conditions:

[0151] Chromatographic column: Pharmacia HI-Trap Protein G;

[0152] Mobile phase: Phase A: 0.05 mol / L phosphate buffer, pH 6.5

[0153] Phase B: 0.05 mol / L phosphate buffer, pH 2.5

[0154] Gradient elution:

[0155] 0~4.5min(100%A~100%A),

[0156] 4.5~5.5min(0%B~100%B),

[0157] 5.5~15.0min(100%B~100%B),

[0158] 15.0~15.5min(100%B~0%B),

[0159] 15.5~22.0min(100%A~100%A);

[0160] Flow rate: 0.4 mL / min;

[0161] Detection wavelength: 280nm, injection volume: 20μL.

[0162] Assay Method: For the reference solution, accurately weigh 0.01 g of immunoglobulin IgG standard, dissolve and dilute to 10.0 mL with mobile phase A, shake well. Take an appropriate amount of the above standard stock solution and dilute with mobile phase A to obtain a series of standard solutions containing 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL of immunoglobulin IgG. Prepare immediately before use. For the test solution, weigh 0.1 g (accurate to 0.001 g) of the sample, dilute to 25.0 mL with mobile phase A, shake well, and inject after passing through a 0.45 μm microporous membrane. Perform the procedure according to the instructions, inject into the high-performance liquid chromatograph, and determine the result. The experimental results are shown below:

[0163] Table 11 Detection data of immunoglobulin IgG content in food

[0164]

[0165] Note: All data here were measured by a third party according to national standards. Due to interference from whole milk powder, the data fluctuates greatly and has poor repeatability.

[0166] To verify the repeatability of the method for determining immunoglobulin IgG in health foods according to GB / T 5009.194-2003, the method was used again to detect the content of immunoglobulin IgG in the same batch of samples from Example 1. The results are as follows:

[0167] Table 12 Detection data of immunoglobulin IgG content in food

[0168]

[0169] In summary, comparing the experimental data of Comparative Example 3 with those of Example 1, it is evident that the method in Comparative Example 3 exhibited poor repeatability in determining immunoglobulin IgG in lipid-containing formula foods, with RSDs of 47.12% and 39.67%, respectively. Furthermore, the detected content differed significantly from the theoretical content, resulting in low accuracy, which did not meet the standard requirements. In contrast, the method in the Example 1 demonstrated better repeatability and higher accuracy in determining IgG in lipid-containing formula foods, with relevant indicators meeting the standard requirements. The experimental data indicate that this method possesses good repeatability and high accuracy, enabling accurate determination of IgG content in lipid-containing formula foods.

[0170] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting IgG in fat-containing formula food, comprising: diluting a sample to be tested with a phosphate buffer, subjecting the sample to enzymatic hydrolysis by lipase, ultrasonic extraction, centrifuging to obtain supernatant, and subjecting the supernatant to HPLC detection; the enzymatic hydrolysis is performed at 35-40 ℃ for 30-120 min, and the mass ratio of lipase to sample is 2:1-20:1; the ultrasonic extraction is performed at a power of 300-500 W, a frequency of 30-50 kHz, a temperature of 10-40 ℃, and a time length of 30-90 min.

2. The detection method according to claim 1, characterized in that, the sample to be tested is at least one of liquid milk, milk powder, milk tablets, milk-containing beverages, or milk-containing candies. 3.The method of claim 1, wherein: the phosphate buffer is a 0.05 mol / L sodium phosphate buffer with pH 7.4; the mass-to-volume ratio of the sample to be tested to the phosphate buffer is 1 g:(50-400) mL.

4. The method of claim 1, wherein, the enzymatic hydrolysis is performed at 37 ℃ for 60 min, and the mass ratio of lipase to sample is 2:

1.

5. The method of claim 1, wherein, the ultrasonic extraction is performed at a power of 400 W, a frequency of 40 kHz, a temperature of 10-40 ℃, and a time length of 60 min.

6. The method of claim 1, wherein the centrifugation is performed at 5000-10000 r / min for 1-10 min.

7. The assay of any one of claims 1 to 6, wherein, the HPLC detection is performed under the following chromatographic conditions: a chromatographic column: Bio-Monolith Protein G; a mobile phase: A phase: a 0.05 mol / L phosphate buffer with pH 7.4; B phase: a 0.5 mol / L aqueous acetic acid solution with pH 2.0; an elution gradient: 0-0.50 min: 100%-100% mobile phase A; 0.51-6.00 min: 0%-0% mobile phase A; 6.01-12.00 min: 100%-100% mobile phase A.

8. The detection method according to claim 7, characterized in that, the HPLC detection is performed under the following chromatographic conditions: a flow rate of 0.1-2.0 mL / min, a column pressure of 40 bar, a column temperature of 25 ℃, a detection wavelength of 280 nm, and an injection volume of 10-50 μL.

9. The detection method according to claim 7, characterized in that, the method further comprises preparation of a reference solution: dissolving IgG in a 0.05 mol / L phosphate buffer with pH 7.4 to obtain the reference solution. the concentration of the reference solution is 0.2-1 mg / mL.

10. The method of claim 7, wherein, the detection method qualitatively and / or quantitatively determines the components of the sample to be tested according to the chromatogram of the reference solution by peak area percentage.

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