A rapid separation and detection method for whey protein components in dairy products

Abstract

The invention provides a fast separation and detection method for whey protein components in a dairy product. The method comprises the following steps: 1 preparing a standard solution or unfreezing the standard solution; 2 preparing a sample solution to be detected; 3 conducting HPLC separation and detection, wherein the standard solution and the sample solution to be detected are separated through gel chromatography columns. According to the method, firstly, a dairy product and / or a standard product to be detected is dissolved or diluted with a phosphate buffer solution; then all types of protein components are separated through the gel chromatography columns and detected through an ultraviolet detector or a diode array detector, and through the comparison with the peak area of the standard area, the content of each type of whey protein in the dairy product is calculated out. When the detection method is used to detect the whey protein in the dairy product, the detection is fast, the operation is simple and convenient, and the reproducibility is good.

Description

technical field

[0001] The invention relates to a detection method for whey protein components in dairy products, in particular to a rapid detection method for whey protein components in milk products for infants and young children. Background technique

[0002] Whey protein is a small, compact globular protein whose unique amino acid sequence and three-dimensional structure endow it with a wide range of functional properties, and its essential amino acid composition fully meets or exceeds the requirements of FAO / WHO. It is low in cholesterol, fat and lactose, easy to digest and easy to absorb. Compared with other proteins, it has the highest bioavailability value. The production process can affect the efficacy of whey protein, and the undenatured whey protein extracted by low temperature and membrane ultrafiltration technology has the best activity and efficacy.

[0003] The functional components of whey protein include: β-lactoglobulin accounts for 48%, α-lactalbumin acc...

Examples

Embodiment 1

[0030] (1) Standard solution preparation

[0031] Weigh 25 mg of bovine milk IgG, 50 mg of α-lactalbumin, 50 mg of bovine serum albumin, 100 mg of β-lactoglobulin, and 2.5 mg of lactoferrin and dissolve them in 500 ml of phosphate buffer. Divide the standard sample into Eppendorph test tubes, freeze and store between -25°C and -18°C, the shelf life can be kept for 3 months, and thaw on the day of use.

[0032] (2) Sample processing

[0033] When the sample is powder, dissolve 100-1000mg powder in 100ml phosphate buffer; when the sample is liquid, dilute the sample with phosphate buffer until the protein content is about 0.1%. If the protein content in the sample is lower than 0.1%, no dilution is required .

[0034] All samples were filtered with a 0.22 μm filter and then determined by high performance liquid chromatography.

[0035] (3) HPLC experimental conditions

[0036] Chromatographic column: 2 TSK G3000PW Xl Gel column series, with pre-column PW Xl (6mm×4cm),

[...

Embodiment 2

[0051] Buffer pH selection: by figure 2 It can be seen that the use of phosphate buffers with different pH values ​​has an impact on component separation and peak eluting time. Through a series of pH gradient experiments, the optimal pH of phosphate buffer was finally determined to be 7.5.

Embodiment 3

[0053] This example uses the method of the present invention to detect whey protein components in a certain infant formula milk powder.

[0054] Reagent and material preparation:

[0055] Mobile phase preparation: add 470.0g deionized water, 413.4g acetonitrile, and 1.0ml trifluoroacetic acid below the liquid level of the chromatographic column.

[0056] The standard solutions were mixed in capped reagent bottles and filtered through a 0.22 μm membrane.

[0057] Sample treatment: Weigh 0.500g sample, dissolve it in phosphate buffer, and dilute to 100ml;

[0058] Preparation and detection of the measurement solution: take the treatment solution and pass it through a 0.22 μm filter membrane, and then use a high-performance liquid chromatograph for measurement.

[0059] The content of each component was calculated according to the announcement, and the results are shown in Table 1.

[0060] Table 1

[0061] component name retention time (min) content Milk I...

technical field

[0001] The invention relates to a detection method for whey protein components in dairy products, in particular to a rapid detection method for whey protein components in milk products for infants and young children. Background technique

[0002] Whey protein is a small, compact globular protein whose unique amino acid sequence and three-dimensional structure endow it with a wide range of functional properties, and its essential amino acid composition fully meets or exceeds the requirements of FAO / WHO. It is low in cholesterol, fat and lactose, easy to digest and easy to absorb. Compared with other proteins, it has the highest bioavailability value. The production process can affect the efficacy of whey protein, and the undenatured whey protein extracted by low temperature and membrane ultrafiltration technology has the best activity and efficacy.

[0003] The functional components of whey protein include: β-lactoglobulin accounts for 48%, α-lactalbumin acc...

Examples

Embodiment 1

[0030] (1) Standard solution preparation

[0031] Weigh 25 mg of bovine milk IgG, 50 mg of α-lactalbumin, 50 mg of bovine serum albumin, 100 mg of β-lactoglobulin, and 2.5 mg of lactoferrin and dissolve them in 500 ml of phosphate buffer. Divide the standard sample into Eppendorph test tubes, freeze and store between -25°C and -18°C, the shelf life can be kept for 3 months, and thaw on the day of use.

[0032] (2) Sample processing

[0033] When the sample is powder, dissolve 100-1000mg powder in 100ml phosphate buffer; when the sample is liquid, dilute the sample with phosphate buffer until the protein content is about 0.1%. If the protein content in the sample is lower than 0.1%, no dilution is required .

[0034] All samples were filtered with a 0.22 μm filter and then determined by high performance liquid chromatography.

[0035] (3) HPLC experimental conditions

[0036] Chromatographic column: 2 TSK G3000PW Xl Gel column series, with pre-column PW Xl (6mm×4cm),

[...

Embodiment 2

[0051] Buffer pH selection: by figure 2 It can be seen that the use of phosphate buffers with different pH values ​​has an impact on component separation and peak eluting time. Through a series of pH gradient experiments, the optimal pH of phosphate buffer was finally determined to be 7.5.

Embodiment 3

[0053] This example uses the method of the present invention to detect whey protein components in a certain infant formula milk powder.

[0054] Reagent and material preparation:

[0055] Mobile phase preparation: add 470.0g deionized water, 413.4g acetonitrile, and 1.0ml trifluoroacetic acid below the liquid level of the chromatographic column.

[0056] The standard solutions were mixed in capped reagent bottles and filtered through a 0.22 μm membrane.

[0057] Sample treatment: Weigh 0.500g sample, dissolve it in phosphate buffer, and dilute to 100ml;

[0058] Preparation and detection of the measurement solution: take the treatment solution and pass it through a 0.22 μm filter membrane, and then use a high-performance liquid chromatograph for measurement.

[0059] The content of each component was calculated according to the announcement, and the results are shown in Table 1.

[0060] Table 1

[0061] component name retention time (min) content Milk I...

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