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Cow milk beta-lactoglobulin quantitative determination kit and application thereof

A lactoglobulin, quantitative detection technology, applied in biological testing, microbial determination/inspection, material inspection products, etc., can solve problems such as inability to measure

Active Publication Date: 2013-09-11
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ren Renping (2010) et al. established a method for the determination of milk β-lactoglobulin in infant formula by LC-ESI-MS. The sample was directly extracted, and according to the principle that the protein would generate multiply charged ions under electrospray ionization conditions, The selected ion scanning mode (SIR) is used for detection. This method is limited to the quantitative detection of non-denatured milk β-lactoglobulin in the sample, and cannot determine the denatured milk β-lactoglobulin due to heat treatment.
After inquiry, no quantitative method has been found so far that uses isotope-labeled internal standard peptide dilution method combined with RPLC-ESI-MS / MS to simultaneously determine heat-denatured and non-denatured milk β-lactoglobulin in milk and dairy products

Method used

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  • Cow milk beta-lactoglobulin quantitative determination kit and application thereof
  • Cow milk beta-lactoglobulin quantitative determination kit and application thereof
  • Cow milk beta-lactoglobulin quantitative determination kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] 1. Design and determination of the isotope-specific peptide of the present invention:

[0051] According to the amino acid sequence of bovine milk β-lactoglobulin specific peptide determined by experiments, fully considering its relevant physical and chemical properties, interference and ionization efficiency in the process of mass spectrometry detection, and taking into account the cost and feasibility of actual production and application, the introduction of stable isotopes Labeled valine (V * ) and leucine (L * ) designed and synthesized bovine milk β-lactoglobulin isotope-specific peptide, whose amino acid sequence is TPEV * DDEAAL * EK.

[0052] Parameters such as retention time, linearity, and matrix effect can reflect the chromatographic separation behavior and mass spectrometric ionization efficiency of the measured substance to a certain extent. Whether the behaviors during separation and mass spectrometry ionization are similar, the experimental design co...

Embodiment 2

[0057] Embodiment 2: kit preparation and instructions for use

[0058] 1. Reagent preparation:

[0059] 1. Preparation of standard stock solution of milk β-lactoglobulin specific peptide: Accurately pipette 5mL of ultrapure water and add it to 1 tube of standard substance (the tube contains the pre-accurately weighed milk β-lactoglobulin specific peptide) , ultrasonically dissolved (30s), the obtained solution is 500 μmol / L milk β-lactoglobulin specific peptide standard stock solution;

[0060] 2. Preparation of isotope-specific peptide standard stock solution: Accurately pipette 5mL of ultrapure water, add it into 2 tubes of standard substance (the tube contains isotope-specific peptide that has been accurately weighed in advance), and dissolve it ultrasonically (30s), and the obtained solution is ready 500 μmol / L isotope-specific peptide standard stock solution;

[0061] 3. Preparation of isotope internal standard standard stock solution: Accurately pipette 5mL of ultrapur...

Embodiment 3

[0074] Sample type: Commercial infant formula milk powder.

[0075] Weigh 0.5g of the sample into a 250mL volumetric flask, add warm water to dissolve, wait to cool to room temperature, add water to the mark, accurately draw 500μL, add 20μL 20μmol / L isotope internal standard and 480μL 50mmol / L NH 4 HCO 3 solution, add 10μL 500mmol / L DTT solution, react at a constant temperature of 50°C for 30 minutes, take it out and cool to room temperature, add 30μL 500mmol / L IAA solution, let it stand in the dark for 30 minutes, add 10μL 100mmol / L CaCl 2 Solution and 50 μL of 1 mg / mL trypsin solution, 37 ° C constant temperature for overnight enzymolysis, take out the next day and add 10 μL of formic acid, let it stand at room temperature for 1 hour, finally dilute the enzymolysis solution to 2 mL, take the obtained sample solution according to the method of Example 2 for detection Analysis, and calculate the result with internal standard method, the content of milk β-lactoglobulin in the ...

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Abstract

The invention provides a cow milk beta-lactoglobulin quantitative determination kit, which mainly comprises a cow milk beta-lactoglobulin specific peptide (amino acid sequence is TPEVDDEALE), an isotope marked cow milk beta-lactoglobulin specific peptide (amino acid sequence is TPEV*DDEAL*EK, wherein, V* and L* are carbonic oxide whole isotope marked amino acids) and isotope marked cow milk beta-lactoglobulin internal standard substance (amino acid sequence is CQCLVRTPEV*DDEAAL*EKFDKALKA, wherein, V* and L* are carbonic oxide whole isotope marked amino acids); the quantitative limit of the kit is 0.004g / 100g, and the reappearance is RSD (6.2%, n=11 day, recovery rate:93.6-99.8% (n=6), and the sample pretreatment is simple and rapid with low cost, and the kit can be used for accurate quantification of non-denaturation cow milk beta-lactoglobulin and heat modification cow milk beta-lactoglobulin in various samples.

Description

(1) Technical field [0001] The invention relates to a bovine milk β-lactoglobulin quantitative detection kit capable of measuring heat-denatured and non-denatured constant or trace amounts in milk or dairy products and an application thereof. (2) Background technology [0002] Cow's milk is one of the eight categories of food that cause human food allergy identified by FAO / WHO. It is a common food that causes food allergy in children. Its incidence rate in children is 0.1-7.5%, which seriously endangers the health of infants and young children. Milk β-lactoglobulin is considered to be the main allergen. [0003] Bovine β-lactglobulin (β-LG) is a kind of protein in milk, accounting for about 7-12% of fresh milk protein. It has been found in cell and animal experiments that the hydrolyzate or molecular modification of β-lactoglobulin has physiological activities such as lowering cholesterol and anti-oxidation. Previous studies have found that there are many forms of genetic ...

Claims

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Application Information

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IPC IPC(8): G01N33/68C12Q1/37
Inventor 任一平赖世云张京顺
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION