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Bovine beta-casein quantitative determination kit and application thereof

A quantitative detection and casein technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of allergen spatial structure requirements, inaccurate test results, and inability to detect allergens

Active Publication Date: 2014-01-22
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, antibodies have requirements on the spatial structure of allergens, and cannot detect denatured allergens
And the ELISA method has a false positive phenomenon, which will lead to inaccurate detection results

Method used

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  • Bovine beta-casein quantitative determination kit and application thereof
  • Bovine beta-casein quantitative determination kit and application thereof
  • Bovine beta-casein 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 β-casein specific peptide determined by experiments, fully considering its related 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, stable isotope labeling was introduced Leucine (L * ) and valine (V * ) designed and synthesized the isotope-specific peptide of bovine β-casein specific peptide, whose amino acid sequence is VL * PV * PQK.

[0052] The purpose of introducing the isotope-specific peptide in the present invention is to overcome the matrix effect caused by the extraction reagent and the matrix. In order to verify the consistency of the isotope-specific peptide designed in the present invention to the bovine β-casein specific peptide under the same matrix, the e...

Embodiment 2

[0058] Embodiment 2: kit preparation and instructions for use

[0059] 1. Reagent preparation:

[0060]1. Preparation of bovine β-casein-specific peptide standard solution: Accurately pipette 1mL of ultrapure water, add it to standard substance 1 tube (the tube contains bovine β-casein-specific peptide that has been accurately weighed in advance), and ultrasonically dissolve (30s), the resulting solution is 1 μg / mL bovine β-casein specific peptide solution;

[0061] 2. Preparation of isotope-specific peptide standard solution: Accurately pipette 1mL of ultrapure water, add it into the standard substance 2 tube (the tube is filled with isotope-specific peptide that has been accurately weighed in advance), ultrasonically dissolve (30s), and the obtained solution is 1 μg / mL isotope-specific peptide solution;

[0062] 3. Preparation of isotope internal standard standard stock solution: Accurately pipette 1mL of ultrapure water, add it into the standard substance 3 tube (the tube...

Embodiment 3

[0076] Sample type: commercially available biscuits;

[0077] Accurately weigh about 10g sample, add 100mL ammonium bicarbonate solution and homogenize, dilute the sample suspension 1:100 with ammonium bicarbonate solution, take 1mL suspension, add 10μL isotope internal standard solution and 10μL dithiothreo Sugar alcohol solution, react at a constant temperature of 50°C for 30 minutes, take it out and cool it to room temperature, add 10 μL of iodoacetamide solution, let it stand in the dark at room temperature for 30 minutes, add 10 μL of trypsin solution, and then add 5 μL of pure formic acid for 2 hours at 37°C. After standing at room temperature for 1 h, the resulting solution was filtered through a 0.22 μm microporous membrane and then injected for analysis. It is measured that it contains 192.51±15.42mg / 100g of bovine β-casein.

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Abstract

The invention provides a bovine beta-casein quantitative determination kit. The kit mainly comprises a bovine beta-casein specific peptide of which the amino acid sequence is VLPVPQK, an isotope labeled bovine beta-casein specific peptide (VL*PV*PQK) and an isotope labeled bovine beta-casein internal standard substance of which the amino acid sequence is QSVLSLSQSKVL*PV*PQKAVPYPQRD). According to the kit, the ration is limited to be 1mg / 100g, the reproducibility RSD less than 9.50 percent (n is equal to 11), the recovery in a food substrate is 73.41-93.88 percent (n is equal to 6), and the recovery in a powdered milk substrate is 91.62-107.28 percent (n is equal to 6); a sample is pre-treated simply and quickly, and is low in cost. The bovine beta-casein quantitative determination kit is applicable to multiple food substrates, and can be used for accurately quantitating macro and trace bovine beta-casein.

Description

(1) Technical field [0001] The invention relates to a bovine beta-casein quantitative detection kit and the application of the kit in quantitative detection of bovine beta-casein content in dairy foods or dairy products. (2) Background technology [0002] Casein is one of the main proteins in mammalian milk, responsible for the transport of nutrients and energy. Casein will coagulate and precipitate under acidic conditions (pH=4.6), and the precipitate can be made into cheese, hence the name casein. Proteins other than casein are collectively referred to as whey proteins. In different mammalian milks, the ratio of whey protein to casein protein varies from about 2:8 in bovine milk to about 6:4 in human milk. Due to the different amino acid sequences of various proteins, casein can be subdivided into α s1 、α s2 , β and κ casein, the ratio in milk is about 38:10:36:13. The content of casein in breast milk is quite different from that of cow's milk, and there is no alpha i...

Claims

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Application Information

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IPC IPC(8): G01N30/02
Inventor 任一平陈启张京顺赖世云章宇
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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