Method and kit for quantitatively detecting human beta-casein content

A quantitative detection and casein technology, applied in the field of analytical chemistry, can solve the problem of inability to accurately quantitatively detect breast milk protein, and achieve high accuracy.

Active Publication Date: 2014-03-05
贝因美(杭州)食品研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of standard products and the cross-reactivity of antibodies to other breast milk proteins, ELISA cannot be applied to the accurate quantitative detection of breast milk proteins

Method used

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  • Method and kit for quantitatively detecting human beta-casein content
  • Method and kit for quantitatively detecting human beta-casein content
  • Method and kit for quantitatively detecting human beta-casein content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Identity of Isotope-Specific Peptides and β-Casein-Specific Peptides

[0045] 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 results of the isotope-specific peptides designed in the present invention to β-casein-specific peptides under the same matrix, the experimental design compared the isotope-specific peptides to β-casein-specific peptides under the same liquid chromatography and mass spectrometry conditions. Retention time, fragmentation of product ions, and linearity.

[0046] Standard series working solutions of specific peptides and isotope-specific peptides in the present invention were respectively prepared, and samples were injected and analyzed under the same chromatographic mass spectrometry conditions to obtain their retention time and linear regression equation. Wherein the retention time...

Embodiment 2

[0049] Example 2: Comparison of isotope internal standard and human β-casein enzymatic efficiency

[0050] In order to verify whether the isotopic internal standard in the present invention and human β-casein have more similar enzymolysis efficiency in various matrices, the following experiments were designed:

[0051] Dilute the breast milk with ammonium bicarbonate solution 1:9, accurately measure 10 μL of the diluted breast milk into the reaction tube, add 0, 5, 20, 50, 200 μL of skim milk solution (1g of skim milk powder to dissolve in 100mL50mM ammonium bicarbonate solution) as a matrix, which can simulate various nutrients in breast milk without affecting the quantitative detection. Then add ammonium bicarbonate solution to a total volume of 945 μL, add 10 μL isotope internal standard solution and 10 μL dithiothreitol solution, shake well and react at a constant temperature of 60 °C for 30 min, take it out and cool to room temperature, add 10 μL iodoacetamide solution ,...

Embodiment 3

[0053] Embodiment 3: the preparation of reagent

[0054] 1. Preparation of β-casein-specific peptide standard solution: Accurately pipette 1mL of ultrapure water, add it to the pre-accurately weighed β-casein-specific peptide tube, dissolve it by ultrasonic for 30s, and the obtained solution is 1mmol / L β-casein specific peptide standard solution;

[0055] 2. Preparation of isotope-specific peptide standard solution: Accurately pipette 1mL of ultrapure water, add it to the tube of isotope-specific peptide that has been accurately weighed in advance, and dissolve it by ultrasonic for 30s, the resulting solution is 20μmol / L isotope-specific peptide standard solution;

[0056] 3. Preparation of the isotope internal standard solution: Accurately pipette 1mL of ultrapure water, add it into the tube of the isotope internal standard that has been accurately weighed in advance, and ultrasonically dissolve it for 30s. The resulting solution is a 20μmol / L isotope internal standard solut...

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Abstract

The invention relates to the field of analytical chemistry, and discloses a method and a kit for quantitatively detecting human beta-casein content. According to the method and the kit, by utilizing a specific peptide sequence VMPVLK obtained after enzymolysis of human beta-casein, isotope labeled specific peptide and a sequence of an isotope labeling internal standard substance are designed, and a new accurate quantitative detection method is provided based on an internal standard method for quantitatively detecting the human beta-casein, with high accuracy, high precision and high sensitivity.

Description

technical field [0001] The invention relates to the field of analytical chemistry, in particular to a method and a kit for quantitatively detecting the content of human beta-casein. Background technique [0002] As early as the 1980s, the advantages of breastfeeding have been discovered by many academic institutions in the world, and many papers have confirmed its superiority. Breast milk provides babies with balanced protein, fat, carbohydrates, minerals and vitamins, enabling babies to grow up healthily. [0003] Breast milk is the gold standard for formula. Due to the difference between the nutritional components of milk and breast milk, various nutrients must be added to milk powder to make the content close to that of breast milk. Among these nutrients, protein is the most important, which plays a pivotal role in the growth and development of infants. Breast milk protein is composed of 40% casein and 60% whey protein, of which casein is mainly composed of β-casein. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
Inventor 任一平陈启黄小强赖世云张京顺
Owner 贝因美(杭州)食品研究院有限公司
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