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Method for detecting different beta-casein variants in milk

A detection method and casein technology are applied in the fields of animal breeding and food detection, which can solve problems such as false positive results, and achieve the effects of simple operation, reasonable design and high detection accuracy.

Active Publication Date: 2019-01-29
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the invention patent (application number: 2016107841806; application number 2016109833819) discloses methods for detecting A1-type β-casein and A2-type β-casein in dairy products, these two protein detection methods are only based on the detection of a β-casein Casein amino acid mutation sites are used to distinguish A1 and A2 milk. Since there are common mutation sites in A2 and A3, E, H1, and I variants, if only one site is detected, it will lead to false positive results.

Method used

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  • Method for detecting different beta-casein variants in milk
  • Method for detecting different beta-casein variants in milk
  • Method for detecting different beta-casein variants in milk

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Construction of a method for detecting different β-casein variants

[0073] 1. Protein extraction and quality control of milk or milk powder samples.

[0074] 8M urea was used to fully dissolve the protein in the sample, the protein and oil in the sample were separated by centrifugation, and the obtained protein sample was subjected to dithiothreitol (DTT) reduction and iodoacetamide (IAM) alkylation protection. Coomassie brilliant blue staining (bradford method) was then used to quantify the protein, and sodium dodecylsulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to identify whether the protein band was clear without degradation.

[0075] 2. Carry out enzymatic digestion to the above-mentioned extracted protein;

[0076] Get appropriate amount of above-mentioned sample, utilize the method for ultracentrifugation, the buffer solution in the sample is changed into 25mM ammonium bicarbonate (NH 4 HCO 3 ), in order to facilitate subsequent ...

Embodiment 2

[0081] Embodiment 2: The accuracy and repeatability detection of the method.

[0082] The dairy cows whose β-casein genotypes have been identified through direct DNA sequencing are used as detection objects, and the method of Example 1 is used to identify different variants of β-casein in milk, and the accuracy rate reaches 100%. It shows that the detection method is accurate and effective.

[0083] Taking milk samples with known β-casein variants as the detection object, this detection method was used for repeated tests, and the results of multiple repeated tests were consistent, indicating that the method has good stability.

Embodiment 3

[0084] Example 3: Detection of actual samples.

[0085] The method of Example 1 of the present invention was used to detect two raw milk samples.

[0086] 1. Protein extraction and quality control;

[0087] (1) Take 50 μl of raw milk sample and add 500 μl of lysate (8M urea, 30mM 4-hydroxyethylpiperazineethanesulfonic acid (Hepes)). Lyse on ice for 10 min. Centrifuge at 4°C, 20000g, 30min, take the supernatant, avoid absorbing oil.

[0088] (2) Add dithiothreitol (DTT) to a final concentration of 10 mM. 56°C water bath for 1h. After taking it out, add iodoacetamide (IAM) quickly to a final concentration of 55mM, and let it stand in a dark room for 1h.

[0089] (3) Proteins were quantified using the Coomassie blue staining method (Bradford method).

[0090] (4) Carry out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to the protein after quantification ( figure 2 ).

[0091] 2. Carry out proteolytic digestion of the above-mentioned extracted prote...

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Abstract

The invention provides a method for detecting different beta-casein variants in milk. The method comprises the following steps: S1. extracting proteins from a to-be-detected sample; S2. performing enzymatic digestion on the extracted proteins by adopting trypsin and V8 protease; and S3. performing mass spectrometry detection on peptide fragments generated by protein enzymolysis to identify the beta-casein variants. The detection method disclosed by the invention is reasonable in design and can effectively detect different beta-casein variants (A1, A2, A3, B, C, E, F, H1 and I) in a dairy product, and is high in detection accuracy and easy to operate. The method can judge A2 type beta-casein variants of a terminal dairy product of a cow, and can regulate the A2 milk dairy product market. The method is a good application to a protein detection technology in production practice and can generate enormous economic and social benefits.

Description

technical field [0001] The invention belongs to the technical field of animal breeding and food detection, and in particular relates to a method for simultaneously detecting β-casein A1, A2, A3, B, C, E, F, H1 and I variants in dairy products. Background technique [0002] The statements in this section merely provide background information related to the present disclosure and do not necessarily constitute prior art. [0003] Casein and whey protein are the two main proteins in milk. Among them, casein accounts for about 80% of the total protein in milk, including αs1, αs2, β, and κ four types. β-casein (β-casein, CSN2) is an important source of amino acids, accounting for about 30% of the total protein. Because the β-casein gene contains multiple single nucleotide polymorphism (SNP) sites, the β-casein presents different protein variants. According to reports, it has at least 12 different amino acid sequence composition forms, such as A1, A2, A3, B, C, D, E, F, H1, H2, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/62C07K14/47C12P21/06
CPCG01N27/62C12P21/06C07K14/4732
Inventor 黄金明王秀革魏晓超姜强王玲玲张亚冉高亚平鞠志花高运东仲跻峰
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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