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Method for separating and detecting whey proteins

A technology for whey protein and albumin, which is applied in the field of separation and determination of whey protein, and can solve problems such as separation and quantification of alpha-lactalbumin that are not involved.

Active Publication Date: 2009-12-30
贝因美(杭州)食品研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, Czerwenka et al. only measured the total bovine β-lactoglobulin content, and did not involve the separation and quantification of α-lactalbumin in milk (see Christoph C. et al, Analytical Chemistry, 2007, 79 (14), 5165-5172)

Method used

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  • Method for separating and detecting whey proteins
  • Method for separating and detecting whey proteins
  • Method for separating and detecting whey proteins

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preparation example Construction

[0038]

[0039] In analytical assays, the composition of the sample matrix can have a significant impact on the sensitivity and stability of mass spectrometry detection. It is known that bovine α-La has good thermal stability, while bovine βb-Lg and bovine βa-Lg have relatively poor thermal stability. Therefore, in the present invention, except for the standard sample solution and other instructions, the whey protein in the sample is destroyed by microwave treatment to obtain a matrix-negative sample.

[0040] The method for preparing the sample blank matrix solution to be tested (without milk α-lactalbumin and milk β-lactoglobulin) is as follows:

[0041] Weigh 1.00g of the sample to be tested in a 100mL beaker, dissolve it with about 70mL of 0.3mol / L sodium chloride solution (containing 0.2% Triton X-100), adjust the pH to 4.6 with 2% trifluoroacetic acid solution, and transfer it into a 100mL volumetric flask In , use the above-mentioned sodium chloride solution (4.3.4) ...

Embodiment 1

[0065] Use Beingmate’s “Champion Baby” newborn infant formula to prepare a blank matrix solution according to the method shown in the aforementioned , and then use the blank matrix solution and water to prepare internal standard and bovine α - La, bovine βa-Lg and bovine βb-Lg three whey protein concentrations of 60 μg / mL standard solution, after sample injection determination, UV detection of the peak area of ​​the peak, the calculation results are shown in Table 2.

[0066] Table 2 The influence of different solution matrices on the ionization effect (n=3)

[0067] Bovine α-La peak area

Human α-La peak area

Bovine βa-Lg peak area

Bovine βb-Lg peak area

water (A1)

211175

151136

5484

30855

Negative matrix (A2)

217384

153957

13457

81467

Area ratio of A2 / A1

1.03

1.02

2.45

2.64

[0068] The results in the above table show that the matrix of the sample digesti...

Embodiment 2

[0071] Prepare standard solutions of bovine α-La, βa-Lg and βb-Lg (concentration: 100 μg / mL) with water, separate and measure them under the aforementioned conditions of chromatography and mass spectrometry, and use MaxEnt.xyz software to calculate α-La The molecular weight of βb-Lg is 14178Da, βb-Lg is 18276Da, and βa-Lg is 18363Da (see Figure 1. In the figure, a is the scan image of bovine α-La, b is the scan image of bovine βb-Lg, and c is the scan image of bovine βa-Lg The scanning picture of each protein; the molecular weight of each protein is calculated by MaxEnt.xyz software), which is completely consistent with the theoretical average molecular weight. From this, it can be confirmed that it is completely feasible to select 2357-2368m / z, 1656-1666m / z and 1665-1675m / z as the interval scanning mass ranges of bovine α-La, bovine βb-Lg and bovine βa-Lg respectively.

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Abstract

The invention discloses a method for separating and detecting whey proteins, which comprises the following steps: pretreating the whey protein; and completely separating bovine alpha-lactalbumin, bovine beta a-lactoglobulin, and bovine beta b-lactoglobulin in a sample by an ultra-performance liquid chromatography column taking 1.7 mu m ethylidene bridged hybrid (BEH) particles as a filler, and performing section scan by a mass analyzer to quantitate the unmodified bovine lactalbumin and lactoglobulin. In the method, human alpha-lactalbumin is added as internal standard. The method can accurately measure the contents of the three types of unmodified bovine whey proteins in multiple kinds of food, and has simple pretreatment, high sensitivity, quick detection speed and good selectivity.

Description

technical field [0001] The invention relates to a method for separating and measuring whey protein. More specifically, it relates to a method capable of efficiently and completely separating and quantitatively determining bovine lactalbumin and bovine lactoglobulin in milk and dairy products. Background technique [0002] Generally, milk contains 2.2% to 4.4% protein, mainly casein, whey protein, fat globule membrane protein, etc. The content of whey protein is second only to casein, accounting for 18% to 20% of the protein content in milk, including β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulin, etc. and some growth factors, of which β -Lactoglobulin and α-lactalbumin are the main components of whey protein, accounting for about 75% of it. Therefore, accurate quantification of whey protein in bovine milk and bovine milk products is of great significance for the quality evaluation and control of bovine milk and bovine milk products. [0003] Existing detec...

Claims

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Application Information

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IPC IPC(8): C07K14/76C07K14/47C07K1/16G01N30/72
Inventor 任一平谢宏蔡增轩储小军林晓黄百芬张京顺
Owner 贝因美(杭州)食品研究院有限公司
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