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Method for analyzing glucosamine content in whey protein glycosylation product by reverse-phase high performance liquid chromatography (RP-HPLC)

A reverse high-performance liquid phase and glucosamine technology, applied in the field of detection and analysis, can solve the problems of cumbersome processing and achieve the effects of convenient and simple operation, high sensitivity and good reproducibility

Active Publication Date: 2015-09-02
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many derivatization reagents for the determination of amino sugars. The sample pretreatment of some derivatization reagents is cumbersome. The key to determining the analytical method is to select the appropriate derivatization reagent and develop the pretreatment method.

Method used

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  • Method for analyzing glucosamine content in whey protein glycosylation product by reverse-phase high performance liquid chromatography (RP-HPLC)
  • Method for analyzing glucosamine content in whey protein glycosylation product by reverse-phase high performance liquid chromatography (RP-HPLC)
  • Method for analyzing glucosamine content in whey protein glycosylation product by reverse-phase high performance liquid chromatography (RP-HPLC)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] (1) Accurately weigh 60mg of whey protein glucosamine glycosylation product, add it to a hydrolysis bottle, add 5ml of 6mol / L hydrochloric acid to seal it, hydrolyze it at 100°C for 4 hours, take 1ml of the hydrolyzate, adjust the pH to 7.0, and Dilute to 25ml with pure water.

[0022] (2) Take 60 mg of raw whey protein, put it in a hydrolysis bottle, add 5 ml of 6 mol / L hydrochloric acid to seal it, hydrolyze it at 100°C for 4 hours, take 1 ml of the hydrolyzate, adjust the pH to 7.0, and dilute to 25 ml with ultrapure water.

[0023] (3) Add 0.7ml of boric acid buffer solution and 0.3ml of derivatization reagent to the sample and blank treatment solution obtained from step (1) and step (2), and mix well. Buffer and 0.1ml 3-mercaptopropionic acid, derivatized at 25°C for 15min in the dark.

[0024] (4) The derivatized sample liquid is subjected to reverse high-performance liquid chromatography analysis, the chromatographic column is Elite C18, and the chromatographic ...

Embodiment 2

[0026] (1) Accurately weigh 60mg of whey protein glucosamine glycosylation product, add it to a hydrolysis bottle, add 5ml of 6mol / L hydrochloric acid to seal it, hydrolyze it at 100°C for 3h, take 1ml of the hydrolyzate, adjust the pH to 7.0, and Dilute to 25ml with pure water.

[0027] (2) Take 60 mg of raw whey protein, put it in a hydrolysis bottle, add 5 ml of 6 mol / L hydrochloric acid to seal it, hydrolyze it at 100°C for 3 hours, take 1 ml of the hydrolyzed solution, adjust the pH to 7.0, and dilute to 25 ml with ultrapure water.

[0028] (3) Add 0.7ml of boric acid buffer solution and 0.3ml of derivatization reagent to the sample and blank treatment solution obtained from step (1) and step (2), and mix well. Buffer and 0.1mL 3-mercaptopropionic acid, derivatized at 25°C for 12min in the dark.

[0029] (4) The derivatized sample liquid is subjected to reverse high-performance liquid chromatography analysis, the chromatographic column is Elite C18, and the chromatograph...

Embodiment 3

[0031] (1) Accurately weigh 60mg of whey protein glucosamine glycosylation product, add it to a hydrolysis bottle, add 5ml of 6mol / L hydrochloric acid to seal, hydrolyze at 100°C for 5h, take 1ml of hydrolyzate, adjust the pH to 7.0, and Dilute to 25ml with pure water.

[0032] (2) Take 60 mg of raw whey protein, put it in a hydrolysis bottle, add 5 ml of 6 mol / L hydrochloric acid to seal it, hydrolyze it at 100°C for 5 hours, take 1 ml of the hydrolyzed solution, adjust the pH to 7.0, and dilute to 25 ml with ultrapure water.

[0033] (3) Add 0.7ml of boric acid buffer solution and 0.3ml of derivatization reagent to the sample and blank treatment solution obtained from step (1) and step (2), and mix well. Buffer and 0.1mL 3-mercaptopropionic acid, derivatized at 25°C for 18min in the dark.

[0034](4) The derivatized sample liquid is subjected to reverse high-performance liquid chromatography analysis, the chromatographic column is Elite C18, and the chromatographic column p...

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Abstract

The invention relates to a quantitative detection method for glucosamine content in a whey protein glycosylation product, belonging to the technical field of detection and analysis. According to the method, hydrochloric acid is adopted for hydrolysis of glucosamine in glycosylation whey protein and precolumn derivatization, and then a reverse-phase high performance liquid chromatography (RP-HPLC) is utilized for determining glucosamine content in a protein hydrolysate. The method is rapid, simple and convenient, high in sensitivity, and suitable for determination of glucosamine content in various protein glucosamine glycosylation products.

Description

technical field [0001] The invention belongs to detection and analysis technology, and mainly relates to a quantitative detection method for glucosamine content in whey protein glycosylation products. Background technique [0002] Whey protein is the remaining protein in raw milk except casein precipitated at the pH isoelectric point, which is collectively referred to as whey protein, accounting for about 18% to 20% of milk protein. Glycosylation modification of whey protein can improve many functional properties, including emulsification, water holding capacity, oil absorption and allergenicity, because the glycosyl endows glycosylated protein with important functional properties. However, the release and quantitative detection of glucosamine in glycosylated proteins is the key to the development of glycosylation modification technology. [0003] The analysis of amino sugars can be carried out by colorimetric method, in which the primary amino group of glucosamine is acety...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/89
Inventor 张英华赵新淮刘艳乐李远
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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