High-flux quantitative determination method for free oligosaccharide in milk

A quantitative determination and oligosaccharide technology, applied in the field of chemical analysis, can solve the problems of ion source, mass analyzer, high detection cost, high milk fat volume content that are easy to contaminate mass spectrometry, achieve stable chromatographic baseline, good separation effect, The effect of good separation

Pending Publication Date: 2019-08-23
BEIJING SANYUAN FOOD
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  • Claims
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Problems solved by technology

In this method, there are the following deficiencies: First, when using mass spectrometry to detect breast milk oligosaccharides, acidic oligosaccharides and neutral oligosaccharides inhibit each other during ionization, and if positive ion mode is used, neutral oligosaccharides inhibit acidic oligosaccharides. Ionization of oligosaccharides, if negative ion mode is used, acidic oligosaccharides inhibit the ionization of neutral oligosaccharides, thus affecting detection sensitivity
The second, adopt the method of ultra-high performance liquid chromatography-tandem mass spectrometry in the prior art, one of reasons is that only relying on ultra-high performance liquid chromatography to the separation effect of breast milk oligosaccharides can not meet the analytical requirements under current conditions, only The high selectivity of mass spectrometry can make up for this deficiency
Third, breast milk generally contains...

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  • High-flux quantitative determination method for free oligosaccharide in milk
  • High-flux quantitative determination method for free oligosaccharide in milk
  • High-flux quantitative determination method for free oligosaccharide in milk

Examples

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experiment example 1

[0057] A high-throughput quantitative assay method for free oligosaccharides in milk, comprising the following steps:

[0058] 1) Separation of free oligosaccharides in breast milk

[0059] Thaw breast milk (colostrum) to room temperature, vortex to mix, take 10 microliters into a 2 ml centrifuge tube, add 990 microliters of ultra-pure water, vortex to mix, and follow the following two steps Either of:

[0060] The first method: transfer the solution obtained after vortex mixing to an ultrafiltration centrifuge tube (ultrafiltration membrane molecular cut-off 10kDa), centrifuge for 20min, and the obtained permeate is a free oligosaccharide sample solution;

[0061] The second method: transfer the solution obtained after vortexing to a centrifuge tube, and centrifuge at 8000 g for 10 min at 4° C., and the lower layer is the free oligosaccharide sample solution.

[0062] 2) 2-AB fluorescently labeled oligosaccharides (derivatized)

[0063] Take 100 microliters of the free oli...

experiment example 2

[0075] A high-throughput quantitative assay method for free oligosaccharides in milk, comprising the following steps:

[0076] 1) Separation of free oligosaccharides in breast milk

[0077] Thaw breast milk (colostrum) to room temperature, vortex to mix, take 50 microliters into a 2 ml centrifuge tube, add 950 microliters of ultra-pure water, vortex to mix, and follow the following two steps Either of:

[0078] The first method: the solution obtained after vortex mixing is transferred to an ultrafiltration centrifuge tube (ultrafiltration membrane molecular cut-off 10kDa), centrifuged for 25min, and the obtained permeate is a free oligosaccharide sample solution;

[0079] The second method: transfer the solution obtained after vortexing to a centrifuge tube, and centrifuge at 10000 g for 10 min at 4° C., and the lower layer is the free oligosaccharide sample solution.

[0080] 2) 2-AB fluorescently labeled oligosaccharides (derivatized)

[0081]Take 100 microliters of the f...

experiment example 3

[0094] A high-throughput quantitative assay method for free oligosaccharides in milk, comprising the following steps:

[0095] 1) Separation of free oligosaccharides in breast milk

[0096] Thaw the breast milk (colostrum) to room temperature, vortex to mix, take 100 microliters into a 2 ml centrifuge tube, add 900 microliters of ultra-pure water, vortex to mix, and follow the following two steps Either of:

[0097] The first method: the solution obtained after vortex mixing is transferred to an ultrafiltration centrifuge tube (ultrafiltration membrane molecular cut-off 10kDa), centrifuged for 30min, and the obtained permeate is a free oligosaccharide sample solution;

[0098] The second method: transfer the solution obtained after vortexing to a centrifuge tube, and centrifuge at 6000 g for 10 min at 4° C., and the lower layer is the free oligosaccharide sample solution.

[0099] 2) 2-AB fluorescently labeled oligosaccharides (derivatized)

[0100] Take 100 microliters of ...

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Abstract

An embodiment of the invention provides a high-flux quantitative determination method for free oligosaccharide in milk; the method comprises the following steps of preparing a sample, and carrying outultra-high performance liquid chromatography detection on the prepared sample, wherein the preparation method of the sample comprises the following steps of diluting the milk sample with ultrapure water, and centrifugally removing milk fat and/or lactoprotein to obtain free oligosaccharide, carrying out fluorescent labeling on the free oligosaccharide, and diluting the concentration of the fluorescent labeled oligosaccharide into a standard curve range by using ultrapure water before detection on a machine. The sample preparation method disclosed by the invention has the advantages that the demand on the sample is small (the lowest to 10 [mu]L), and the previous reported liquid chromatography-fluorescence detection method is simplified, complex operation is not needed in the whole preparation process, only dilution and fixed centrifugation operation is needed, and the oligosaccharide is not subjected to purification treatment after being derived, so that the loss of the oligosaccharide is reduced, the generation of human random errors is greatly reduced, the operability and the reliability of the method are improved, and the flux of the sample preparation is improved.

Description

technical field [0001] The invention relates to the technical field of chemical analysis, in particular to a high-throughput quantitative determination method for free oligosaccharides in milk. Background technique [0002] Free oligosaccharides of structural complexity (breast milk oligosaccharides) are abundant in breast milk, and these oligosaccharides are the carbohydrates found in the highest concentration in the infant gut. This type of oligosaccharide cannot be directly digested and utilized by the human body. One of its most important biological functions is to promote the colonization of bifidobacteria in the intestinal tract of infants and maintain the dominance of the bifidobacteria flora. One of the biggest differences between breast milk and infant formula is the complex structure and high concentration of free oligosaccharides in breast milk. Infant formula milk powder is an important source of nutrition for insufficiently breastfed infants, and its main compo...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06
Inventor 陈历俊黄训文杨春英姜铁民乔为仓刘彦品周伟明
Owner BEIJING SANYUAN FOOD
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