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Method for detecting oligosaccharide in breast milk

A technology of breast milk oligosaccharide and detection method, applied in the detection field of breast milk oligosaccharide, can solve the problems of limited separation of oligosaccharide isomers and high limit of quantification, and achieves effective separation of various types and low limit of quantification , Pre-processing simple effects

Pending Publication Date: 2022-07-12
AUSNUTRIA DAIRY CHINA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Liquid chromatography-mass spectrometry is currently widely used in the qualitative and quantitative analysis of breast milk oligosaccharides. However, if an amino column is used, the separation of oligosaccharide isomers is limited and the limit of quantification is high.

Method used

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  • Method for detecting oligosaccharide in breast milk
  • Method for detecting oligosaccharide in breast milk
  • Method for detecting oligosaccharide in breast milk

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Step 1. Take 200ul of breast milk sample, 13000r / min, centrifuge at 4°C for 15min, remove the fat in the upper layer, remove the supernatant, pass the supernatant through a 10kDa molecular weight filter, and centrifuge at 13000rpm / min for 30min; take 100μL of filtrate, add 100 μL of sodium borohydride (0.5M) was reacted in a water bath at 60°C for 30 minutes, 100 μL of acetic acid (0.5M) was added to stop the reaction, and the sample was filtered through a 0.22 μm membrane to obtain the sample.

[0086] Step 2. Take 1 mg of the purchased 21 kinds of oligosaccharide standard products, dissolve them in 2 mL of ultrapure water, and mix them to obtain a primary standard solution, which is stored at -80°C.

[0087] Step 3, take group 1 standard products including lactoyl-N-tetrasaccharide, lactoyl-N-neoctaose, 2'-fucose lactose, 3-fucose lactose, difucosyllactose, lactoyl -N-fucopentaose I, lactoyl-N-difucohexaose I, lactose-N-difucosyl-octasaccharide, 6'-sialylated lactose,...

Embodiment 2

[0093] Same as Example 1, the difference is only in the sample pretreatment, specifically:

[0094] Step 1. Take 300ul of breast milk sample, centrifuge at 13000r / min for 15min at 4°C, remove the fat in the upper layer, remove the supernatant, pass the supernatant through a 10kDa molecular weight filter, centrifuge at 13000rpm / min for 30min, take 150μL of filtrate, and then Add 150 μL of sodium borohydride (0.75M) to react in a 60°C water bath for 30 min, add 150 μL of acetic acid (0.75M) to stop the reaction, and filter through a 0.22 μm filter to obtain the sample.

[0095] Step 2. Take 1 mg of the purchased 21 kinds of oligosaccharide standard products, dissolve them in 2 mL of ultrapure water, and mix them to obtain a primary standard solution, which is stored at -80°C. .

[0096] Step 3, take group 1 standard products including lactoyl-N-tetrasaccharide, lactoyl-N-neoctaose, 2'-fucose lactose, 3-fucose lactose, difucosyllactose, lactoyl -N-fucopentaose I, lactoyl-N-difu...

Embodiment 3

[0102] Same as Example 1, the difference is only in the sample pretreatment, specifically:

[0103] Step 1. Take a breast milk sample of 400ul, 13000r / min, centrifuge at 4°C for 15min, remove the fat in the upper layer, remove the supernatant, pass the supernatant through a 10kDa molecular weight filter, centrifuge at 13000rpm / min for 30min, take 200μL of filtrate, add 200 μL of sodium borohydride (1.0 M) was reacted in a 60°C water bath for 30 min, 200 μL of acetic acid (1.0 M) was added to terminate the reaction, and the sample was filtered through a 0.22 μm filter membrane.

[0104] Step 2. Take 1 mg of the purchased 21 kinds of oligosaccharide standard products, dissolve them in 2 mL of ultrapure water, and mix them to obtain a primary standard solution, which is stored at -80°C. .

[0105] Step 3, take group 1 standard products including lactoyl-N-tetrasaccharide, lactoyl-N-neoctaose, 2'-fucose lactose, 3-fucose lactose, difucosyllactose, lactoyl -N-fucopentaose I, lact...

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Abstract

The invention provides a breast milk oligosaccharide detection method, which comprises: pretreating a milk sample, and reducing with sodium borohydride to obtain a liquid to be detected; the method comprises the following steps: reducing a breast milk oligosaccharide standard substance by adopting sodium borohydride to obtain a standard substance solution; determining the standard substance solution by adopting high performance liquid chromatography-mass spectrometry, and establishing a standard curve; carrying out qualitative analysis on the to-be-detected liquid by adopting high performance liquid chromatography, and carrying out quantitative analysis by adopting mass spectrometry and a standard curve; the chromatographic conditions are as follows: a chromatographic column is a Hypercarb porous graphite carbon chromatography; the mobile phase A is an aqueous solution containing 0.05-0.15% of formic acid; the mobile phase B is an acetonitrile solution containing 0.05-0.15% of formic acid; and gradient elution. By adopting the detection method, the isomeride can be effectively separated, the oligosaccharide with extremely low concentration in the breast milk can be quantified, the quantitation limit is low, various isomeride can be effectively separated, and various breast milk oligosaccharides can be accurately quantified.

Description

technical field [0001] The invention relates to the technical field of food detection, in particular to a detection method of breast milk oligosaccharides. Background technique [0002] Human milk oligosaccharides (HMOs) are the third largest component in human breast milk after lactose and fat. The content of human milk oligosaccharides is as high as 22-25 g / L in colostrum and 12-13 g / L in mature milk. HMOs have important biological functions, not only to resist the infection of intestinal pathogenic microorganisms, but also to maintain the intestinal microecological balance. Breast milk is the gold standard for the preparation of infant milk powder. Therefore, establishing a detection method for breast milk oligosaccharides is a necessary prerequisite for analyzing the composition of breast milk and developing more nutritious and healthy infant milk powder. [0003] The pretreatment method of samples in the detection of HMOs is to remove fat and protein from breast milk. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/34G01N30/72B01J20/282
CPCG01N30/02G01N30/06G01N30/34G01N30/7266B01J20/282G01N2030/067
Inventor 张兵董玲邓泽元潘丽娜汪家琦李静朱柳颖彭小雨高宇戴智勇颜卫彬
Owner AUSNUTRIA DAIRY CHINA
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