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A Rapid Qualitative and Quantitative Method for Human Milk Oligosaccharides

A breast milk oligosaccharide, rapid technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of complex structure, long time, lag, etc., achieve high sensitivity and separation, shorten analysis time, and ensure accuracy. Effect

Active Publication Date: 2019-07-02
INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, some studies have pointed out that oligosaccharides are closely related to nerve synapses and nerve conduction, which can promote the cognitive development of infants and enhance learning and memory abilities; Twelve per thousand, and the composition of breast milk oligosaccharides exceeds 200 kinds, the structure is complex, which is not conducive to the research of breast milk oligosaccharides, and it is also very difficult to detect breast milk oligosaccharides. The research on polysaccharides is relatively lagging behind. The method of detecting breast milk oligosaccharides is not only time-consuming, but also cannot quantify breast milk oligosaccharides. To provide a method for rapid detection of breast milk oligosaccharides and quantification of oligosaccharides is very necessary

Method used

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  • A Rapid Qualitative and Quantitative Method for Human Milk Oligosaccharides
  • A Rapid Qualitative and Quantitative Method for Human Milk Oligosaccharides
  • A Rapid Qualitative and Quantitative Method for Human Milk Oligosaccharides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Step 1. Take 150 μL of breast milk and add an equal volume of ultrapure water, centrifuge at 5000r / min at 2°C for 8 minutes, remove the upper layer of fat, then add 400 μL of pure acetonitrile, vortex and mix well, then sonicate for 8 minutes, centrifuge at 8000r / min at 2°C After 12 minutes, take the supernatant, then centrifuge the supernatant at 2°C at a speed of 8000r / min. After centrifuging the supernatant for 12 minutes, take the final supernatant, add ultrapure water to dilute and then use it Sample.

[0044] Step 2. Take 0.5 mg of the purchased 12 kinds of standard products, dissolve them in 0.5 mL of ultrapure water, mix well to obtain a primary standard product solution, and store them at -50°C.

[0045] Step 3. Take 8 μL each of the primary standard solution of 2-fucosyl lactose and 3-fucosyllactose, and add 60 μL of ultrapure water respectively to obtain two mixed standard samples with a concentration of 100 mg / L. Take the two The mixed standard samples were...

Embodiment 2

[0051] Step 1. Take 200 μL of breast milk and add an equal volume of ultrapure water, centrifuge at 6000r / min at 4°C for 10 minutes, remove the upper layer of fat, then add 600 μL of pure acetonitrile, vortex and mix well, then sonicate for 10 minutes, centrifuge at 10,000r / min at 4°C After 10 minutes, take the supernatant, then centrifuge the supernatant at 4°C at a speed of 10,000 r / min. After centrifuging the supernatant for 10 minutes, take the final supernatant, dilute it with ultrapure water, and then use it Sample.

[0052] Step 2. Take 1 mg of the purchased 12 kinds of standard products, dissolve them in 1 mL of ultrapure water, mix well to obtain primary standard product solutions, and store them at -80°C.

[0053] Step 3. Take 10 μL each of the primary standard solution of 2-fucosyl lactose and 3-fucosyllactose, and add 80 μL of ultrapure water respectively to obtain two mixed standard samples, both of which have a concentration of 100 mg / L. Take the two The mixed s...

Embodiment 3

[0059] Step 1. Take 250 μL of breast milk and add an equal volume of ultrapure water, centrifuge at 7000 r / min at 2°C for 8 minutes, remove the upper layer of fat, then add 700 μL of pure acetonitrile, vortex and mix well, then sonicate for 12 minutes, centrifuge at 12000 r / min at 8°C After 8 minutes, take the supernatant, then centrifuge the supernatant, and centrifuge the supernatant at 8°C at a speed of 12000r / min. Sample.

[0060] Step 2. Take 3 mg of the purchased 12 kinds of standard products, dissolve them in 3 mL of ultrapure water, mix well to obtain primary standard product solutions, and store them at -80°C.

[0061] Step 3. Take 12 μL each of the primary standard solution of 2-fucosyl lactose and 3-fucosyl lactose, and add 100 μL of ultrapure water to obtain two mixed standard samples, both of which have a concentration of 100 mg / L. Take the two The mixed standard samples were diluted step by step to different concentrations; the 3-sialyllactose, 6-sialyllactose, ...

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Abstract

The invention discloses a quick qualitative and quantitative method for oligosaccharide in breast milk. The quick qualitative and quantitative method mainly includes steps of 1, pretreating samples, to be more specific, removing fat and proteins from 150-250 micro-l of breast milk to obtain ultimate supernatant, adding ultra-pure water into the supernatant and diluting the supernatant to obtain the loaded samples; 2, establishing standard curves for standard substances by the aid of ultrahigh-performance liquid chromatography and mass spectrometry; 3, separating different components of the oligosaccharide in the breast milk in the loaded samples by the aid of ultrahigh-performance liquid mass spectrometry and carrying out quantitative analysis by the aid of mass spectrometry combined with the standard curves so as to obtain the content of the oligosaccharide in the breast milk. The ultrahigh-performance liquid chromatography is implemented by the aid of amino chromatographic columns with the sizes of 2.1*100 mm and 1.7 micrometers, 8-10 mmol / L of ammonium formate solution (A) and acetonitrile (B), and the ammonium formate solution (A) and the acetonitrile (B) are used as mobile phases; gradient elution programs are carried out by the aid of 95%-75% of B for 0-10 min or are carried out by the aid of 75% of B for 10-15 min or are carried out by the aid of 75%-65% of B for 15-20 min or are carried out by the aid of 65%-10% of B for 20-21 min or are carried out by the aid of 10% of B for 21-24 min or are carried out by the aid of 10%-95% of B for 24-25 min or are carried out by the aid of 95% of B for 25-35 min; the flow rates are 0.3 mL / min, and the column temperatures are 40-60 DEG C. The quick qualitative and quantitative method has the advantage that 12 types of oligosaccharide in the breast milk can be quickly detected by the aid of the quick qualitative and quantitative method and can be quantified by the aid of the quick qualitative and quantitative method.

Description

technical field [0001] The invention relates to the detection field of breast milk oligosaccharides, in particular to a rapid qualitative and quantitative method for breast milk oligosaccharides. Background technique [0002] Breast milk oligosaccharides have a variety of physiological functions, which can not only reduce the occurrence of infection, but also effectively promote the proliferation of beneficial intestinal flora, indirectly inhibit the growth of harmful flora, in order to maintain the intestinal micro-ecological balance, thereby protecting the infant intestinal immunity. Invaded by pathogenic bacteria, it also has the effect of promoting the maturity of the baby's intestinal local and systemic immune system. A small part of breast milk oligosaccharides can also reduce immune stress and prevent chronic inflammation. In addition, some studies have pointed out that oligosaccharides are closely related to nerve synapses and nerve conduction, which can promote the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 芦晶吕加平陈新新刘鹭逄晓阳张书文
Owner INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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