High-flux quantitative determination method for free oligosaccharide in milk

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...

AI Technical Summary

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 3 to 4.6% fat. Although the above method first dilutes breast milk by 2 times with ultrapure water and then centrifuges to remove milk fat, the volume content of milk fat is still high, which will affect the final oligomerization. Immediate impact on sugar quantitation results

Fourth, although the effect of acetonitrile to precipitate milk protein is better, acetonitrile will reduce the recovery rate of breast milk oligosaccharides

Fifth, liquid chromatography-mass spectrometry equipment is a relatively expensive precision instrument, and the content of lactose in breast milk is very high (60-70mg / mL), which is easy to contaminate the ion source and mass analyzer of mass spectrometry, and the maintenance and maintenance costs are high, which is harmful to the test The technical requirements of the personnel are much higher than the operation requirements of liquid chromatography, which makes the overall detection cost higher

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