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CN-121994899-A - Detection method of breast milk oligosaccharide

CN121994899ACN 121994899 ACN121994899 ACN 121994899ACN-121994899-A

Abstract

The invention discloses a method for detecting breast milk oligosaccharide. The invention provides a method for detecting breast milk oligosaccharide, which comprises the step of detecting a sample to be detected by adopting a detection method based on capillary electrophoresis-laser induced fluorescence, wherein an operation buffer used by the detection method based on capillary electrophoresis-laser induced fluorescence comprises Tris-base, EDTA, urea, PVP (polyvinyl pyrrolidone) with the concentration of more than 2% (m/V) and less than 4% (m/V) and boric acid with the concentration of more than 5% (m/V) and less than 15% (m/V). The detection method provided by the invention can be applied to separation detection of acidic and neutral HMOs isomers in samples to be detected (such as breast milk and infant formula samples).

Inventors

  • ZHANG XUGUANG
  • WANG XIANGXIN
  • Wei Qiaosi
  • DU YUTONG
  • MA XINMING
  • XIE QINGGANG
  • JIANG SHILONG
  • LU SIYU

Assignees

  • 飞鹤(阿鲁科尔沁旗)乳品有限公司
  • 黑龙江飞鹤乳业有限公司

Dates

Publication Date
20260508
Application Date
20260409

Claims (10)

  1. 1. A method for detecting breast milk oligosaccharide is characterized in that the method for detecting breast milk oligosaccharide comprises the step of detecting a sample to be detected by adopting a detection method based on capillary electrophoresis-laser induced fluorescence, Wherein the running buffer used in the detection method based on capillary electrophoresis-laser induced fluorescence comprises Tris-base, EDTA, urea, PVP of more than 2% (m/V) and less than 4% (m/V) and boric acid of more than 5% (m/V) and less than 15% (m/V).
  2. 2. The method for detecting breast milk oligosaccharides as claimed in claim 1, wherein said running buffer contains 70-100 mmol/L Tris-base,1-5 mmol/L EDTA and/or 2-10 mol/L urea.
  3. 3. The method for detecting breast milk oligosaccharide according to claim 1 or 2, further comprising the step of pretreating the sample to be detected before the step of detecting the sample to be detected by a detection method based on capillary electrophoresis-laser induced fluorescence; Wherein the step of pre-treating comprises a step of sample purification and/or a step of sample derivatization.
  4. 4. The method for detecting human milk oligosaccharide according to claim 3, wherein in the step of purifying the sample, the sample to be detected is purified by using an alcohol precipitation and solid phase extraction column, and/or, In the sample derivatization step, a reductive amination derivatization method is adopted to carry out derivatization reaction on the sample to be detected.
  5. 5. The method for detecting breast milk oligosaccharide according to claim 1 or 2, wherein in the step of detecting the sample to be detected by the detection method based on capillary electrophoresis-laser induced fluorescence, the capillary electrophoresis is performed at a voltage of not less than 15 kV.
  6. 6. The method for detecting breast milk oligosaccharide according to claim 1 or 2, wherein in the step of detecting the sample to be detected by the detection method based on capillary electrophoresis-laser induced fluorescence, the capillary is operated at a temperature of more than 25 ℃ in the capillary electrophoresis, and/or the sample tray is kept at a temperature of 10 to 25 ℃.
  7. 7. The method for detecting breast milk oligosaccharide according to claim 1 or 2, wherein the capillary is a capillary having chemical inertness and hydrophobicity.
  8. 8. The method for detecting breast milk oligosaccharides according to claim 1 or 2, wherein said sample to be detected comprises milk and/or a dairy product.
  9. 9. The method for detecting breast milk oligosaccharide according to claim 1 or 2, wherein the breast milk oligosaccharide comprises neutral breast milk oligosaccharide and acidic breast milk oligosaccharide composed of sialic acid.
  10. 10. The method for detecting human milk oligosaccharides as recited in claim 9, wherein said human milk oligosaccharides include at least one of lactose-N-fucopentaose I, lactose-N-tetraose, 2 '-fucosyllactose, lactose-N-neotetraose, 3,2' -difucosyllactose, disialyllactose-N-tetraose, 3-fucosyllactose, 3 '-sialyllactose, and 6' -sialyllactose.

Description

Detection method of breast milk oligosaccharide Technical Field The invention belongs to the field of detection of breast milk oligosaccharide, in particular relates to a detection method of breast milk oligosaccharide, and more particularly relates to a CE-LIF method for simultaneously separating and detecting the contents of 9 breast milk oligosaccharides in a dairy product. Background Breast milk oligosaccharides (HMOs) are important bioactive compounds in breast milk, and are the third largest solid component after lactose and lipids, at concentrations typically of 5-20 g/L. These complex carbohydrates play a versatile role in infant health, including promoting beneficial intestinal flora and regulating immune responses. HMOs are increasingly being added to infant formulas and dietary supplements to mimic the protective effects of breast milk due to their unique functional properties. Currently, over 200 breast milk oligosaccharides (HMOs) have been identified, of which more than 140 have been elucidated in structure. The structure of HMOs mainly comprises 5 monosaccharide building units, the core skeleton of the HMOs is lactose (Lac) with a reducing end, and fucose (Fuc) or sialic acid (Neu 5 Ac) residues can be further introduced for derivatization through different glycosidic bond connection modes, so that isomers with the same molecular weight and different structures are formed. These isomers include mainly positional isomers such as 2 '-fucosyllactose (2' -FL) and 3-fucosyllactose (3-FL), and 3 '-sialyllactose (3' -SL) and 6 '-sialyllactose (6' -SL), and further include linking isomers such as lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT). This high structural complexity and heterogeneous diversity present significant technical challenges for analytical detection of HMOs. Currently, researchers have used a variety of analytical techniques to characterize HMOs, but because of their complex structure, lack of ultraviolet absorbing groups, and the electroneutrality of some components, they present special challenges for analytical detection. In recent years, the application of liquid chromatography-mass spectrometry (LC-MS) technology in HMOs analysis is increasingly wide, and especially, an LC-MS method using porous graphitized carbon as a stationary phase can realize the synchronous quantitative analysis of neutral and acidic HMOs, thereby remarkably improving the analysis flux and the structural analysis capability. However, this approach still presents challenges for the discrimination of structurally very similar linked isomers. In addition, the traditional chromatographic method generally uses a large amount of organic mobile phase, and the generated waste liquid not only increases the running cost, but also reduces the environmental friendliness of the method. In general, despite the significant progress made in HMOs detection by existing analytical methods, how to achieve efficient separation of key isomers in complex matrices while compromising analytical throughput, sensitivity and green chemistry requirements remains a problem to be solved by current research. In view of the extremely large concentration span of HMOs in breast milk, the detection method needs to have a wide linear dynamic range and high sensitivity. Capillary Electrophoresis (CE) has become an effective supplementary platform for chromatographic methods due to its high separation efficiency, short analysis time, low sample consumption, simple pretreatment, etc. The introduction of Laser Induced Fluorescence (LIF) detection techniques provides a solution to the problem of HMOs lacking chromophores or fluorophores that are difficult to optically detect. Derivatization of HMOs by using fluorescent markers (e.g., trisodium 8-aminopyrene-1, 3, 6-trisulfonate, APTS) not only provides necessary charge for their migration in an electric field, but also significantly improves the detection sensitivity of CE. The particular buffer is critical to the separation effect of the CE. Citation 1 discloses that a conventional CE substrate (e.g., HR-NCHO) is capable of separating LNT from LNnT, but is not effective in distinguishing 2' -FL from 3-FL. The borate-added background electrolyte provides a higher degree of separation by borate-diol complexation. The difference of the three-dimensional structures of the 2' -FL and the 3-FL causes the difference of complexing ability of the 2' -FL and the 3-FL with borates, so that the apparent mobility difference is generated, and the effective separation of the 2' -FL and the 3-FL is realized. In addition, the CE technology has the characteristics of miniaturization and less waste liquid amount, and meets the requirements of green analytical chemistry better. Although studies have been conducted to detect HMOs using CE, limitations remain. The separation effect is poor, and reference 2 has developed a laser-induced fluorescence detection capillary electrophoresis (CE-LIF) method, but i