WO2020252677A1 - Procédé de détermination quantitative à haut rendement d'oligosaccharides libres dans le lait - Google Patents

Procédé de détermination quantitative à haut rendement d'oligosaccharides libres dans le lait Download PDF

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WO2020252677A1
WO2020252677A1 PCT/CN2019/091827 CN2019091827W WO2020252677A1 WO 2020252677 A1 WO2020252677 A1 WO 2020252677A1 CN 2019091827 W CN2019091827 W CN 2019091827W WO 2020252677 A1 WO2020252677 A1 WO 2020252677A1
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milk
oligosaccharides
lactose
sample
solution
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PCT/CN2019/091827
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Chinese (zh)
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陈历俊
黄训文
杨春英
姜铁民
乔为仓
刘彦品
周伟明
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北京三元食品股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/04—Dairy products

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  • the invention relates to the technical field of chemical analysis, in particular to a high-throughput quantitative determination method of free oligosaccharides in milk.
  • oligosaccharides There are abundant free oligosaccharides (breast milk oligosaccharides) with complex structures in breast milk. These oligosaccharides are the highest concentration of carbohydrates in the intestinal tract of infants. Such oligosaccharides cannot be directly digested and utilized by the human body. One of its most important biological functions is to promote the colonization of infant intestinal bifidobacteria and maintain the advantage of the bifidobacteria flora. One of the biggest differences between breast milk and infant formula milk powder is the complex structure and high concentration of free oligosaccharides in breast milk. Infant formula milk powder is an important source of nutrition for infants with insufficient breastfeeding. Its main component is cow's milk.
  • main oligosaccharides contained in it contain a variety of free oligosaccharides with the same structure as breast milk oligosaccharides, the content is extremely low. . Therefore, accurate and quantitative detection of the main free oligosaccharide content in breast milk is important for obtaining statistical data on the main oligosaccharides in breast milk of a representative population, developing breast-emulsified oligosaccharide milk powder for infants and young children, and product development and supervision. Significance.
  • UPLC Ultra Performance Liquid Chromatography
  • HPLC High Performance Liquid Chromatography
  • the existing method for the determination of 12 kinds of breast milk oligosaccharides in breast milk using ultra-high performance liquid chromatography-tandem mass spectrometry the main steps include: 2 times dilution with ultrapure water-centrifugal degreasing-acetonitrile precipitation of protein-centrifugal removal of protein ——Removal of protein by second centrifugation——Dilution with ultrapure water——Test on the machine.
  • this method there are the following shortcomings: First, when using mass spectrometry to detect breast milk oligosaccharides, acidic oligosaccharides and neutral oligosaccharides inhibit each other during ionization.
  • 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.
  • the sugar quantification results have a direct impact.
  • acetonitrile has a better effect on precipitating milk proteins, acetonitrile will reduce the recovery rate of breast milk oligosaccharides.
  • the LC/MS equipment is a relatively expensive precision instrument, and the lactose content in breast milk is very high (60-70mg/mL), which is easy to contaminate the ion source and mass analyzer of the mass spectrometer, and the maintenance and maintenance costs are high.
  • the technical requirements of the staff are much higher than those for the operation of liquid chromatography, which makes the overall detection cost higher.
  • the purpose of the embodiments of the present invention is to provide a high-throughput quantitative determination method of free oligosaccharides in milk.
  • the method for high-throughput quantitative determination of free oligosaccharides in milk includes the following steps: sample preparation and the use of ultra-high performance liquid chromatography on the prepared samples instead of quantitative analysis by liquid mass spectrometry;
  • sample preparation includes the following steps: adjust the milk sample with ultrapure water to a total carbohydrate concentration of less than 20mM, centrifuge to remove milk fat and/or milk protein to obtain free oligosaccharides, perform fluorescent labeling on the free oligosaccharides, Use ultrapure water to dilute the concentration of fluorescently labeled oligosaccharides to within the range of the standard curve before testing.
  • the sample includes a liquid sample or a solid sample
  • the liquid sample includes at least one of breast milk, cow's milk or other liquid dairy products
  • the solid sample includes skimmed milk powder, whole milk powder Or at least one of whey powder.
  • the solid sample can be dissolved and diluted with appropriate ultrapure water, and then subjected to the separation and fluorescent labeling of free oligosaccharides after being mixed with ultrasonic treatment.
  • free oligosaccharides in milk that can be simultaneously detected include 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 4'- Galactosyl lactose (4'-GOS), 3'-galactosyl lactose (3'-GOS), 6'-galactosyl lactose (6'-GOS), 3'-sialyllactose (3'-SL) ), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-sialyllactose (6'-SL), lacto-N-fucosylpentaose V (LNFP V ), Milk-N-Fucosyl Pentaose I (LNFP I), Milk-N-Fucosyl Pentaose II (LNFP II), Milk
  • the free oligosaccharides in breast milk include 2'-fucosyllactose, 3-fucosyllactose, 4'-galactosyllactose, 3'-galactosyl Lactose, 6'-galactosyllactose, 3'-sialyllactose, lactose-N-tetraose, lact-N-neotetraose, 6'-sialyllactose, lactose-N-fucosylpentaose V , Milk-N-Fucosyl Pentaose I, Milk-N-Fucosyl Pentaose II, Milk-N-Difucosyl Hexose I, Sialyl Milk-N-tetraose C, Di-saliva At least one of yogurt.
  • the free oligosaccharides in the milk, whey powder, skimmed milk powder or whole milk powder include 2-fucosyllactose, 3-fucosyllactose, 4' -Galactosyl lactose, 3'-galactosyl lactose, 6'-galactosyl lactose, 3'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, 6'-sialyllactose , At least one of Sialyl Milk-N-tetrasaccharide c.
  • the centrifugal removal of milk fat and/or milk protein to obtain free oligosaccharides includes the use of either of the following two centrifugal methods: ultrafiltration centrifugation or low-temperature centrifugal degreasing.
  • the ultrafiltration centrifugation method includes: transferring the sample solution to an ultrafiltration centrifuge tube, centrifuging for 20-30 minutes, the molecular retention of the ultrafiltration membrane is 3-10kDa, and the obtained permeate is free Glycan sample solution.
  • the low-temperature centrifugal degreasing method includes: centrifuging the sample solution at 6000-10000 g at 4° C. for 10 minutes, and removing the lower clear liquid as the free oligosaccharide sample solution.
  • the fluorescent labeling of free oligosaccharides includes the following steps: Take 100 microliters of the separated free oligosaccharide sample solution, add 2-aminobenzamide (2-AB) solution and 2- 50 microliters of picoline-N-borane solution each makes 2-AB and 2-picoline-N-borane excess relative to free oligosaccharides, among which 2-aminobenzamide (2-AB)
  • the solvents used in the solution and 2-methylpyridine-N-borane solution are both dimethyl sulfoxide and acetic acid mixed solvents with a volume ratio of 85:15. After vortexing and mixing, incubate at a constant temperature of 2.5 in a 65°C water bath. h. Take out and cool to room temperature to obtain a free oligosaccharide sample after derivatization.
  • the ultra-high performance liquid chromatography adopts a hydrophilic interaction chromatography column and/or a fluorescence detector.
  • Detector fluorescence detector; detection wavelength: excitation wavelength 250nm, emission wavelength 425nm;
  • Chromatographic column Waters Acquity UPLC BEH Amide chromatographic column (2.1mm ⁇ 100mm, 1.7 ⁇ m); Chromatographic column temperature: 45 ⁇ 55°C;
  • Constant flow rate mode 0.400 ⁇ 0.500mL/min
  • N ranges from 63.0 to 64.5.
  • the above table shows: from 1.00 to 2.00 minutes from the start of gradient elution, the mobile phase is stable to reach 14-16% of phase A, and the remaining components are phase B; after reaching 14 to 16%, to 34.00 to 35.00 minutes from the start of elution, The mobile phase is stable up to 20% of the phase A, and the remaining components are phase B; and so on.
  • the above-mentioned high-throughput quantitative determination method uses an external standard method to establish a linear regression equation to quantify free oligosaccharides based on the peak area.
  • the above-mentioned high-throughput quantitative determination method includes the following steps:
  • 2-AB solution and 2-picoline-N-borane solution to fluorescently label the free oligosaccharides in the separated free oligosaccharide solution, including: 2-aminobenzamide solution and 2
  • the solvent used in the -picoline-N-borane solution is a mixed solvent of dimethyl sulfoxide and acetic acid with a volume ratio of 85:15, and then dilute the oligosaccharide concentration to within the linear range with ultrapure water, and then Detect with ultra-high performance liquid chromatography-fluorescence detection method, and calculate the content of free oligosaccharides in the sample to be tested according to the linear regression equation established by the external standard method according to the peak area.
  • the high-throughput quantitative determination method in the embodiment of the present invention can detect 15 kinds of acidic and neutral free oligosaccharides at the same time when there is a high concentration of lactose in the milk by using only liquid chromatography. And it has high sensitivity and good separation effect for 15 kinds of free oligosaccharides, the chromatographic baseline is relatively stable, and the detection limit can reach 0.8-7.6pg, especially for 2'-fucosyllactose. Sensitivity, the detection limit can reach 0.8pg.
  • the LOD using ultra-high performance liquid chromatography-tandem mass spectrometry in the prior art is higher than 230 pg, for example, the detection limits of 2'-fucosyllactose and 3-fucosyllactose are higher than 460 pg.
  • the high-throughput quantitative determination method in the embodiment of the present invention has a good separation effect on various structural isomers of oligosaccharides, such as 2'-FL and 3-FL, LNT and LNnT baseline separation It also has a good resolution for structural isomers such as LNFP I, LNFP II, LNFP III, and LNFP V.
  • structural isomers such as LNFP I, LNFP II, LNFP III, and LNFP V.
  • high performance liquid chromatography-tandem mass spectrometry cannot completely separate these structural isomers. open.
  • a hydrophobic group (benzene ring) is added to the reducing end of the oligosaccharide, which increases the hydrophobicity, which can effectively inhibit the formation of anomers during the separation process, and obtain better results.
  • the chromatographic separation effect As the concentration of acetonitrile in the mobile phase decreases on the hydrophilic interaction chromatography, the 2-AB-derived oligosaccharides are basically separated in the order of molecular weight from small to large (the retention time of acid oligosaccharides is relatively prolonged), which is more compatible with low molecular weight.
  • the glycans are qualitatively determined to eliminate the interference between oligosaccharides.
  • the high-throughput quantitative determination method in the embodiment of the present invention has high accuracy and high recovery rate.
  • the sample is diluted by more than 10 times before ultrafiltration centrifugation or low-temperature centrifugal degreasing to reduce the influence of the volume effect of milk fat on the quantitative detection result of milk oligosaccharide to less than 0.5%.
  • the high-throughput quantitative determination method in the embodiment of the present invention has high precision.
  • the RSD of the peak area and retention time measured within the batch are lower than 3.0% and 0.08%, respectively; the RSD of the peak area and retention time measured between the batches It is lower than 3.0% and 0.14% respectively.
  • the sample preparation method is simple, easy to operate, high-throughput, and has good reliability, and can complete the preparation of large quantities of samples within 3 hours.
  • the sample preparation method of the present invention requires a small amount of sample (at least 10 ⁇ L), a simple preparation method, no complicated operations in the entire preparation process, only dilution and fixation centrifugal operations, and no purification treatment after derivatization of oligosaccharides. The loss of oligosaccharides is reduced, the generation of man-made random errors is greatly reduced, the operability and reliability of the method are improved, and the throughput of sample preparation is improved.
  • sample preparation does not use organic solvents such as chloroform, methanol, ethanol, acetonitrile, and does not use toxic sodium boron cyanide reagents when the sample is derived, which has a good environmental protection effect and can better protect the bodies of operators and other personnel health.
  • the high-throughput quantitative determination method in the embodiment of the present invention has low requirements on the performance of analytical instruments, and the price of ultra-high performance liquid chromatography is moderate. It is a conventional precision instrument equipped in most laboratories, which is easy to maintain and operate, and reduces Training time and investment for testing personnel.
  • Figure 1 is a chromatogram of 2-AB derivatives of free oligosaccharides in human milk (colostrum) in Experimental Example 2;
  • Figure 2 is the chromatogram of the 2-AB derivative of the mixed standard oligosaccharides (15 species) in Example 4.
  • breast milk collected from lactating mothers
  • cow milk purchased from large supermarkets
  • 15 standard oligosaccharides purchased from Carbosynth and Sigma
  • deionized water homemade
  • 2-aminobenzamide 2-methyl Pyridine-N-borane was purchased from Sigma-Aldrich
  • Acetonitrile chromatographically pure
  • ammonium formate mass pure
  • Ultra-high performance liquid chromatograph Thermo Fisher company, equipped with fluorescence detector; Ultrasonic cleaner purchased from Kunshan Ultrasonic Instrument Co., Ltd.; Constant temperature water bath was purchased from Yuyao Dongfang Electrical Instrument Factory; Vortex oscillator was purchased from SCILOGEX Company ; The high-speed refrigerated centrifuge was purchased from Sigma; the ultrapure water meter was purchased from Millipore; the acidity meter was purchased from Sartorius.
  • oligosaccharides are lost to a certain extent, and the stability is not high. Therefore, a simple and easy-to-operate sample preparation method is more suitable for the quantitative detection of breast milk oligosaccharides.
  • a high-throughput quantitative determination method of free oligosaccharides in milk includes the following steps:
  • the first method transfer the solution obtained after vortexing to an ultrafiltration centrifuge tube (ultrafiltration membrane molecular cut-off 10kDa), centrifuge for 20 minutes, and the obtained permeate is a free oligosaccharide sample solution;
  • the second method transfer the solution obtained after vortexing to a centrifuge tube, centrifuge at 8000g for 10 min at 4°C, and the lower layer of the solution is the free oligosaccharide sample solution.
  • the solvent used in the picoline-N-borane solution is a mixture of dimethyl sulfoxide and acetic acid with a volume ratio of 85:15.
  • the detection conditions are as follows:
  • Detector fluorescence detector; detection wavelength: excitation wavelength 250nm, emission wavelength 425nm;
  • Chromatographic column Waters Acquity UPLC BEH Amide chromatographic column (2.1mm ⁇ 100mm, 1.7 ⁇ m); Column temperature: 45°C;
  • Constant flow rate mode 0.400mL/min
  • a high-throughput quantitative determination method of free oligosaccharides in milk includes the following steps:
  • the first type transfer the solution obtained after vortexing to an ultrafiltration centrifuge tube (ultrafiltration membrane molecular cut-off 10kDa), centrifuge for 25 minutes, and the obtained permeate is a free oligosaccharide sample solution;
  • the second method transfer the solution obtained after vortex mixing to a centrifuge tube, centrifuge at 10000g for 10 minutes at 4°C, and the lower clear liquid is the free oligosaccharide sample solution.
  • the solvent used in the picoline-N-borane solution is a mixture of dimethyl sulfoxide and acetic acid with a volume ratio of 85:15.
  • the detection conditions are as follows:
  • Detector fluorescence detector; detection wavelength: excitation wavelength 250nm, emission wavelength 425nm;
  • Chromatographic column Waters Acquity UPLC BEH Amide chromatographic column (2.1mm ⁇ 100mm, 1.7 ⁇ m); Chromatographic column temperature: 55°C;
  • Constant flow rate mode 0.450mL/min
  • a high-throughput quantitative determination method of free oligosaccharides in milk includes the following steps:
  • the first method transfer the solution obtained after vortexing to an ultrafiltration centrifuge tube (ultrafiltration membrane molecular cut-off 10kDa), centrifuge for 30 minutes, and the obtained permeate is a free oligosaccharide sample solution;
  • the second method transfer the solution obtained after vortexing to a centrifuge tube, centrifuge at 6000g for 10 min at 4°C, and the lower layer of the solution is the free oligosaccharide sample solution.
  • the solvent used in the picoline-N-borane solution is a mixture of dimethyl sulfoxide and acetic acid with a volume ratio of 85:15.
  • the detection conditions are as follows:
  • Detector fluorescence detector; detection wavelength: excitation wavelength 250nm, emission wavelength 425nm;
  • Chromatographic column Waters Acquity UPLC BEH Amide chromatographic column (2.1mm ⁇ 100mm, 1.7 ⁇ m); Chromatographic column temperature: 50°C;
  • Constant flow rate mode 0.500mL/min
  • the oligosaccharide mixed standard solution was prepared with ultrapure water so that the concentration of each oligosaccharide was 40mg/mL, and it was gradually diluted with ultrapure water to prepare a series of standard solutions with a concentration of 20-800ppg.
  • the linear regression equation of the standard curve established with the concentration as the abscissa versus the peak area is shown in the following table. All 15 oligosaccharides have good linearity, and R 2 is all above 0.999.
  • the above-mentioned 40mg/mL oligosaccharide mixed standard solution was used to prepare a series of standard solutions with a concentration of 0.5-30ppb according to the gradient dilution method, and each sample was measured 3 times in parallel, using the chromatographic integral that comes with the chromatograph
  • the software analyzes the limit of detection (LOD) of each oligosaccharide. This method detects 15 free oligosaccharides with a LOD of 0.8-7.6 pg, which has high sensitivity.
  • LOD limit of detection
  • the ultra-high performance liquid chromatogram of the standard oligosaccharide mixture (15 species) of 2-AB derivatives is shown in Figure 2.
  • Blank substrate Take 50 microliters of breast milk colostrum and add 950 microliters of ultrapure water as a blank substrate.
  • Standard addition sample Take the blank matrix solution and add the appropriate volume of the oligosaccharide mixed standard solution in Example 4 to prepare the concentration of each oligosaccharide to 80ppb (low concentration), 360ppb (medium concentration), 720ppb (High concentration) Three concentration levels of the spiked sample solution, and then according to the method described in Example 2, the same spiked sample was separated by one of two methods: ultrafiltration centrifugation and low-temperature centrifugal degreasing. sugar.
  • Ultrafiltration centrifugation method transfer the spiked solution to an ultrafiltration centrifuge tube (ultrafiltration membrane molecular cut-off 10kDa), centrifuge for 25 minutes, and take the permeate as the free oligosaccharide sample solution; low-temperature centrifugal degreasing method: add The labeled solution was placed in a 2mL centrifuge tube, centrifuged at 10,000g at 4°C for 10 minutes, and the lower layer was taken as the free oligosaccharide sample solution (both methods were tested).
  • the concentration of the fluorescently labeled oligosaccharide is diluted with ultrapure water to the linear range, filtered through a 0.22 ⁇ m filter membrane before injection, and the samples to be analyzed are stored in a refrigerator at -40°C.
  • Blank matrix When cow milk and whey powder are used as the matrix, the cow milk is diluted 10 times with ultrapure water, and the whey powder is prepared with ultrapure water to prepare a 50mg/mL whey powder solution as the blank matrix.
  • Spiked samples Prepare 80ppb (low concentration) and 720ppb (high concentration) two spiked samples with a blank matrix of milk and whey powder solution, and separate free oligosaccharides by ultrafiltration and centrifugation. Other processes are the same as those of breast milk spiked samples The preparation method is the same.
  • the blank matrix and the spiked samples processed by the two sample preparation methods (referring to ultrafiltration centrifugation and/or low-temperature centrifugal degreasing method) (repeated determination 5 times) were measured at the same time, and the recovery rate was calculated.
  • the recovery rate of 14 oligosaccharides except DSLNT was 73.3-111%; when the breast milk spiked sample was prepared by low-temperature centrifugal degreasing method, the recovery rate of 15 oligosaccharides was better It is 88-107%.
  • the milk or whey powder samples were processed by ultrafiltration and centrifugation, and the accuracy of the detection method was verified by the low-concentration and high-concentration milk or whey powder samples.
  • the recovery rate of these 10 kinds of oligosaccharides was 78.1 ⁇ 101%. Very high accuracy.
  • the peak area (Area) and retention time (RT) RSD of the 15 oligosaccharides in the same test batch were respectively less than 2.8% And 0.08%; the RSD of the peak area and retention time of 15 oligosaccharides among three different detection batches are less than 3.0% and 0.14%, respectively, which has good repeatability and reproducibility.
  • the peak area and retention time RSD of the 15 oligosaccharides in the same test batch were less than 4.3% and 0.06%, respectively;
  • the RSD of the peak area and retention time of 15 kinds of oligosaccharides between different detection batches were less than 4.0% and 0.43%, respectively.
  • the method has good repeatability and reproducibility.
  • the milk or whey powder obtained by the ultrafiltration and centrifugation process in Example 5 was spiked with the sample solution (80ppb (low concentration), 720ppb (high concentration)) after derivatization
  • the same test batch was repeated 7 times for each sample to verify the precision (repeatability) of the method, and analyze the relative standard deviation of the peak area and retention time of oligosaccharides in each sample (RSD), to obtain the repeatability of the method.
  • the RSD of the peak area and retention time of 10 oligosaccharides in the same test batch Respectively less than 3.3% and 0.05%, the method has good precision.
  • the method for high-throughput quantitative determination of free oligosaccharides in milk includes the following steps: sample preparation and quantitative analysis of the prepared sample using ultra-high performance liquid chromatography instead of liquid mass spectrometry; Among them: sample preparation includes the following steps: adjust the milk sample with ultrapure water to a total carbohydrate concentration of less than 20mM, centrifuge to remove milk fat and/or milk protein to obtain free oligosaccharides, perform fluorescent labeling on the free oligosaccharides, Use ultrapure water to dilute the concentration of fluorescently labeled oligosaccharides to within the range of the standard curve before testing.
  • the sample preparation method is simple, easy to operate, high throughput, and has good reliability, and the preparation of large quantities of samples can be completed within 3 hours.
  • the sample preparation method of the present invention requires a small amount of sample (as low as 10 ⁇ L), and simplifies the cumbersome sample preparation method in the previously reported liquid chromatography-fluorescence detection method. There is no complicated operation in the entire sample preparation process, only The centrifugal operation of dilution and fixation does not undergo purification treatment after derivatization of oligosaccharides, which reduces the loss of oligosaccharides, greatly reduces the generation of man-made random errors, improves the operability and reliability of the method, and improves the sample preparation Flux.

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Abstract

L'invention concerne un procédé de détermination quantitative à haut rendement d'oligosaccharides libres dans le lait, ledit procédé comprenant : la préparation d'échantillons et la détection des échantillons préparés à l'aide d'une chromatographie liquide à ultra-haute efficacité ; la préparation d'échantillon comportant : la dilution d'un échantillon de lait avec de l'eau ultra pure, la centrifugation afin d'éliminer la graisse du lait et/ou la protéine du lait afin d'obtenir des oligosaccharides libres, la mise en œuvre d'un marquage par fluorescence des oligosaccharides libres et, avant la détection par machine, l'utilisation d'eau ultra pure pour diluer la concentration d'oligosaccharides marqués par fluorescence dans les limites de l'amplitude d'une courbe standard.
PCT/CN2019/091827 2019-06-19 2019-06-19 Procédé de détermination quantitative à haut rendement d'oligosaccharides libres dans le lait WO2020252677A1 (fr)

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