WO2023000769A1 - Qualitative and quantitative detection method for human milk oligosaccharides in dairy product - Google Patents

Qualitative and quantitative detection method for human milk oligosaccharides in dairy product Download PDF

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WO2023000769A1
WO2023000769A1 PCT/CN2022/091597 CN2022091597W WO2023000769A1 WO 2023000769 A1 WO2023000769 A1 WO 2023000769A1 CN 2022091597 W CN2022091597 W CN 2022091597W WO 2023000769 A1 WO2023000769 A1 WO 2023000769A1
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human milk
milk oligosaccharides
lactose
qualitative
organic solvent
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PCT/CN2022/091597
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French (fr)
Chinese (zh)
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方诩
牛康乐
姜福娇
黄爱玲
宋江辉
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山东恒鲁生物科技有限公司
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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

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  • the invention belongs to the field of detection of human milk oligosaccharides in dairy products, and relates to a qualitative and quantitative detection method for human milk oligosaccharides in dairy products, in particular to the qualitative and quantitative detection of lactose-N-tetrasaccharide and lactose-N-trisaccharide in dairy products method.
  • Human milk oligosaccharides also known as human milk oligosaccharides (HMOs) were found to be a unique class of oligosaccharides present in breast milk, and the undigested oligosaccharides in breast milk partially stimulated the growth of bifidobacteria in the colon, And these bifidobacteria are beneficial to the protection and prevention of intestinal infection, therefore, breast milk oligosaccharides are the main components of the innate immune system.
  • the difference in human milk oligosaccharide content is one of the important reasons why breastfeeding is more advantageous than milk formula feeding.
  • lactose-N-tetraose (LNT) and lactose-N-triose (Lacto-N-tetriaose II, LNTII) are the main components in human milk oligosaccharides (HMOs), which have various Physiological functions can reduce the occurrence of infection, promote the proliferation and activity of beneficial intestinal flora, indirectly inhibit the growth of harmful flora, maintain the balance of intestinal micro-ecology, and thus protect the intestinal tract of infants from the invasion of pathogenic bacteria.
  • HMOs human milk oligosaccharides
  • Chinese patent document CN107621399A discloses a method for detecting oligosaccharides in breast milk, which includes the step of pre-processing the breast milk sample.
  • the pre-treatment specifically includes: taking a breast milk sample, centrifuging to remove the upper layer of fat, Take the lower layer liquid and add alcohol solvent, freeze at -40°C ⁇ -100°C for 5-15min, centrifuge, and take the supernatant; Quantitative detection.
  • the detection time is 70-145 minutes.
  • the present invention provides a qualitative and quantitative detection method for human milk oligosaccharides in dairy products, providing an accurate and rapid detection method for the quality control of dairy products.
  • Room temperature It has a well-known meaning in the technical field, and generally refers to 25 ⁇ 2°C.
  • a pretreatment method for the detection of human milk oligosaccharides in dairy products First, the protein in the dairy product is removed by heating. For dairy products containing multiple complex components, the protein can be further removed with a water-miscible organic solvent, or the protein can be removed again. Utilize organic solvent extraction method to remove lipid, obtain dairy product for test solution, specifically as follows:
  • a pretreatment method for detecting human milk oligosaccharides in dairy products comprising: Step A. Take a sample of dairy products, add 1-10 times the volume of ultrapure water to mix, heat to 80-100 ° C for 10-30 minutes, Cool down to room temperature, centrifuge at 2000-20000 ⁇ g for 5-50min in the range of 0-10°C, and collect the supernatant as the test solution.
  • step A heat treatment at 85-100°C for 20-30 minutes; more preferably heat at 90-100°C.
  • the centrifugation condition in step A is within the range of 0-5°C, 5000-15000 ⁇ g for 5-30 minutes; more preferably 4°C, 10000-15000 ⁇ g for 10-20 minutes.
  • the pretreatment method for detecting human milk oligosaccharides in dairy products of the present invention further includes: step B. adding an organic solvent to the test solution described in step A, shaking or vortexing Mix well, centrifuge in the range of 0-10°C, centrifuge at 2000-20000 ⁇ g for 5-50min to remove the precipitate, collect the supernatant as the test solution, the organic solvent is miscible with water, selected from methanol, ethanol, propanol, iso One or more of propanol and acetonitrile; preferably, the amount of the organic solvent added is 0.5-1 times the volume of the ultrapure water in step A.
  • the centrifugation condition is 0-5°C, 5000-15000 ⁇ g for 5-30 minutes; more preferably 4°C, 10000-15000 ⁇ g for 10-20 minutes.
  • the pretreatment method for detecting human milk oligosaccharides in dairy products of the present invention further includes: step C. adding an organic solvent to the test solution obtained in step A or step B, shaking or Vortex and mix well, centrifuge in the range of 0-10°C, centrifuge at 2000-20000 ⁇ g for 5-50min, collect the aqueous phase as the test solution;
  • the organic solvent is selected from butanol, methylene chloride, chloroform or ethyl acetate One or more of them; preferably, the amount of the organic solvent added is 0.7-1.5 times the volume of the ultrapure water in step A.
  • the human milk oligosaccharides of the present invention include but not limited to lactose-N-tetraose and lactose-N-triose.
  • a qualitative and/or quantitative detection method for human milk oligosaccharides in dairy products including the above pretreatment method, and further comprising:
  • Step D Qualitative and/or quantitative analysis of the human milk oligosaccharides in the test solution by high performance liquid chromatography, that is, qualitative and/or quantitative analysis of the test solution obtained in step C.
  • the chromatographic column is selected from a hydrophilic chromatographic column or a hydrophobic chromatographic column.
  • the hydrophilic chromatographic column filler is silica gel, polar group bonded phase silica gel or dextran; the polar group in the polar group bonded phase silica gel is amide group, urea group One or more of cyano, amino, carboxyl, sugar and zwitterions;
  • the chromatographic column filler is aminopropyl bonded silica gel.
  • the chromatographic column is an aminopropyl bonded silica gel chromatographic column, referred to as an aminopropyl chromatographic column or an amino chromatographic column, selected from Cosmosil Sugar-D/ of Nacalai Company or AsahipakNH2P- of Shodex Company 504E.
  • the detector used in the high performance liquid chromatography is a differential refractive index detector, an ultraviolet detector or a diode array detector; preferably an ultraviolet detector or a diode array detector, and the detection wavelength is 180-280nm.
  • the mobile phase of the high-performance liquid chromatography is water and an organic solvent, and its volume ratio is 10/90 to 90/10, using isocratic elution or gradient elution;
  • the organic solvent is selected from methanol, ethanol , propanol, isopropanol, butanol, acetonitrile in one or more.
  • the mobile phase is 60-75% (v/v) acetonitrile aqueous solution; preferably 65-70% (v/v) acetonitrile aqueous solution.
  • the flow rate of the high performance liquid chromatography is 0.2-1 mL/min
  • the column temperature is 20-60° C.
  • the flow rate of the mobile phase is 0.4-0.8 mL/min.
  • the injection volume is preferably 10 ⁇ L, the column temperature is 20-60°C, and the inner diameter of the chromatographic column is 2.0-6.0 mm.
  • the human milk oligosaccharide of the present invention is one of lactose-N-tetraose and lactose-N-triose.
  • a qualitative or quantitative detection method of lactose-N-tetrasaccharide or lactose-N-triose in dairy products is high performance liquid chromatography; including the above pretreatment method, also includes the following steps:
  • step C Including establishing a lactose-N-tetrasaccharide or lactose-N-triose standard curve, and detecting the test solution obtained in step C, and performing qualitative or quantitative analysis of the lactose-N-tetraose or lactose-N-triose in the test solution Analysis steps.
  • the detector used in the high performance liquid chromatography is a differential refractive index detector, an ultraviolet detector or a diode array detector; more preferably an ultraviolet detector or a diode array detector; the detection wavelength is 180-280nm .
  • the mobile phase of the high-performance liquid chromatography in the present invention is 60-75% (v/v) acetonitrile aqueous solution, using isocratic elution or gradient elution.
  • the concentration of the acetonitrile aqueous solution in the mobile phase is lower than 60% (v/v) or higher than 75% (v/v)
  • the problem of chromatographic peak interference will be caused, and human milk oligosaccharides in dairy products cannot be detected normally. content.
  • the invention provides a qualitative and/or quantitative detection method of human milk oligosaccharides contained in dairy products.
  • the present invention mainly uses the heating method to remove protein, etc. If the ingredients are complex, step B and/or step C can be selected to process dairy products.
  • the method of the present invention can effectively remove protein, lipid, etc., and other components in dairy product samples Compared with the commonly used pretreatment methods of centrifugation to remove lipids and ethanol/acetonitrile precipitation to remove proteins, the existence of interference peaks is greatly reduced, and the accuracy and reliability of the detection results are guaranteed. Dairy products are detected by high performance liquid chromatography content of human milk oligosaccharides.
  • the qualitative and/or quantitative detection method of the present invention adopts, and the pretreatment liquid is separated and analyzed by high performance liquid chromatography, and the preferred scheme of the high performance liquid chromatography adopts Cosmosil Sugar-D of Nacalai Company and/or Asahipak of Shodex Company NH2P-504E is beneficial to the separation and detection of human milk oligosaccharides.
  • the high-performance liquid chromatography system has fast detection speed and high sensitivity, and the detection result can be obtained within 1 hour, which greatly shortens the analysis time. It greatly reduces the detection cost and detection difficulty.
  • the invention provides a qualitative and quantitative detection method for human milk oligosaccharides in dairy products, which can accurately and rapidly detect human milk oligosaccharides in dairy products.
  • the provision of detection methods for lactose-N-tetrasaccharide and lactose-N-triose has positive significance for the quality control of dairy products and is conducive to the rapid promotion and application of human milk oligosaccharides in the dairy industry.
  • Figure 1 is a schematic diagram of the qualitative and quantitative detection and analysis process of human milk oligosaccharides in dairy products.
  • Fig. 3 HPLC chromatogram of determination of lactose-N-triose in the whole milk sample of embodiment 2.
  • Fig. 4 The HPLC chromatogram of the content determination of lactose-N-triose and lactose-N-tetrasaccharide in the yogurt sample of comparative example 1.
  • Fig. 6 The HPLC chromatogram of the content determination of lactose-N-triose and lactose-N-tetrasaccharide in the yogurt sample of comparative example 3.
  • Fig. 7 The HPLC chromatogram of the content determination of lactose-N-triose and lactose-N-tetrasaccharide in the yogurt sample of comparative example 4.
  • Lactose-N-tetrasaccharide and lactose-N-triose standard products were purchased from Shanghai Zhenzhun Biotechnology Co., Ltd.; other commercial standard products can also be used in the present invention.
  • Yogurt, milk, and milk powder used in the examples are all commercially available dairy products that do not contain human milk oligosaccharides.
  • lactose-N-triose standard product dissolve it in 100 ⁇ L of ultrapure water, mix well to obtain a 1 mg/mL primary standard solution, and store it at -20°C for later use; take 10 ⁇ L of the primary standard solution, add 90 ⁇ L of ultrapure water to obtain a standard solution with a concentration of 100 mg/L, which was diluted step by step to 20 mg/L, 40 mg/L, 60 mg/L, and 80 mg/L respectively to obtain a standard gradient solution of lactose-N-triose.
  • the high-performance liquid chromatography conditions of the standard solution are: adopt 4.6 ⁇ 250mm, 5 ⁇ m Cosmosil Sugar-D or AsahipakNH2P-504E amino chromatographic column, and use 60%-75% (v/v) acetonitrile aqueous solution as the mobile equipotential elution ;
  • the flow rate is 0.5mL/min
  • the column temperature is 25°C or 30°C
  • the injection volume is 10 ⁇ L
  • the detection wavelength is 195nm.
  • the concentration of the lactose-N-triose standard solution used in the examples and comparative examples of the present invention is 0.6 mg/mL; the concentration of the lactose-N-tetrasaccharide standard solution is 0.6 mg/mL.
  • Embodiment 1 Qualitative and quantitative detection of human milk oligosaccharides in yogurt samples, the flow diagram is shown in Figure 1, and the steps are as follows:
  • Step A Take 1mL yogurt sample (the sample does not contain human milk oligosaccharides), add 1mL lactose-N-tetrasaccharide standard solution to it, add 1ml ultrapure water and mix well, and treat it in a water bath at 100°C for 20min. Cool down to room temperature, centrifuge at 12000 ⁇ g for 10 min at 4°C, and collect the supernatant;
  • Step B Add 0.5 ml of isopropanol to the supernatant obtained in step A, shake and mix, centrifuge at 12000 ⁇ g for 10 min at 4°C to further remove protein, and collect the supernatant;
  • Step C Add 1ml of butanol to the supernatant obtained in step B, vortex or vortex to mix, centrifuge at 5°C, 10000 ⁇ g for 25min, collect the water phase, and obtain protein and lipid-free dairy products For test solution;
  • the condition of high-performance liquid chromatography is: adopt 4.6 * 250mm, the Cosmosil Sugar-D amino chromatographic column of 5 ⁇ m, take the acetonitrile aqueous solution of 65% (v/v) as flow equal degree elution; Flow velocity is 0.5mL/min , the column temperature is 25°C, the injection volume is 10 ⁇ L, the diode array detector, and the detection wavelength is 195 nm.
  • the high-performance liquid chromatogram of lactose-N-tetrasaccharide content detection in the yoghurt sample is as shown in Figure 2, in conjunction with standard curve, calculates the content of lactose-N-tetrasaccharide in the described dairy product test solution to be 0.576mg, the recovery rate 96%.
  • Step A Take 1mL whole milk sample (excluding human milk oligosaccharides), add 1mL lactose-N-triose standard solution to the milk sample, add 3ml ultrapure water and mix well, and treat it in a water bath at 90°C for 30min , lowered to room temperature, centrifuged at 10,000 ⁇ g for 20 min at 4°C, and collected the supernatant;
  • Step C Add 4.5ml of ethyl acetate to the supernatant obtained in step A, shake and mix well, centrifuge at 15000 ⁇ g for 10min at 4°C, collect the water phase, and obtain a dairy product test solution with protein and lipid removed ;
  • the condition of high-performance liquid chromatography is: adopt the Cosmosil Sugar-D amino chromatographic column of 4.6 * 250mm, 5 ⁇ m, take the acetonitrile aqueous solution of 70% (v/v) as flow equal degree elution; Flow velocity is 0.5mL/min , the column temperature is 25°C, the injection volume is 10 ⁇ L, the diode array detector, and the detection wavelength is 195 nm.
  • Example 3 Qualitative and quantitative detection of human milk oligosaccharides in whole milk powder samples, the steps are as follows:
  • Step A Take 1g of milk powder sample (excluding human milk oligosaccharides), add 1mL of lactose-N-triose standard solution to the milk powder sample, add 10ml of ultrapure water and mix evenly, and treat it in a water bath at 90°C for 30min. Centrifuge at 10,000 ⁇ g for 10 min at 4°C to room temperature, and collect the supernatant;
  • Step B Add 5 ml of acetonitrile to the supernatant obtained in step A, shake and mix well, centrifuge at 15000 ⁇ g for 10 min at 4°C to further remove protein, and collect the supernatant;
  • Step C Add 10 ml of ethyl acetate to the supernatant obtained in Step B, shake and mix, centrifuge at 15000 ⁇ g for 10 min at 4°C, collect the aqueous phase, and obtain a dairy product test solution from which protein and lipid have been removed;
  • the condition of high-performance liquid chromatography is: adopt the Cosmosil Sugar-D amino chromatographic column of 4.6 * 250mm, 5 ⁇ m, take the acetonitrile aqueous solution of 75% (v/v) as flow equal degree elution; Flow velocity is 0.5mL/min , the column temperature is 30° C., the injection volume is 10 ⁇ L, a diode array detector, and the detection wavelength is 195 nm.
  • the content of lactose-N-triose in the test solution of the product is calculated to be 0.598 mg, and the recovery rate is 99.67%.
  • step B Without pretreatment step B, other operations were the same, the content of lactose-N-triose in the sample was measured to be 0.567, and the recovery rate was 94.5%.
  • Example 4 Qualitative and quantitative detection of human milk oligosaccharides in yogurt samples, the steps are as follows:
  • Step A Take 200 ⁇ L yogurt sample (excluding human milk oligosaccharides), add 200 ⁇ L lactose-N-tetrasaccharide standard solution and 200 ⁇ L lactose-N-triose standard solution to the yogurt sample, add 1ml ultrapure water and mix well , treated in a water bath at 100°C for 30 minutes, cooled to room temperature, centrifuged at 15,000 ⁇ g for 50 minutes at 4°C, and collected the supernatant;
  • Step B Add 0.5 ml of isopropanol to the supernatant obtained in step A, shake and mix, centrifuge at 15000 ⁇ g for 50 min at 4°C, and collect the supernatant;
  • Step C Add 1ml of dichloromethane to the supernatant obtained in step B, shake and mix well, centrifuge at 15000 ⁇ g for 10min at 4°C, collect the water phase, and obtain a dairy product test solution with protein and lipid removed
  • the condition of high-performance liquid chromatography is: adopt the Cosmosil Sugar-D amino chromatographic column of 4.6 * 250mm, 5 ⁇ m, take the acetonitrile aqueous solution of 65% (v/v) as flow equal degree elution; Flow velocity is 0.5mL/min , the column temperature is 25°C, the injection volume is 10 ⁇ L, the diode array detector, and the detection wavelength is 195 nm.
  • step B If there is no step B, his operation is the same, and the content of lactose-N-tetrasaccharide in the yogurt sample measured is 0.113mg, and the recovery rate is 94.17; the content of lactose-N-triose sugar is 0.110mg, and the recovery rate is 91.67% .
  • Step A Take 200 ⁇ L yogurt sample (excluding human milk oligosaccharides), add 200 ⁇ L lactose-N-tetrasaccharide standard solution and 200 ⁇ L lactose-N-triose standard solution to the yogurt sample, add 1ml ultrapure water and mix well , treated in a water bath at 80°C for 10 minutes, cooled to room temperature, centrifuged at 5000 ⁇ g for 5 minutes at 4°C, and collected the supernatant;
  • Step B Add 0.5 ml of isopropanol to the supernatant obtained in step A, shake and mix, centrifuge at 5000 ⁇ g for 5 min at 4°C, and collect the supernatant;
  • Step C Add 1 ml of dichloromethane to the supernatant obtained in step B, shake and mix, centrifuge at 15000 ⁇ g for 10 min at 4°C, collect the aqueous phase, and obtain a dairy product test solution from which protein and lipid have been removed;
  • the condition of high-performance liquid chromatography is: adopt the Cosmosil Sugar-D amino chromatographic column of 4.6 * 250mm, 5 ⁇ m, take the acetonitrile aqueous solution of 65% (v/v) as flow equal degree elution; Flow velocity is 0.5mL/min , the column temperature is 25° C., the injection volume is 10 ⁇ L, and the ultraviolet detector has a detection wavelength of 195 nm.
  • the content of lactose-N-tetrasaccharide in the dairy product test solution is calculated to be 0.123mg, and the recovery rate is 102.5%; the content of lactose-N-triose is 0.118mg , the recovery rate was 98.3%.
  • Example 6 Qualitative and quantitative detection of human milk oligosaccharides in yogurt samples, the steps are as follows:
  • Pretreatment Take 1mL yogurt sample (excluding human milk oligosaccharides), add 1mL lactose-N-triose standard solution to it, add 5ml ultrapure water and mix well, and treat it in a water bath at 100°C for 20min.
  • the conditions of high-performance liquid chromatography are: adopt 4.6 ⁇ 250mm, 5 ⁇ m Cosmosil Sugar-D amino chromatographic column, use 60% acetonitrile solution as mobile equipotential elution; flow rate is 0.5mL/min, column temperature is 30°C , with an injection volume of 10 ⁇ L, a differential refractive index detector, and a detection wavelength of 195 nm.
  • the peak at 14.477min was lactose-N-triose.
  • the content of lactose-N-triose in the yogurt sample was calculated to be 0.576mg, and the recovery rate was 96%.
  • Embodiment 7 Qualitative and quantitative detection of human milk oligosaccharides in milk-containing beverages, the steps are as follows:
  • Pretreatment Take 2mL milk beverage sample (excluding human milk oligosaccharides), add 1mL lactose-N-triose standard solution to it, add 2ml ultrapure water to mix, and treat in 100°C water bath for 20min After cooling down to room temperature, centrifuge at 12000 ⁇ g for 10 min at 4°C, and collect the supernatant as the test solution;
  • the conditions of high performance liquid chromatography are as follows: 4.6 ⁇ 250 mm, 5 ⁇ m Asahipak NH2P-504E amino chromatographic column is used, and 75% acetonitrile solution is used as the mobile equipotential elution; the flow rate is 0.7 mL/min, and the column temperature is 30 ° C.
  • the injection volume is 10 ⁇ L, and the detection wavelength is 195 nm with a differential refractive index detector.
  • the peak at 14.477min was lactose-N-triose. According to its linear equation, the content of lactose-N-triose in the yogurt sample was calculated to be 0.583mg, and the recovery rate was 97.17%.
  • Comparative example 1 The sample is pretreated by the method of patent CN2017201710308499.6
  • the pretreatment method adopts centrifugation to remove lipids and acetonitrile precipitation to remove proteins.
  • the steps are as follows:
  • Pretreatment Take 200 ⁇ L yogurt sample (excluding human milk oligosaccharides), add 200 ⁇ L lactose-N-tetrasaccharide standard solution and 200 ⁇ L lactose-N-triose standard solution to the yogurt sample, add an equal volume of ultrapure water Mix well, centrifuge at 5000r/min, 2°C for 8min, remove the upper layer of fat, then add 400 ⁇ L of pure acetonitrile, vortex and mix well, then sonicate for 8min, centrifuge at 8000r/min, 2°C for 12min, take the supernatant, and then Centrifuge, centrifuge the supernatant at 2°C at a speed of 8000r/min for 12min, collect the final supernatant, and obtain a dairy product test solution from which proteins and lipids have been removed;
  • HPLC detection was carried out according to the method described in Example 4.
  • the high-performance liquid chromatogram of yoghurt sample is as shown in Figure 4, and in conjunction with standard curve, calculates that the content of lactose-N-tetrasaccharide in described dairy product test solution is 0.057mg, and recovery rate is 47.5%; Lactose-N-tetrasaccharide Sugars did not elute and were not detected.
  • Comparative example 2 The sample is pretreated by the method of patent CN201610556457.X
  • Pretreatment Take 200 ⁇ L yogurt sample (excluding human milk oligosaccharides), add 200 ⁇ L lactose-N-tetrasaccharide standard solution and 200 ⁇ L lactose-N-triose standard solution to the yogurt sample, add an equal volume of ultrapure water Mix well, remove the upper layer of fat by centrifugation at 2000g, 20min, 2°C, take the lower layer sample solution and add twice the volume of 66.7% ethanol, freeze at -80°C for 10min, take it out and centrifuge, the centrifugation condition is 10000r/min, 10min, take the upper layer after centrifugation Clear liquid, obtains the dairy product that removes protein and lipid for test solution;
  • HPLC detection was carried out according to the method described in Example 4.
  • Pretreatment Take 200 ⁇ L yogurt sample (excluding human milk oligosaccharides), add 200 ⁇ L lactose-N-tetrasaccharide standard solution and 200 ⁇ L lactose-N-triose standard solution to the yogurt sample, add an equal volume of ultrapure water Mix well, add an equal volume of isopropanol, shake and mix well, centrifuge at 5000 ⁇ g for 5 min at 4°C, collect the supernatant, and obtain the dairy product test solution;
  • HPLC detection was carried out according to the method described in Example 5.
  • the HPLC chromatogram of the yogurt sample is shown in Figure 6, combined with the standard curve, the calculated content of lactose-N-tetrasaccharide in the dairy product test solution is 0.076mg, and the recovery rate is 63.33%; lactose-N-tetrasaccharide The sugar content was 0.057 mg, and the recovery rate was 47.5%.
  • Pretreatment Take 200 ⁇ L yogurt sample (excluding human milk oligosaccharides), add 200 ⁇ L lactose-N-tetrasaccharide standard solution and 200 ⁇ L lactose-N-triose standard solution to the yogurt sample, add an equal volume of ultrapure water Mix evenly, treat in a water bath at 80°C for 10 minutes, cool down to room temperature, centrifuge at 5000 ⁇ g for 5 minutes at 4°C, collect the supernatant, and obtain the test solution for dairy products;
  • HPLC detection was carried out according to the method described in Example 5.
  • the high-performance liquid chromatogram of the yogurt sample is shown in Figure 7, combined with the standard curve, the content of lactose-N-tetrasaccharide in the dairy product for the test solution is calculated to be 0.080mg, and the recovery rate is 66.67%; lactose-N-tetrasaccharide The sugar content was 0.064 mg, and the recovery rate was 53.33%.
  • the qualitative and quantitative detection method of the present invention can effectively detect lactose-N-tetrasaccharide or lactose-N-triose in dairy products, or simultaneously detect lactose-N-tetraose and lactose-N-triose in dairy products Sugar, the detection process is fast and accurate.
  • Comparative example 1 and comparative example 2 respectively adopt the method of patent CN2017201710308499.6 and CN201610556457.X to carry out qualitative and quantitative detection of human milk oligosaccharides in yogurt samples. It may be because yogurt products are used in the present invention. As far as breast milk is concerned, the content of protein, lipid, etc. is more, and only simple centrifugation pretreatment to remove impurities will cause too much interference and affect the detection effect.
  • the detection method of the present invention has the characteristics of accurate results and high sensitivity for the qualitative and quantitative detection of lactose-N-tetrasaccharide and/or lactose-N-triose in dairy products.
  • LC/MS LC/MS, it greatly reduces the detection cost and detection difficulty, and can realize the rapid qualitative and quantitative detection of human milk oligosaccharides in dairy samples, especially lactose-N-tetrasaccharide and lactose-N-triose.

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Abstract

A qualitative and quantitative detection method for human milk oligosaccharides in a dairy product. Proteins are removed by a heating method; if necessary, interference components or lipids are then further removed by using an organic solvent to obtain a dairy product test solution; the dairy product test solution is detected by using a high performance liquid chromatography method; human milk oligosaccharides in a dairy product are qualitatively and quantitatively analyzed according to a standard curve. The qualitative and quantitative detection method for human milk oligosaccharides in a dairy product can accurately and quickly detect human milk oligosaccharides in a dairy product.

Description

一种乳制品中人乳寡糖的定性定量检测方法A qualitative and quantitative detection method for human milk oligosaccharides in dairy products 技术领域technical field
本发明属于乳制品中人乳寡糖的检测领域，涉及乳制品中人乳寡糖的定性定量检测方法，具体涉及乳制品中乳糖-N-四糖和乳糖-N-三糖的定性定量检测方法。The invention belongs to the field of detection of human milk oligosaccharides in dairy products, and relates to a qualitative and quantitative detection method for human milk oligosaccharides in dairy products, in particular to the qualitative and quantitative detection of lactose-N-tetrasaccharide and lactose-N-trisaccharide in dairy products method.
背景技术Background technique
人乳寡糖又称母乳低聚糖(HMOs)，其被发现是一类存在于母乳中的独特低聚糖，母乳中未被消化的低聚糖部分刺激了结肠中双歧杆菌的生长，并且这些双歧杆菌对保护、预防肠道感染有益，因此，母乳低聚糖是先天免疫系统的主要成分。人乳寡糖含量的差别是导致母乳喂养较奶粉喂养更有优势的重要原因之一。其中，乳糖-N-四糖(Lacto-N-tetraose，LNT)和乳糖-N-三糖(Lacto-N-tetriaose II，LNTII)是母乳低聚糖(HMOs)中的主要成分，具有多种生理功能，可以减少感染的发生，促进肠道有益菌群的增殖和活性，间接抑制有害菌群生长，维持肠道微生态平衡，从而保护婴儿肠道免受致病菌侵袭。Human milk oligosaccharides, also known as human milk oligosaccharides (HMOs), were found to be a unique class of oligosaccharides present in breast milk, and the undigested oligosaccharides in breast milk partially stimulated the growth of bifidobacteria in the colon, And these bifidobacteria are beneficial to the protection and prevention of intestinal infection, therefore, breast milk oligosaccharides are the main components of the innate immune system. The difference in human milk oligosaccharide content is one of the important reasons why breastfeeding is more advantageous than milk formula feeding. Among them, lactose-N-tetraose (LNT) and lactose-N-triose (Lacto-N-tetriaose II, LNTII) are the main components in human milk oligosaccharides (HMOs), which have various Physiological functions can reduce the occurrence of infection, promote the proliferation and activity of beneficial intestinal flora, indirectly inhibit the growth of harmful flora, maintain the balance of intestinal micro-ecology, and thus protect the intestinal tract of infants from the invasion of pathogenic bacteria.
值得注意的是，在山羊奶、绵羊奶以及牛奶等乳制品中几乎没有发现这些人乳寡糖，并且。目前，市售乳制品并不含有人乳寡糖，婴儿配方奶粉中添加的大都是低聚乳糖(Galactooligosaccharides，GOS)和低聚果糖(fructooligosaccharide，FOS)，以此来模拟人乳寡糖的部分功能。然而，低聚乳糖和低聚果糖对婴儿的健康生长作用远不及人乳寡糖。所以，向人造奶或乳制品中添加具有生理功能的人乳寡糖，具有明显的社会和经济效益。It is worth noting that these human milk oligosaccharides are hardly found in dairy products such as goat's milk, sheep's milk and cow's milk, and. At present, commercially available dairy products do not contain human milk oligosaccharides, and most infant formulas are added with oligosaccharides (Galactooligosaccharides, GOS) and fructooligosaccharides (FOS), so as to simulate the part of human milk oligosaccharides Features. However, lacto-oligosaccharides and fructo-oligosaccharides are far less effective on the healthy growth of infants than human milk oligosaccharides. Therefore, adding human milk oligosaccharides with physiological functions to artificial milk or dairy products has obvious social and economic benefits.
人乳寡糖的种类超过200多种，结构复杂，不同种类人乳寡糖之间的检测方法差异较大。目前，人乳寡糖的检测方法耗时较长，操作复杂，如中国专利文献CN111239313A(申请号CN201811441066.9)公开了一种人乳寡糖的快速轮廓分析方法，该发明样品经过低温离心脱脂，加入有机溶剂沉淀除蛋白后，经亲水作用色谱柱分离，以质谱为检测器，得到人乳寡糖组成、结构和含量信息。但是此方法存在着分析仪器昂贵、分析工序耗时不低于3小时、对操作人员要求高等问题。There are more than 200 types of human milk oligosaccharides with complex structures, and the detection methods of different types of human milk oligosaccharides are quite different. At present, the detection method of human milk oligosaccharides is time-consuming and complicated to operate. For example, Chinese patent document CN111239313A (application number CN201811441066.9) discloses a rapid profile analysis method of human milk oligosaccharides. The samples of this invention are degreased by low temperature centrifugation , adding an organic solvent to precipitate and remove protein, then separated by a hydrophilic interaction chromatographic column, and using mass spectrometry as a detector to obtain information on the composition, structure and content of human milk oligosaccharides. However, this method has problems such as expensive analytical instruments, time-consuming analysis process of not less than 3 hours, and high requirements for operators.
乳制品中人乳寡糖含量的测定尚没有成熟的分析方法，天然寡糖对人乳寡糖的检测也存在一定的干扰。中国专利文献CN107192771A(申请号CN201710308499.6)公开了一种母乳低聚糖快速定性定量的方法，主要包括以下步骤：步骤1、样品前处理：取150-250μL母乳，去除脂肪和蛋白质，得最终上清液，加超纯水稀释，得上样样品；步骤2、对标准品采用超高效液相色谱、质谱，建立标准曲线；步骤3、采用超高效液相色谱将所述上样样品内母乳低聚糖进行不同组分的分离，并利用质谱结合标准曲线，进行定量分析，即得母乳低聚糖含量。上述发明在94～106min范围内可检测12种母乳低聚糖，并对12种母乳低聚糖进行了定量。There is no mature analytical method for the determination of human milk oligosaccharide content in dairy products, and natural oligosaccharides also interfere to a certain extent with the detection of human milk oligosaccharides. Chinese patent document CN107192771A (application number CN201710308499.6) discloses a method for rapid qualitative and quantitative breast milk oligosaccharides, which mainly includes the following steps: Step 1, sample pretreatment: take 150-250 μ L of breast milk, remove fat and protein, and obtain the final The supernatant is diluted with ultrapure water to obtain a loading sample; step 2, adopt ultra-high performance liquid chromatography and mass spectrometry to the standard, and establish a standard curve; The different components of breast milk oligosaccharides are separated, and the mass spectrometry combined with the standard curve is used for quantitative analysis to obtain the content of breast milk oligosaccharides. The above invention can detect 12 kinds of breast milk oligosaccharides in the range of 94-106 minutes, and quantify the 12 kinds of breast milk oligosaccharides.
中国专利文献CN107621399A(申请号CN201610556457.X)公开了一种检测母乳中低聚糖的方法，包括对母乳样品进行前处理的步骤，所述前处理具体为：取母乳样品，离心除去上层脂肪，取下层液体并加入醇类溶剂，于﹣40℃～﹣100℃冷冻5-15min后，离心，取上清液即得；采用液质法对前处理后的母乳中的低聚糖进行定性和定量检测。检测时间为 70～145min。Chinese patent document CN107621399A (application number CN201610556457.X) discloses a method for detecting oligosaccharides in breast milk, which includes the step of pre-processing the breast milk sample. The pre-treatment specifically includes: taking a breast milk sample, centrifuging to remove the upper layer of fat, Take the lower layer liquid and add alcohol solvent, freeze at -40°C ~ -100°C for 5-15min, centrifuge, and take the supernatant; Quantitative detection. The detection time is 70-145 minutes.
综上，现有技术中，对母乳及动物乳的前处理方法，脱除蛋白质的方法主要有两种，分别是乙醇沉淀法和乙腈沉淀法，脱除脂质的方法主要为离心沉淀法，上述前处理方法对于乳制品中人乳寡糖的检测效果不佳，不能快速准确地对乳制品中的人乳寡糖进行定性定量分析，相对于乳制品中的天然寡糖，乳制品中添加的人乳寡糖含量相对较少，检测其含量时，需避免天然寡糖的干扰，以提高检测准确率。对于人乳及动物乳的后处理检测方法，多采用液质联用法，但是该检测方法存在分析仪器昂贵、分析耗时长、对操作人员要求高等问题。到目前为止还没有报道快速地对乳制品中人乳寡糖进行定性定量的检测方法。To sum up, in the prior art, there are mainly two methods for removing protein in the pretreatment method of breast milk and animal milk, namely ethanol precipitation method and acetonitrile precipitation method, and the method for removing lipid is mainly centrifugal precipitation method. The above pretreatment methods are not effective for the detection of human milk oligosaccharides in dairy products, and cannot quickly and accurately perform qualitative and quantitative analysis of human milk oligosaccharides in dairy products. Compared with natural oligosaccharides in dairy products, the addition of The content of human milk oligosaccharides is relatively small. When detecting its content, it is necessary to avoid the interference of natural oligosaccharides to improve the detection accuracy. For the post-processing detection method of human milk and animal milk, liquid chromatography-mass spectrometry is mostly used, but this detection method has problems such as expensive analytical instruments, long analysis time, and high requirements for operators. So far, no rapid qualitative and quantitative detection method for human milk oligosaccharides in dairy products has been reported.
发明内容Contents of the invention
发明目的：针对现有技术的不足，本发明提供了一种乳制品中人乳寡糖的定性定量检测方法，为乳制品的质量控制提供准确、快速的检测方法。Purpose of the invention: Aiming at the deficiencies of the prior art, the present invention provides a qualitative and quantitative detection method for human milk oligosaccharides in dairy products, providing an accurate and rapid detection method for the quality control of dairy products.
术语说明：室温：具有本技术领域公知的含义，一般是指25±2℃。Explanation of terms: Room temperature: It has a well-known meaning in the technical field, and generally refers to 25±2°C.
本发明的技术方案：Technical scheme of the present invention:
一种乳制品中人乳寡糖检测的预处理方法，首先通过加热法去除乳制品中的蛋白质，对于含有多重复杂成分的乳制品，可以再用与水互溶的有机溶剂进一步除蛋白，或再利用有机溶剂提取法去除脂质，得乳制品供试液，具体如下：A pretreatment method for the detection of human milk oligosaccharides in dairy products. First, the protein in the dairy product is removed by heating. For dairy products containing multiple complex components, the protein can be further removed with a water-miscible organic solvent, or the protein can be removed again. Utilize organic solvent extraction method to remove lipid, obtain dairy product for test solution, specifically as follows:
一种乳制品中人乳寡糖检测的预处理方法，包括：步骤A.取乳制品样品，加入1-10倍体积的超纯水混匀，加热至80-100℃热处理10-30min后，降至室温，在0-10℃范围内，2000-20000×g离心5-50min，收集清液作为供试液。A pretreatment method for detecting human milk oligosaccharides in dairy products, comprising: Step A. Take a sample of dairy products, add 1-10 times the volume of ultrapure water to mix, heat to 80-100 ° C for 10-30 minutes, Cool down to room temperature, centrifuge at 2000-20000×g for 5-50min in the range of 0-10°C, and collect the supernatant as the test solution.
优选的，步骤A加入超纯水后，加热至85-100℃热处理20-30min；更优选加热至90-100℃。Preferably, after adding ultrapure water in step A, heat treatment at 85-100°C for 20-30 minutes; more preferably heat at 90-100°C.
优选的，步骤A离心条件为0-5℃范围内，5000-15000×g离心5-30min；更优选为4℃，10000-15000×g离心10-20min。Preferably, the centrifugation condition in step A is within the range of 0-5°C, 5000-15000×g for 5-30 minutes; more preferably 4°C, 10000-15000×g for 10-20 minutes.
优选的，如果乳制品成分复杂，本发明所述乳制品中人乳寡糖检测的预处理方法，还包括：步骤B.向步骤A所述供试液中，加入有机溶剂，振荡或涡旋混匀，于0-10℃范围内离心，2000-20000×g离心5-50min除去沉淀，收集清液作为供试液，所述有机溶剂与水互溶，选自甲醇、乙醇、丙醇、异丙醇、乙腈中的一种或多种；优选的，所述有机溶剂的加入量为步骤A超纯水体积的0.5-1倍。Preferably, if the ingredients of dairy products are complex, the pretreatment method for detecting human milk oligosaccharides in dairy products of the present invention further includes: step B. adding an organic solvent to the test solution described in step A, shaking or vortexing Mix well, centrifuge in the range of 0-10°C, centrifuge at 2000-20000×g for 5-50min to remove the precipitate, collect the supernatant as the test solution, the organic solvent is miscible with water, selected from methanol, ethanol, propanol, iso One or more of propanol and acetonitrile; preferably, the amount of the organic solvent added is 0.5-1 times the volume of the ultrapure water in step A.
优选的，所述离心条件为0-5℃，5000-15000×g离心5-30min；更优选为4℃，10000-15000×g离心10-20min。Preferably, the centrifugation condition is 0-5°C, 5000-15000×g for 5-30 minutes; more preferably 4°C, 10000-15000×g for 10-20 minutes.
优选的，如果乳制品成分复杂，本发明所述乳制品中人乳寡糖检测的预处理方法，还包括：步骤C.向步骤A或步骤B所得供试液中，加入有机溶剂，振荡或涡旋混匀，于0-10℃范围内离心，2000-20000×g离心5-50min，收集水相作为供试液；所述有机溶剂选自丁醇、二氯甲烷、氯仿或乙酸乙酯中的一种或多种；优选的，所述有机溶剂的加入量为步骤A超纯水体积的0.7-1.5倍。Preferably, if the ingredients of dairy products are complex, the pretreatment method for detecting human milk oligosaccharides in dairy products of the present invention further includes: step C. adding an organic solvent to the test solution obtained in step A or step B, shaking or Vortex and mix well, centrifuge in the range of 0-10°C, centrifuge at 2000-20000×g for 5-50min, collect the aqueous phase as the test solution; the organic solvent is selected from butanol, methylene chloride, chloroform or ethyl acetate One or more of them; preferably, the amount of the organic solvent added is 0.7-1.5 times the volume of the ultrapure water in step A.
本发明所述人乳寡糖包括但不限于乳糖-N-四糖和乳糖-N-三糖。The human milk oligosaccharides of the present invention include but not limited to lactose-N-tetraose and lactose-N-triose.
一种乳制品中人乳寡糖的定性和/或定量检测方法，包括上述预处理方法，还包括：A qualitative and/or quantitative detection method for human milk oligosaccharides in dairy products, including the above pretreatment method, and further comprising:
步骤D.通过高效液相色谱法对供试液中的人乳寡糖进行定性和/或定量分析，即对步骤C所得供试液进行定性和/或定量分析。Step D. Qualitative and/or quantitative analysis of the human milk oligosaccharides in the test solution by high performance liquid chromatography, that is, qualitative and/or quantitative analysis of the test solution obtained in step C.
优选的，所述高效液相色谱法，色谱柱选自亲水性色谱柱或疏水性色谱柱。Preferably, in the high performance liquid chromatography, the chromatographic column is selected from a hydrophilic chromatographic column or a hydrophobic chromatographic column.
进一步优选的，所述亲水性色谱柱填料为硅胶、极性基团键合相硅胶或葡聚糖；所述极性基团键合相硅胶中的极性基团为酰胺基、脲基、氰基、氨基、羧基、糖基和两性离子中的一种或多种；Further preferably, the hydrophilic chromatographic column filler is silica gel, polar group bonded phase silica gel or dextran; the polar group in the polar group bonded phase silica gel is amide group, urea group One or more of cyano, amino, carboxyl, sugar and zwitterions;
进一步优选的，所述色谱柱填料为氨丙基键合硅胶。Further preferably, the chromatographic column filler is aminopropyl bonded silica gel.
优选的，所述高效液相色谱法，色谱柱为氨丙基键合硅胶色谱柱，简称氨丙基色谱柱或者氨基色谱柱，选自Nacalai公司的Cosmosil Sugar-D/或Shodex公司的AsahipakNH2P-504E。Preferably, in the high performance liquid chromatography, the chromatographic column is an aminopropyl bonded silica gel chromatographic column, referred to as an aminopropyl chromatographic column or an amino chromatographic column, selected from Cosmosil Sugar-D/ of Nacalai Company or AsahipakNH2P- of Shodex Company 504E.
优选的，所述高效液相色谱法采用的检测器为示差折光检测器、紫外检测器或二极管阵列检测器；优选紫外检测器或二极管阵列检测器，检测波长为180-280nm。Preferably, the detector used in the high performance liquid chromatography is a differential refractive index detector, an ultraviolet detector or a diode array detector; preferably an ultraviolet detector or a diode array detector, and the detection wavelength is 180-280nm.
优选的，所述高效液相色谱法的流动相为水和有机溶剂，其体积比为10/90～90/10，使用等度洗脱或梯度洗脱；所述有机溶剂选自甲醇、乙醇、丙醇、异丙醇、丁醇、乙腈中的一种或多种。Preferably, the mobile phase of the high-performance liquid chromatography is water and an organic solvent, and its volume ratio is 10/90 to 90/10, using isocratic elution or gradient elution; the organic solvent is selected from methanol, ethanol , propanol, isopropanol, butanol, acetonitrile in one or more.
进一步优选的，所述流动相为60-75％(v/v)的乙腈水溶液；优选65-70％(v/v)的乙腈水溶液。Further preferably, the mobile phase is 60-75% (v/v) acetonitrile aqueous solution; preferably 65-70% (v/v) acetonitrile aqueous solution.
优选的，所述高效液相色谱法的流速为0.2-1mL/min，柱温为20-60℃，优选的，流动相流速为0.4-0.8mL/min。Preferably, the flow rate of the high performance liquid chromatography is 0.2-1 mL/min, the column temperature is 20-60° C., preferably, the flow rate of the mobile phase is 0.4-0.8 mL/min.
进样体积以10μL为宜，柱温为20-60℃，色谱柱内径为2.0-6.0mm。The injection volume is preferably 10 μL, the column temperature is 20-60°C, and the inner diameter of the chromatographic column is 2.0-6.0 mm.
优选的，本发明所述人乳寡糖为乳糖-N-四糖、乳糖-N-三糖中的一种。Preferably, the human milk oligosaccharide of the present invention is one of lactose-N-tetraose and lactose-N-triose.
一种乳制品中乳糖-N-四糖或乳糖-N-三糖的定性或定量检测方法，所述方法为高效液相色谱法；包括上述预处理方法，还包括如下步骤：A qualitative or quantitative detection method of lactose-N-tetrasaccharide or lactose-N-triose in dairy products, the method is high performance liquid chromatography; including the above pretreatment method, also includes the following steps:
包括建立乳糖-N-四糖或乳糖-N-三糖标准曲线，并检测步骤C所得供试液，对供试液中的乳糖-N-四糖或乳糖-N-三糖进行定性或定量分析的步骤。Including establishing a lactose-N-tetrasaccharide or lactose-N-triose standard curve, and detecting the test solution obtained in step C, and performing qualitative or quantitative analysis of the lactose-N-tetraose or lactose-N-triose in the test solution Analysis steps.
根据本发明优选的，所述高效液相色谱法采用的检测器为示差折光检测器、紫外检测器或二极管阵列检测器；进一步优选为紫外检测器或二极管阵列检测器；检测波长为180-280nm。Preferably according to the present invention, the detector used in the high performance liquid chromatography is a differential refractive index detector, an ultraviolet detector or a diode array detector; more preferably an ultraviolet detector or a diode array detector; the detection wavelength is 180-280nm .
本发明所述高效液相色谱法的流动相为60-75％(v/v)的乙腈水溶液，使用等度洗脱或梯度洗脱。The mobile phase of the high-performance liquid chromatography in the present invention is 60-75% (v/v) acetonitrile aqueous solution, using isocratic elution or gradient elution.
本发明技术方案中，如果流动相乙腈水溶液浓度低于60％(v/v)或高于75％(v/v)，都会导致色谱峰干扰问题，不能正常检测乳制品中的人乳寡糖含量。In the technical solution of the present invention, if the concentration of the acetonitrile aqueous solution in the mobile phase is lower than 60% (v/v) or higher than 75% (v/v), the problem of chromatographic peak interference will be caused, and human milk oligosaccharides in dairy products cannot be detected normally. content.
有益效果：Beneficial effect:
本发明提供了一种乳制品中所含有的人乳寡糖的定性和/或定量检测方法。本发明以加热法除蛋白等为主，如果成分复杂，可以选择步骤B和/或步骤C处理乳制品，本发明所述方法能够有效去除乳制品样品中的蛋白质、脂质等，以及其他成分，相对于常用的离心除脂质和 乙醇/乙腈沉淀除蛋白的预处理方法，大大减少了干扰峰的存在，保证了检测结果的准确度和可靠性，并通过高效液相色谱法检测乳制品中人乳寡糖的含量。The invention provides a qualitative and/or quantitative detection method of human milk oligosaccharides contained in dairy products. The present invention mainly uses the heating method to remove protein, etc. If the ingredients are complex, step B and/or step C can be selected to process dairy products. The method of the present invention can effectively remove protein, lipid, etc., and other components in dairy product samples Compared with the commonly used pretreatment methods of centrifugation to remove lipids and ethanol/acetonitrile precipitation to remove proteins, the existence of interference peaks is greatly reduced, and the accuracy and reliability of the detection results are guaranteed. Dairy products are detected by high performance liquid chromatography content of human milk oligosaccharides.
本发明所述定性和/或定量检测方法采，用高效液相色谱对预处理液进行分离分析，所述高效液相色谱的优选方案采用Nacalai公司的Cosmosil Sugar-D和/或Shodex公司的Asahipak NH2P-504E，有利于人乳寡糖的分离与检测，所述高效液相色谱系统检测速度快、灵敏度高，可在1h即可得到检测结果，大大缩短了分析时间，相较于液质联用，大大降低了检测成本与检测难度。The qualitative and/or quantitative detection method of the present invention adopts, and the pretreatment liquid is separated and analyzed by high performance liquid chromatography, and the preferred scheme of the high performance liquid chromatography adopts Cosmosil Sugar-D of Nacalai Company and/or Asahipak of Shodex Company NH2P-504E is beneficial to the separation and detection of human milk oligosaccharides. The high-performance liquid chromatography system has fast detection speed and high sensitivity, and the detection result can be obtained within 1 hour, which greatly shortens the analysis time. It greatly reduces the detection cost and detection difficulty.
本发明提供了一种乳制品中人乳寡糖的定性定量检测方法，该方法能够准确、快速的检测乳制品中的人乳寡糖。尤其是对乳糖-N-四糖和乳糖-N-三糖检测方法的提供，对乳制品的质量控制有着积极的意义，有利于人乳寡糖在乳制品行业中进行迅速的推广与运用。The invention provides a qualitative and quantitative detection method for human milk oligosaccharides in dairy products, which can accurately and rapidly detect human milk oligosaccharides in dairy products. In particular, the provision of detection methods for lactose-N-tetrasaccharide and lactose-N-triose has positive significance for the quality control of dairy products and is conducive to the rapid promotion and application of human milk oligosaccharides in the dairy industry.
附图说明Description of drawings
图1.为乳制品中人乳寡糖的定性定量检测分析流程示意图。Figure 1 is a schematic diagram of the qualitative and quantitative detection and analysis process of human milk oligosaccharides in dairy products.
图2.实施例1酸奶样品中乳糖-N-四糖测定的HPLC色谱图。Fig. 2. HPLC chromatogram of determination of lactose-N-tetrasaccharide in the yogurt sample of embodiment 1.
图3.实施例2全脂牛奶样品中乳糖-N-三糖测定的HPLC色谱图。Fig. 3. HPLC chromatogram of determination of lactose-N-triose in the whole milk sample of embodiment 2.
图4.对比例1酸奶样品中乳糖-N-三糖、乳糖-N-四糖含量测定的HPLC色谱图。Fig. 4. The HPLC chromatogram of the content determination of lactose-N-triose and lactose-N-tetrasaccharide in the yogurt sample of comparative example 1.
图5.对比例2酸奶样品中乳糖-N-三糖、乳糖-N-四糖含量测定的HPLC色谱图Figure 5. The HPLC chromatogram of the content determination of lactose-N-triose and lactose-N-tetrasaccharide in the yogurt sample of comparative example 2
图6.对比例3酸奶样品中乳糖-N-三糖、乳糖-N-四糖含量测定的HPLC色谱图。Fig. 6. The HPLC chromatogram of the content determination of lactose-N-triose and lactose-N-tetrasaccharide in the yogurt sample of comparative example 3.
图7.对比例4酸奶样品中乳糖-N-三糖、乳糖-N-四糖含量测定的HPLC色谱图。Fig. 7. The HPLC chromatogram of the content determination of lactose-N-triose and lactose-N-tetrasaccharide in the yogurt sample of comparative example 4.
具体实施方式detailed description
下面结合实施例和说明书附图对本发明的技术方案作进一步说明，但是本发明的保护范围并不仅限于此。实施例中涉及的样品及试剂，若无特殊说明，均为普通市售产品。The technical solution of the present invention will be further described below in conjunction with the embodiments and the accompanying drawings, but the protection scope of the present invention is not limited thereto. The samples and reagents involved in the examples are common commercially available products unless otherwise specified.
(1)标准品来源：(1) Source of standard product:
乳糖-N-四糖和乳糖-N-三糖标准品购买自上海甄准生物科技有限公司；其他市购标准品也可用于本发明。Lactose-N-tetrasaccharide and lactose-N-triose standard products were purchased from Shanghai Zhenzhun Biotechnology Co., Ltd.; other commercial standard products can also be used in the present invention.
(2)样品来源(2) Sample source
实施例中使用的酸奶、牛奶、奶粉均为普通市售且不含人乳寡糖的乳制品商品。Yogurt, milk, and milk powder used in the examples are all commercially available dairy products that do not contain human milk oligosaccharides.
(3)标准品梯度溶液制备：(3) Preparation of standard gradient solution:
取乳糖-N-四糖标准品0.1mg，溶于100μL的超纯水中，混匀后得1mg/mL的初级标准品溶液，放置-20℃下储藏备用；取初级标准品溶液10μL，加入90μL超纯水，得浓度为100mg/L标准品溶液，分别逐级稀释为20mg/L、40mg/L、60mg/L、80mg/L浓度，得到乳糖-N-四糖的标准品梯度溶液；Take 0.1 mg of the lactose-N-tetrasaccharide standard product, dissolve it in 100 μL of ultrapure water, mix well to obtain a 1 mg/mL primary standard solution, and store it at -20°C for later use; take 10 μL of the primary standard solution, add 90 μL of ultrapure water to obtain a standard solution with a concentration of 100 mg/L, which was diluted step by step to 20 mg/L, 40 mg/L, 60 mg/L, and 80 mg/L respectively to obtain a standard gradient solution of lactose-N-tetrasaccharide;
取乳糖-N-三糖标准品0.1mg，溶于100μL的超纯水中，混匀后得1mg/mL的初级标准品溶液，放置-20℃下储藏备用；取初级标准品溶液10μL，加入90μL超纯水，得浓度为100mg/L标准品溶液，分别逐级稀释为20mg/L、40mg/L、60mg/L、80mg/L浓度，得到乳糖-N-三糖的标准品梯度溶液。Take 0.1 mg of lactose-N-triose standard product, dissolve it in 100 μL of ultrapure water, mix well to obtain a 1 mg/mL primary standard solution, and store it at -20°C for later use; take 10 μL of the primary standard solution, add 90 μL of ultrapure water to obtain a standard solution with a concentration of 100 mg/L, which was diluted step by step to 20 mg/L, 40 mg/L, 60 mg/L, and 80 mg/L respectively to obtain a standard gradient solution of lactose-N-triose.
(4)上述标准品溶液经高效液相色谱分析后，建立的标准曲线如下：(4) After the above-mentioned standard solution was analyzed by high performance liquid chromatography, the standard curve established was as follows:
乳糖-N-四糖：二极管阵列检测器线性方程y＝4693150.00x-113708.75，R 2＝0.99，流动相65％(v/v)乙腈水溶液，柱温25℃，线性范围0.2-1mg/mL，出峰时间为第14.907min； Lactose-N-tetrasaccharide: Diode array detector linear equation y=4693150.00x-113708.75, R 2 =0.99, mobile phase 65% (v/v) acetonitrile aqueous solution, column temperature 25°C, linear range 0.2-1mg/mL, The peak time is 14.907 minutes;
紫外检测器线性方程y＝9676605.8571x-94905.4286，R2＝0.99，流动相65％(v/v)乙腈水溶液，柱温25℃，线性范围0.2-1mg/mL，出峰时间为第14.623min。The linear equation of the ultraviolet detector is y=9676605.8571x-94905.4286, R2=0.99, the mobile phase is 65% (v/v) acetonitrile aqueous solution, the column temperature is 25°C, the linear range is 0.2-1 mg/mL, and the peak time is 14.623 minutes.
乳糖-N-三糖：二极管阵列检测器线性方程y＝8868477x-250735，R 2＝0.96，流动相75％(v/v)乙腈水溶液，柱温30℃，线性范围0.2-1mg/mL，出峰时间为第13.327min； Lactose-N-triose: Diode array detector linear equation y=8868477x-250735, R 2 =0.96, mobile phase 75% (v/v) acetonitrile aqueous solution, column temperature 30°C, linear range 0.2-1mg/mL, output The peak time is 13.327 minutes;
二极管阵列检测器线性方程y＝37898702x-6499662.9，R 2＝0.98，流动相65％(v/v)乙腈水溶液，柱温25℃，线性范围0.2-1mg/mL，出峰时间为第12.240min； Diode array detector linear equation y=37898702x-6499662.9, R 2 =0.98, mobile phase 65% (v/v) acetonitrile aqueous solution, column temperature 25°C, linear range 0.2-1mg/mL, peak eluting time is 12.240min;
紫外检测器线性方程y＝37898702x-6499662.9，R 2＝0.98，流动相65％(v/v)乙腈水溶液，柱温25℃，线性范围0.2-1mg/mL，出峰时间为第12.573min； The linear equation of the ultraviolet detector is y=37898702x-6499662.9, R 2 =0.98, the mobile phase is 65% (v/v) acetonitrile aqueous solution, the column temperature is 25°C, the linear range is 0.2-1 mg/mL, and the peak eluting time is 12.573 minutes;
示差折光检测器线性方程y＝137467x+200，R 2＝1，流动相60％(v/v)乙腈水溶液，柱温30℃，线性范围0.2-1mg/mL，出峰时间为第14.477min。 The linear equation of the differential refractive index detector is y=137467x+200, R 2 =1, the mobile phase is 60% (v/v) acetonitrile aqueous solution, the column temperature is 30°C, the linear range is 0.2-1mg/mL, and the peak eluting time is 14.477min.
标准品溶液的高效液相色谱条件为：采用4.6×250mm，5μm的Cosmosil Sugar-D或AsahipakNH2P-504E氨基色谱柱，以60％-75％(v/v)的乙腈水溶液为流动相等度洗脱；流速为0.5mL/min，柱温为25℃或30℃，进样体积为10μL，二极管阵列检测器，或紫外检测器，或示差折光检测器，检测波长195nm。The high-performance liquid chromatography conditions of the standard solution are: adopt 4.6×250mm, 5 μm Cosmosil Sugar-D or AsahipakNH2P-504E amino chromatographic column, and use 60%-75% (v/v) acetonitrile aqueous solution as the mobile equipotential elution ; The flow rate is 0.5mL/min, the column temperature is 25°C or 30°C, the injection volume is 10μL, a diode array detector, or an ultraviolet detector, or a differential refractive index detector, and the detection wavelength is 195nm.
本发明实施例及对照例中所用乳糖-N-三糖标准品溶液的浓度为0.6mg/mL；乳糖-N-四糖标准品溶液的浓度为0.6mg/mL。分别取乳糖-N-三糖和乳糖-N-四糖标准品6mg，溶于10mL的超纯水中，混匀后得两种标准品溶液。The concentration of the lactose-N-triose standard solution used in the examples and comparative examples of the present invention is 0.6 mg/mL; the concentration of the lactose-N-tetrasaccharide standard solution is 0.6 mg/mL. Take 6 mg of standard lactose-N-triose and lactose-N-tetraose respectively, dissolve them in 10 mL of ultrapure water, and mix well to obtain two standard solutions.
实施例1.酸奶样品中人乳寡糖的定性定量检测，流程示意图如图1所示，步骤如下：Embodiment 1. Qualitative and quantitative detection of human milk oligosaccharides in yogurt samples, the flow diagram is shown in Figure 1, and the steps are as follows:
(1)预处理：(1) Preprocessing:
步骤A.取1mL酸奶样品(样品中不含人乳寡糖)，向其中加入1mL乳糖-N-四糖标准品溶液，加入1ml的超纯水混匀，于100℃水浴中处理20min后，降至室温，4℃条件下12000×g离心10min，收集上清液；Step A. Take 1mL yogurt sample (the sample does not contain human milk oligosaccharides), add 1mL lactose-N-tetrasaccharide standard solution to it, add 1ml ultrapure water and mix well, and treat it in a water bath at 100°C for 20min. Cool down to room temperature, centrifuge at 12000×g for 10 min at 4°C, and collect the supernatant;
步骤B.向步骤A所得上清液中，加入0.5ml异丙醇，振荡混匀，于4℃条件下12000×g离心10min进一步除蛋白，收集上清液；Step B. Add 0.5 ml of isopropanol to the supernatant obtained in step A, shake and mix, centrifuge at 12000×g for 10 min at 4°C to further remove protein, and collect the supernatant;
步骤C.向步骤B所得上清液中，加入1ml丁醇，振荡或涡旋混匀，于5℃范围内离心，10000×g离心25min，收集水相，得到除蛋白质和脂质的乳制品供试液；Step C. Add 1ml of butanol to the supernatant obtained in step B, vortex or vortex to mix, centrifuge at 5°C, 10000×g for 25min, collect the water phase, and obtain protein and lipid-free dairy products For test solution;
(2)HPLC检测：将预处理步骤得到的乳制品供试液稀释后，经0.22μm滤膜过滤，采用高效液相色谱法进行样品检测，根据标准曲线对酸奶样品中的乳糖-N-四糖进行定性定量分析；(2) HPLC detection: After diluting the dairy product test solution obtained in the pretreatment step, it was filtered through a 0.22 μm filter membrane, and the sample was detected by high performance liquid chromatography. Qualitative and quantitative analysis of sugar;
其中，高效液相色谱法的条件为：采用4.6×250mm，5μm的Cosmosil Sugar-D氨基色谱柱，以65％(v/v)的乙腈水溶液为流动相等度洗脱；流速为0.5mL/min，柱温为25℃，进样体积为10μL，二极管阵列检测器，检测波长195nm。Wherein, the condition of high-performance liquid chromatography is: adopt 4.6 * 250mm, the Cosmosil Sugar-D amino chromatographic column of 5 μm, take the acetonitrile aqueous solution of 65% (v/v) as flow equal degree elution; Flow velocity is 0.5mL/min , the column temperature is 25°C, the injection volume is 10 μL, the diode array detector, and the detection wavelength is 195 nm.
酸奶样品中乳糖-N-四糖含量检测的高效液相色谱图如图2所示，结合标准曲线，计算所述乳制品供试液中乳糖-N-四糖的含量为0.576mg，回收率96％。The high-performance liquid chromatogram of lactose-N-tetrasaccharide content detection in the yoghurt sample is as shown in Figure 2, in conjunction with standard curve, calculates the content of lactose-N-tetrasaccharide in the described dairy product test solution to be 0.576mg, the recovery rate 96%.
实施例2Example 2
全脂牛奶样品中人乳寡糖的定性定量检测，步骤如下：Qualitative and quantitative detection of human milk oligosaccharides in whole milk samples, the steps are as follows:
(1)预处理：(1) Preprocessing:
步骤A.取1mL全脂牛奶样品(不含人乳寡糖)，向牛奶样品中加1mL乳糖-N-三糖标准品溶液，加入3ml超纯水混匀，于90℃水浴中处理30min后，降至室温，4℃条件下10000×g离心20min，收集上清液；Step A. Take 1mL whole milk sample (excluding human milk oligosaccharides), add 1mL lactose-N-triose standard solution to the milk sample, add 3ml ultrapure water and mix well, and treat it in a water bath at 90°C for 30min , lowered to room temperature, centrifuged at 10,000×g for 20 min at 4°C, and collected the supernatant;
步骤C.向步骤A所得上清液中加入4.5ml的乙酸乙酯，振荡混匀，于4℃条件下15000×g离心10min，收集水相，得到去除蛋白质和脂质的乳制品供试液；Step C. Add 4.5ml of ethyl acetate to the supernatant obtained in step A, shake and mix well, centrifuge at 15000×g for 10min at 4°C, collect the water phase, and obtain a dairy product test solution with protein and lipid removed ;
(2)HPLC检测：将预处理步骤得到的乳制品供试液稀释后，经0.22μm滤膜过滤，采用高效液相色谱法进行样品检测，根据标准曲线对牛奶样品中的乳糖-N-三糖进行定性定量分析；(2) HPLC detection: After diluting the dairy product test solution obtained in the pretreatment step, it was filtered through a 0.22 μm filter membrane, and the sample was detected by high performance liquid chromatography. Qualitative and quantitative analysis of sugar;
其中，高效液相色谱法的条件为：采用4.6×250mm、5μm的Cosmosil Sugar-D氨基色谱柱，以70％(v/v)的乙腈水溶液为流动相等度洗脱；流速为0.5mL/min，柱温为25℃，进样体积为10μL，二极管阵列检测器，检测波长195nm。Wherein, the condition of high-performance liquid chromatography is: adopt the Cosmosil Sugar-D amino chromatographic column of 4.6 * 250mm, 5 μm, take the acetonitrile aqueous solution of 70% (v/v) as flow equal degree elution; Flow velocity is 0.5mL/min , the column temperature is 25°C, the injection volume is 10 μL, the diode array detector, and the detection wavelength is 195 nm.
全脂牛奶样品中乳糖-N-三糖含量检测的高效液相色谱图如图3所示，结合标准曲线，计算得到所述乳制品供试液中乳糖-N-三糖的含量为0.564mg，回收率94％。The high-performance liquid chromatogram of lactose-N-triose content detection in the whole milk sample is shown in Figure 3, combined with the standard curve, it is calculated that the content of lactose-N-triose in the dairy product test solution is 0.564mg , The recovery rate was 94%.
实施例3：全脂奶粉样品中人乳寡糖的定性定量检测，步骤如下：Example 3: Qualitative and quantitative detection of human milk oligosaccharides in whole milk powder samples, the steps are as follows:
方案1：plan 1:
(1)预处理：(1) Preprocessing:
步骤A.取1g奶粉样品(不含人乳寡糖)，向奶粉样品中加1mL乳糖-N-三糖标准品溶液，加入10ml超纯水混匀，于90℃水浴中处理30min后，降至室温，4℃条件下10000×g离心10min，收集上清液；Step A. Take 1g of milk powder sample (excluding human milk oligosaccharides), add 1mL of lactose-N-triose standard solution to the milk powder sample, add 10ml of ultrapure water and mix evenly, and treat it in a water bath at 90°C for 30min. Centrifuge at 10,000×g for 10 min at 4°C to room temperature, and collect the supernatant;
步骤B.向步骤A所得上清液中加入5ml乙腈，振荡混匀，于4℃条件下15000×g离心10min进一步除蛋白，收集上清液；Step B. Add 5 ml of acetonitrile to the supernatant obtained in step A, shake and mix well, centrifuge at 15000×g for 10 min at 4°C to further remove protein, and collect the supernatant;
步骤C.向步骤B所得上清液中加入10ml乙酸乙酯，振荡混匀，于4℃条件下15000×g离心10min，收集水相，得到去除蛋白质和脂质的乳制品供试液；Step C. Add 10 ml of ethyl acetate to the supernatant obtained in Step B, shake and mix, centrifuge at 15000×g for 10 min at 4°C, collect the aqueous phase, and obtain a dairy product test solution from which protein and lipid have been removed;
(2)HPLC检测：将预处理步骤得到的乳制品供试液稀释后，经0.22μm滤膜过滤，采用高效液相色谱法进行样品检测，根据标准曲线对奶粉样品中的乳糖-N-三糖进行定性定量分析；(2) HPLC detection: After diluting the dairy product test solution obtained in the pretreatment step, it was filtered through a 0.22 μm filter membrane, and the sample was detected by high performance liquid chromatography. Qualitative and quantitative analysis of sugar;
其中，高效液相色谱法的条件为：采用4.6×250mm、5μm的Cosmosil Sugar-D氨基色谱柱，以75％(v/v)的乙腈水溶液为流动相等度洗脱；流速为0.5mL/min，柱温为30℃，进样体积为10μL，二极管阵列检测器，检测波长195nm。Wherein, the condition of high-performance liquid chromatography is: adopt the Cosmosil Sugar-D amino chromatographic column of 4.6 * 250mm, 5 μm, take the acetonitrile aqueous solution of 75% (v/v) as flow equal degree elution; Flow velocity is 0.5mL/min , the column temperature is 30° C., the injection volume is 10 μL, a diode array detector, and the detection wavelength is 195 nm.
结合本奶粉样品高效液相图谱和标准曲线，计算所述品供试液中乳糖-N-三糖的含量为0.598mg，回收率99.67％。Combined with the HPLC chromatogram of the milk powder sample and the standard curve, the content of lactose-N-triose in the test solution of the product is calculated to be 0.598 mg, and the recovery rate is 99.67%.
方案2：Scenario 2:
没有预处理步骤B，其他操作相同，测得样品中乳糖-N-三糖的含量为0.567，回收率 94.5％。Without pretreatment step B, other operations were the same, the content of lactose-N-triose in the sample was measured to be 0.567, and the recovery rate was 94.5%.
方案3：没有预处理步骤B和C，其他操作相同，测得样品中乳糖-N-三糖的含量为0.542，回收率90.3％。Scheme 3: No pretreatment steps B and C, other operations are the same, the content of lactose-N-triose in the sample is measured to be 0.542, and the recovery rate is 90.3%.
实施例4：酸奶样品中人乳寡糖的定性定量检测，步骤如下：Example 4: Qualitative and quantitative detection of human milk oligosaccharides in yogurt samples, the steps are as follows:
方案1：plan 1:
(1)预处理：(1) Preprocessing:
步骤A.取200μL酸奶样品(不含人乳寡糖)，向酸奶样品中加入200μL乳糖-N-四糖标准品溶液、200μL乳糖-N-三糖标准品溶液，加入1ml超纯水混匀，于100℃水浴中处理30min后，降至室温，4℃条件下15000×g离心50min，收集上清液；Step A. Take 200 μL yogurt sample (excluding human milk oligosaccharides), add 200 μL lactose-N-tetrasaccharide standard solution and 200 μL lactose-N-triose standard solution to the yogurt sample, add 1ml ultrapure water and mix well , treated in a water bath at 100°C for 30 minutes, cooled to room temperature, centrifuged at 15,000×g for 50 minutes at 4°C, and collected the supernatant;
步骤B.向步骤A所得上清液中加入0.5ml异丙醇，振荡混匀，于4℃条件下15000×g离心50min，收集上清液；Step B. Add 0.5 ml of isopropanol to the supernatant obtained in step A, shake and mix, centrifuge at 15000×g for 50 min at 4°C, and collect the supernatant;
步骤C.向步骤B所得上清液中加入1ml二氯甲烷，振荡混匀，于4℃条件下15000×g离心10min，收集水相，得到去除蛋白质和脂质的乳制品供试液Step C. Add 1ml of dichloromethane to the supernatant obtained in step B, shake and mix well, centrifuge at 15000×g for 10min at 4°C, collect the water phase, and obtain a dairy product test solution with protein and lipid removed
(2)HPLC检测：将预处理步骤得到的乳制品供试液稀释后，经0.22μm滤膜过滤，采用高效液相色谱法进行样品检测，根据标准曲线对酸奶样品中的乳糖-N-四糖、乳糖-N-三糖进行定性定量分析；(2) HPLC detection: After diluting the dairy product test solution obtained in the pretreatment step, it was filtered through a 0.22 μm filter membrane, and the sample was detected by high performance liquid chromatography. Qualitative and quantitative analysis of sugar and lactose-N-trisaccharide;
其中，高效液相色谱法的条件为：采用4.6×250mm、5μm的Cosmosil Sugar-D氨基色谱柱，以65％(v/v)的乙腈水溶液为流动相等度洗脱；流速为0.5mL/min，柱温为25℃，进样体积为10μL，二极管阵列检测器，检测波长195nm。Wherein, the condition of high-performance liquid chromatography is: adopt the Cosmosil Sugar-D amino chromatographic column of 4.6 * 250mm, 5 μm, take the acetonitrile aqueous solution of 65% (v/v) as flow equal degree elution; Flow velocity is 0.5mL/min , the column temperature is 25°C, the injection volume is 10 μL, the diode array detector, and the detection wavelength is 195 nm.
结合本奶粉样品高效液相图谱(附图3所示)和标准曲线，计算所述乳制品供试液中乳糖-N-四糖的含量为0.120mg，回收率为100％；乳糖-N-三糖的含量为0.117mg，回收率为97.5％。In conjunction with this milk powder sample HPLC collection (shown in accompanying drawing 3) and standard curve, calculate the content of lactose-N-tetrasaccharide in the described milk product test solution to be 0.120mg, the rate of recovery is 100%; Lactose-N- The content of trisaccharide was 0.117 mg, and the recovery rate was 97.5%.
方案2：Scenario 2:
如没有步骤B，他操作相同，测得到所述酸奶样品中乳糖-N-四糖的含量为0.113mg，回收率为94.17；乳糖-N-三糖的含量为0.110mg，回收率为91.67％。If there is no step B, his operation is the same, and the content of lactose-N-tetrasaccharide in the yogurt sample measured is 0.113mg, and the recovery rate is 94.17; the content of lactose-N-triose sugar is 0.110mg, and the recovery rate is 91.67% .
实施例5：Example 5:
酸奶样品中人乳寡糖的定性定量检测，步骤如下：Qualitative and quantitative detection of human milk oligosaccharides in yogurt samples, the steps are as follows:
(1)预处理：(1) Preprocessing:
步骤A.取200μL酸奶样品(不含人乳寡糖)，向酸奶样品中加入200μL乳糖-N-四糖标准品溶液、200μL乳糖-N-三糖标准品溶液，加入1ml超纯水混匀，于80℃水浴中处理10min后，降至室温，4℃条件下5000×g离心5min，收集上清液；Step A. Take 200 μL yogurt sample (excluding human milk oligosaccharides), add 200 μL lactose-N-tetrasaccharide standard solution and 200 μL lactose-N-triose standard solution to the yogurt sample, add 1ml ultrapure water and mix well , treated in a water bath at 80°C for 10 minutes, cooled to room temperature, centrifuged at 5000×g for 5 minutes at 4°C, and collected the supernatant;
步骤B.向步骤A所得上清液中加入0.5ml的异丙醇，振荡混匀，于4℃条件下5000×g离心5min，收集上清液；Step B. Add 0.5 ml of isopropanol to the supernatant obtained in step A, shake and mix, centrifuge at 5000×g for 5 min at 4°C, and collect the supernatant;
步骤C.向步骤B所得上清液中加入1ml二氯甲烷，振荡混匀，于4℃条件下15000×g离心10min，收集水相，得到去除蛋白质和脂质的乳制品供试液；Step C. Add 1 ml of dichloromethane to the supernatant obtained in step B, shake and mix, centrifuge at 15000×g for 10 min at 4°C, collect the aqueous phase, and obtain a dairy product test solution from which protein and lipid have been removed;
(2)HPLC检测：将预处理步骤得到的乳制品供试液稀释后，经0.22μm滤膜过滤，采 用高效液相色谱法进行样品检测，根据标准曲线对酸奶样品中的乳糖-N-四糖、乳糖-N-三糖进行定性定量分析；(2) HPLC detection: After diluting the dairy product test solution obtained in the pretreatment step, it was filtered through a 0.22 μm filter membrane, and the sample was detected by high performance liquid chromatography. Qualitative and quantitative analysis of sugar and lactose-N-trisaccharide;
其中，高效液相色谱法的条件为：采用4.6×250mm、5μm的Cosmosil Sugar-D氨基色谱柱，以65％(v/v)的乙腈水溶液为流动相等度洗脱；流速为0.5mL/min，柱温为25℃，进样体积为10μL，紫外检测器，检测波长195nm。Wherein, the condition of high-performance liquid chromatography is: adopt the Cosmosil Sugar-D amino chromatographic column of 4.6 * 250mm, 5 μm, take the acetonitrile aqueous solution of 65% (v/v) as flow equal degree elution; Flow velocity is 0.5mL/min , the column temperature is 25° C., the injection volume is 10 μL, and the ultraviolet detector has a detection wavelength of 195 nm.
结合酸奶样品的高效液相色谱图和标准曲线，计算所述乳制品供试液中乳糖-N-四糖的含量为0.123mg，回收率102.5％；乳糖-N-三糖的含量为0.118mg，回收率98.3％。Combined with the high-performance liquid chromatogram and standard curve of the yogurt sample, the content of lactose-N-tetrasaccharide in the dairy product test solution is calculated to be 0.123mg, and the recovery rate is 102.5%; the content of lactose-N-triose is 0.118mg , the recovery rate was 98.3%.
实施例6：酸奶样品中人乳寡糖的定性定量检测，步骤如下：Example 6: Qualitative and quantitative detection of human milk oligosaccharides in yogurt samples, the steps are as follows:
(1)预处理：取1mL酸奶样品(不含人乳寡糖)，向其中加入1mL乳糖-N-三糖标准品溶液，加入5ml超纯水混匀，于100℃水浴中处理20min后，降至室温，4℃条件下12000×g离心10min，收集上清液；向上述上清液中加入2.5ml异丙醇，振荡混匀，于4℃条件下12000×g离心10min，收集上清液；向上清液中加入3.5ml氯仿，振荡混匀，于4℃条件下15000×g离心10min，收集水相，得到去除蛋白质和脂质的乳制品供试液；(1) Pretreatment: Take 1mL yogurt sample (excluding human milk oligosaccharides), add 1mL lactose-N-triose standard solution to it, add 5ml ultrapure water and mix well, and treat it in a water bath at 100°C for 20min. Cool down to room temperature, centrifuge at 12,000×g for 10 minutes at 4°C, collect the supernatant; add 2.5ml of isopropanol to the above supernatant, shake and mix, and centrifuge at 12,000×g for 10 minutes at 4°C, collect the supernatant solution; add 3.5ml chloroform to the supernatant, shake and mix well, centrifuge at 15,000×g for 10min at 4°C, collect the water phase, and obtain a dairy product test solution from which protein and lipid have been removed;
(2)HPLC检测：将预处理步骤得到的乳制品供试液稀释后，经0.22μm滤膜过滤，采用高效液相色谱法进行样品检测，根据标准曲线对所述乳制品供试液中的乳糖-N-三糖进行定性定量分析；(2) HPLC detection: After the dairy product test solution obtained in the pretreatment step is diluted, it is filtered through a 0.22 μm filter membrane, and the sample is detected by high performance liquid chromatography. Qualitative and quantitative analysis of lactose-N-triose;
其中，高效液相色谱法的条件为：采用4.6×250mm、5μm的Cosmosil Sugar-D氨基色谱柱，以60％乙腈溶液为流动相等度洗脱；流速为0.5mL/min，柱温为30℃，进样体积为10μL，示差折光检测器，检测波长195nm。Among them, the conditions of high-performance liquid chromatography are: adopt 4.6×250mm, 5μm Cosmosil Sugar-D amino chromatographic column, use 60% acetonitrile solution as mobile equipotential elution; flow rate is 0.5mL/min, column temperature is 30°C , with an injection volume of 10 μL, a differential refractive index detector, and a detection wavelength of 195 nm.
对照乳糖-N-三糖标准品酸奶样品的高效液相色谱图，在14.477min的出峰为乳糖-N-三糖。根据其线性方程计算得出酸奶样品中乳糖-N-三糖的含量为0.576mg，回收率96％。Compared with the high-performance liquid chromatogram of the lactose-N-triose standard yogurt sample, the peak at 14.477min was lactose-N-triose. According to its linear equation, the content of lactose-N-triose in the yogurt sample was calculated to be 0.576mg, and the recovery rate was 96%.
这说明当进行HPLC检测时，使用示差折光检测器也能比较灵敏地检测出乳糖-N-三糖。This shows that when performing HPLC detection, the use of a differential refractive index detector can also detect lactose-N-triose more sensitively.
实施例7.含奶饮料中人乳寡糖的定性定量检测，步骤如下：Embodiment 7. Qualitative and quantitative detection of human milk oligosaccharides in milk-containing beverages, the steps are as follows:
(1)预处理：取2mL含奶饮料样品(不含人乳寡糖)，向其中加入1mL乳糖-N-三糖标准品溶液，加入2ml超纯水混匀，于100℃水浴中处理20min后，降至室温，4℃条件下12000×g离心10min，收集上清液作为供试液；(1) Pretreatment: Take 2mL milk beverage sample (excluding human milk oligosaccharides), add 1mL lactose-N-triose standard solution to it, add 2ml ultrapure water to mix, and treat in 100℃ water bath for 20min After cooling down to room temperature, centrifuge at 12000×g for 10 min at 4°C, and collect the supernatant as the test solution;
(2)HPLC检测：供试液经0.22μm滤膜过滤，采用高效液相色谱法检测其中的乳糖-N-三糖含量。(2) HPLC detection: the test solution was filtered through a 0.22 μm filter membrane, and the content of lactose-N-triose in it was detected by high performance liquid chromatography.
其中，高效液相色谱法的条件为：采用4.6×250mm、5μm的AsahipakNH2P-504E氨基色谱柱，以75％乙腈溶液为流动相等度洗脱；流速为0.7mL/min，柱温为30℃，进样体积为10μL，示差折光检测器，检测波长195nm。在14.477min的出峰为乳糖-N-三糖。根据其线性方程计算得出酸奶样品中乳糖-N-三糖的含量为0.583mg，回收率97.17％。Among them, the conditions of high performance liquid chromatography are as follows: 4.6 × 250 mm, 5 μm Asahipak NH2P-504E amino chromatographic column is used, and 75% acetonitrile solution is used as the mobile equipotential elution; the flow rate is 0.7 mL/min, and the column temperature is 30 ° C. The injection volume is 10 μL, and the detection wavelength is 195 nm with a differential refractive index detector. The peak at 14.477min was lactose-N-triose. According to its linear equation, the content of lactose-N-triose in the yogurt sample was calculated to be 0.583mg, and the recovery rate was 97.17%.
对比例1：采用专利CN2017201710308499.6的方法对样品进行预处理Comparative example 1: The sample is pretreated by the method of patent CN2017201710308499.6
酸奶样品中人乳寡糖的定性定量检测，其中预处理方法采用离心除脂质和乙腈沉淀除蛋白的处理方式，步骤如下：Qualitative and quantitative detection of human milk oligosaccharides in yogurt samples. The pretreatment method adopts centrifugation to remove lipids and acetonitrile precipitation to remove proteins. The steps are as follows:
预处理：取200μL酸奶样品(不含人乳寡糖)，向酸奶样品中加入200μL乳糖-N-四糖 标准品溶液、200μL乳糖-N-三糖标准品溶液，加入等体积的超纯水混匀，5000r/min，2℃离心8min后，去除上层脂肪，然后加入400μL的纯乙腈，涡旋混匀后超声8min，8000r/min，2℃离心12min后取上清，再将上清液离心，以8000r/min的速度在2℃下，将所述上清液离心12min后，收集最终上清液，得到去除蛋白质和脂质的乳制品供试液；Pretreatment: Take 200 μL yogurt sample (excluding human milk oligosaccharides), add 200 μL lactose-N-tetrasaccharide standard solution and 200 μL lactose-N-triose standard solution to the yogurt sample, add an equal volume of ultrapure water Mix well, centrifuge at 5000r/min, 2°C for 8min, remove the upper layer of fat, then add 400μL of pure acetonitrile, vortex and mix well, then sonicate for 8min, centrifuge at 8000r/min, 2°C for 12min, take the supernatant, and then Centrifuge, centrifuge the supernatant at 2°C at a speed of 8000r/min for 12min, collect the final supernatant, and obtain a dairy product test solution from which proteins and lipids have been removed;
HPLC检测均按照实施例4所述的方法进行。HPLC detection was carried out according to the method described in Example 4.
酸奶样品的高效液相色谱图如图4所示，结合标准曲线，计算得到所述乳制品供试液中乳糖-N-四糖的含量为0.057mg，回收率47.5％；乳糖-N-三糖没有出峰，未被检测出。The high-performance liquid chromatogram of yoghurt sample is as shown in Figure 4, and in conjunction with standard curve, calculates that the content of lactose-N-tetrasaccharide in described dairy product test solution is 0.057mg, and recovery rate is 47.5%; Lactose-N-tetrasaccharide Sugars did not elute and were not detected.
对比例2：采用专利CN201610556457.X的方法对样品进行预处理Comparative example 2: The sample is pretreated by the method of patent CN201610556457.X
酸奶样品中人乳寡糖的定性定量检测，其中预处理方法采用离心除脂质和乙醇沉淀除蛋白的处理方式，步骤如下：Qualitative and quantitative detection of human milk oligosaccharides in yogurt samples, wherein the pretreatment method adopts centrifugation to remove lipids and ethanol precipitation to remove proteins. The steps are as follows:
预处理：取200μL酸奶样品(不含人乳寡糖)，向酸奶样品中加入200μL乳糖-N-四糖标准品溶液、200μL乳糖-N-三糖标准品溶液，加入等体积的超纯水混匀，经2000g、20min、2℃离心除去上层脂肪，取下层样液加入两倍体积66.7％乙醇，-80℃冷冻10min，取出后离心，离心条件为10000r/min、10min，离心后取上层清液，得到去除蛋白质和脂质的乳制品供试液；Pretreatment: Take 200 μL yogurt sample (excluding human milk oligosaccharides), add 200 μL lactose-N-tetrasaccharide standard solution and 200 μL lactose-N-triose standard solution to the yogurt sample, add an equal volume of ultrapure water Mix well, remove the upper layer of fat by centrifugation at 2000g, 20min, 2°C, take the lower layer sample solution and add twice the volume of 66.7% ethanol, freeze at -80°C for 10min, take it out and centrifuge, the centrifugation condition is 10000r/min, 10min, take the upper layer after centrifugation Clear liquid, obtains the dairy product that removes protein and lipid for test solution;
HPLC检测均按照实施例4所述的方法进行。HPLC detection was carried out according to the method described in Example 4.
酸奶样品的高效液相色谱图如图5所示，结合标准曲线，计算得到所述乳制品供试液中乳糖-N-四糖的含量为0.050mg，回收率41.67％；乳糖-N-三糖的含量为0.052mg，回收率43.33％。The high-performance liquid chromatogram of yoghurt sample is shown in Figure 5, combined with the standard curve, the content of lactose-N-tetrasaccharide in the said dairy product test solution is calculated to be 0.050mg, and the recovery rate is 41.67%; lactose-N-tetrasaccharide The sugar content was 0.052 mg, and the recovery rate was 43.33%.
对比例3：Comparative example 3:
酸奶样品中人乳寡糖的定性定量检测，其中预处理方法仅采用有机溶剂处理，步骤如下：Qualitative and quantitative detection of human milk oligosaccharides in yogurt samples, wherein the pretreatment method only uses organic solvents, and the steps are as follows:
预处理：取200μL酸奶样品(不含人乳寡糖)，向酸奶样品中加入200μL乳糖-N-四糖标准品溶液、200μL乳糖-N-三糖标准品溶液，加入等体积的超纯水混匀，加入等体积的异丙醇，振荡混匀，于4℃条件下5000×g离心5min，收集上清液，得到乳制品供试液；Pretreatment: Take 200 μL yogurt sample (excluding human milk oligosaccharides), add 200 μL lactose-N-tetrasaccharide standard solution and 200 μL lactose-N-triose standard solution to the yogurt sample, add an equal volume of ultrapure water Mix well, add an equal volume of isopropanol, shake and mix well, centrifuge at 5000×g for 5 min at 4°C, collect the supernatant, and obtain the dairy product test solution;
HPLC检测均按照实施例5所述的方法进行。HPLC detection was carried out according to the method described in Example 5.
酸奶样品的高效液相色谱图如图6所示，结合标准曲线，计算得到所述乳制品供试液中乳糖-N-四糖的含量为0.076mg，回收率63.33％；乳糖-N-三糖的含量为0.057mg，回收率47.5％。The HPLC chromatogram of the yogurt sample is shown in Figure 6, combined with the standard curve, the calculated content of lactose-N-tetrasaccharide in the dairy product test solution is 0.076mg, and the recovery rate is 63.33%; lactose-N-tetrasaccharide The sugar content was 0.057 mg, and the recovery rate was 47.5%.
对比例4：Comparative example 4:
酸奶样品中人乳寡糖的定性定量检测，其中预处理方法仅采用加热处理，步骤如下：Qualitative and quantitative detection of human milk oligosaccharides in yogurt samples, wherein the pretreatment method only uses heat treatment, and the steps are as follows:
预处理：取200μL酸奶样品(不含人乳寡糖)，向酸奶样品中加入200μL乳糖-N-四糖标准品溶液、200μL乳糖-N-三糖标准品溶液，加入等体积的超纯水混匀，于80℃水浴中处理10min后，降至室温，4℃条件下5000×g离心5min，收集上清液，得到乳制品供试液；Pretreatment: Take 200 μL yogurt sample (excluding human milk oligosaccharides), add 200 μL lactose-N-tetrasaccharide standard solution and 200 μL lactose-N-triose standard solution to the yogurt sample, add an equal volume of ultrapure water Mix evenly, treat in a water bath at 80°C for 10 minutes, cool down to room temperature, centrifuge at 5000×g for 5 minutes at 4°C, collect the supernatant, and obtain the test solution for dairy products;
HPLC检测均按照实施例5所述的方法进行。HPLC detection was carried out according to the method described in Example 5.
酸奶样品的高效液相色谱图如图7所示，结合标准曲线，计算得到所述乳制品供试液中乳糖-N-四糖的含量为0.080mg，回收率66.67％；乳糖-N-三糖含量为0.064mg，回收率53.33％。The high-performance liquid chromatogram of the yogurt sample is shown in Figure 7, combined with the standard curve, the content of lactose-N-tetrasaccharide in the dairy product for the test solution is calculated to be 0.080mg, and the recovery rate is 66.67%; lactose-N-tetrasaccharide The sugar content was 0.064 mg, and the recovery rate was 53.33%.
总之，本发明所述定性定量检测方法，可以有效检测乳制品中的乳糖-N-四糖或乳糖-N-三糖，或者同时检测乳制品中乳糖-N-四糖和乳糖-N-三糖，检测过程快速、准确。In a word, the qualitative and quantitative detection method of the present invention can effectively detect lactose-N-tetrasaccharide or lactose-N-triose in dairy products, or simultaneously detect lactose-N-tetraose and lactose-N-triose in dairy products Sugar, the detection process is fast and accurate.
对比例1和对比例2分别采用专利CN2017201710308499.6和CN201610556457.X的方法对酸奶样品中人乳寡糖进行定性定量检测，可能由于本发明中采用的是酸奶制品，相较于其发明专利中的母乳而言，蛋白质、脂质等的含量更多，只通过简单的离心预处理除杂，会导致干扰过多，影响检测效果。Comparative example 1 and comparative example 2 respectively adopt the method of patent CN2017201710308499.6 and CN201610556457.X to carry out qualitative and quantitative detection of human milk oligosaccharides in yogurt samples. It may be because yogurt products are used in the present invention. As far as breast milk is concerned, the content of protein, lipid, etc. is more, and only simple centrifugation pretreatment to remove impurities will cause too much interference and affect the detection effect.
综上，由上述检测分析结果可知，本发明的检测方法对于乳制品中乳糖-N-四糖和/或乳糖-N-三糖的定性定量检测检测具有结果准确、灵敏度高的特点，相较于液质联用法，大大降低了检测成本与检测难度，可实现对乳制品样品中人乳寡糖，尤其是乳糖-N-四糖和乳糖-N-三糖的快速定性定量检测。In summary, it can be seen from the above detection and analysis results that the detection method of the present invention has the characteristics of accurate results and high sensitivity for the qualitative and quantitative detection of lactose-N-tetrasaccharide and/or lactose-N-triose in dairy products. Using LC/MS, it greatly reduces the detection cost and detection difficulty, and can realize the rapid qualitative and quantitative detection of human milk oligosaccharides in dairy samples, especially lactose-N-tetrasaccharide and lactose-N-triose.

Claims (10)

  1. 一种乳制品中人乳寡糖检测的预处理方法，其特征在于，通过加热法后离心的方法得供试液，包括：A pretreatment method for detecting human milk oligosaccharides in dairy products, characterized in that the test solution is obtained by centrifuging after heating, including:
    步骤A.取乳制品样品，加入1-10倍体积的超纯水混匀，加热至80-100℃热处理10-30min后，降至室温，在0-10℃范围内，离心，收集清液作为供试液。Step A. Take a dairy product sample, add 1-10 times the volume of ultrapure water, mix well, heat to 80-100°C for 10-30min, then cool down to room temperature, centrifuge at 0-10°C, and collect the supernatant as a test solution.
  2. 如权利要求1所述乳制品中人乳寡糖检测的预处理方法，其特征在于，还包括步骤B：向步骤A所得供试液中，加入有机溶剂，振荡或涡旋混匀，于0-10℃范围内离心，收集清液作为供试液，所述有机溶剂与水互溶；优选的，所述有机溶剂选自甲醇、乙醇、丙醇、异丙醇、乙腈中的一种或多种；优选的，所述有机溶剂的加入量为步骤A超纯水体积的0.5-1倍。The pretreatment method for detecting human milk oligosaccharides in dairy products according to claim 1, further comprising step B: adding an organic solvent to the test solution obtained in step A, shaking or vortex mixing, and Centrifuge in the range of -10°C, collect the clear liquid as the test solution, and the organic solvent is miscible with water; preferably, the organic solvent is selected from one or more of methanol, ethanol, propanol, isopropanol, and acetonitrile species; preferably, the added amount of the organic solvent is 0.5-1 times the volume of the ultrapure water in step A.
  3. 如权利要求1或2所述乳制品中人乳寡糖检测的预处理方法，其特征在于，还包括步骤C.向所述供试液中，加入有机溶剂，振荡或涡旋混匀，于0-10℃范围内离心，收集水相作为供试液；所述有机溶剂选自丁醇、二氯甲烷、氯仿或乙酸乙酯中的一种或多种；优选的，所述有机溶剂的加入量为步骤A超纯水体积的0.7-1.5倍。The pretreatment method for detecting human milk oligosaccharides in dairy products according to claim 1 or 2, further comprising step C. adding an organic solvent to the test solution, shaking or vortex mixing, and then Centrifuge in the range of 0-10°C, collect the aqueous phase as the test solution; the organic solvent is selected from one or more of butanol, methylene chloride, chloroform or ethyl acetate; preferably, the organic solvent The amount added is 0.7-1.5 times the volume of the ultrapure water in step A.
  4. 如权利要求1-3任一项所述的预处理方法，其特征在于，满足以下条件之一项或多项：The pretreatment method according to any one of claims 1-3, wherein one or more of the following conditions are met:
    i.步骤A加入超纯水后，加热至85-100℃热处理20-30min；i. After adding ultrapure water in step A, heat it to 85-100°C for 20-30min;
    ii.步骤A加入超纯水后，加热至90-100℃热处理20-30min；ii. After adding ultrapure water in step A, heat to 90-100°C for 20-30 minutes;
    iii..步骤B所述有机溶剂选自甲醇、乙醇、丙醇、异丙醇、乙腈中的一种或多种；iii.. the organic solvent described in step B is selected from one or more of methanol, ethanol, propanol, isopropanol, acetonitrile;
    iv.步骤A和步骤B所述离心条件为0-5℃，5000-15000×g离心5-30min；iv. The centrifugation conditions described in step A and step B are 0-5°C, 5000-15000×g centrifugation for 5-30min;
    v.步骤A和步骤B所述离心条件为4℃，10000-15000×g离心10-20min；v. The centrifugation conditions described in step A and step B are 4°C, 10000-15000×g centrifugation for 10-20min;
    vi.步骤C所述有机溶剂选自丁醇、二氯甲烷、氯仿或乙酸乙酯中的一种或多种；vi. The organic solvent described in step C is selected from one or more of butanol, methylene chloride, chloroform or ethyl acetate;
    vii.所述人乳寡糖包括乳糖-N-四糖和/或乳糖-N-三糖。vii. The human milk oligosaccharides include lactose-N-tetraose and/or lactose-N-triose.
  5. 一种乳制品中人乳寡糖的定性和/或定量检测方法，其特征在于，包括权利要求1和/或权利要求2和/或权利要求3和/或权利要求4所述预处理方法，还包括如下步骤：A qualitative and/or quantitative detection method for human milk oligosaccharides in dairy products, characterized in that it comprises the pretreatment method described in claim 1 and/or claim 2 and/or claim 3 and/or claim 4, Also include the following steps:
    步骤D.通过高效液相色谱法对供试液中的人乳寡糖进行定性和/或定量分析。Step D. Qualitative and/or quantitative analysis of the human milk oligosaccharides in the test solution by high performance liquid chromatography.
  6. 如权利要求5所述乳制品中人乳寡糖的定性和/或定量检测方法，其特征在于，所述高效液相色谱法，色谱柱选自亲水性色谱柱或疏水性色谱柱；The qualitative and/or quantitative detection method of human milk oligosaccharides in dairy products according to claim 5, characterized in that, in the high performance liquid chromatography, the chromatographic column is selected from a hydrophilic chromatographic column or a hydrophobic chromatographic column;
    所述亲水性色谱柱填料选自硅胶、极性基团键合相硅胶或葡聚糖；所述极性基团键合相硅胶中的极性基团选自酰胺基、脲基、氰基、氨基、羧基、糖基和两性离子中的一种或多种；The hydrophilic chromatographic column filler is selected from silica gel, polar group bonded phase silica gel or dextran; the polar group in the polar group bonded phase silica gel is selected from amido, ureido, cyanide One or more of groups, amino groups, carboxyl groups, sugar groups and zwitterions;
    所述色谱柱填料选自氨丙基键合硅胶。The chromatographic column filler is selected from aminopropyl bonded silica gel.
  7. 如权利要求5或6所述乳制品中人乳寡糖的定性和/或定量检测方法，其特征在于，所述高效液相色谱法所用色谱柱选自氨丙基键合硅胶色谱柱，是指硅胶基质键合氨丙基，所述高效液相色谱法采用的检测器选自示差折光检测器、紫外检测器或二极管阵列检测器；检测波长为180-280nm。The qualitative and/or quantitative detection method of human milk oligosaccharides in dairy products as claimed in claim 5 or 6, is characterized in that, the chromatographic column used in the high performance liquid chromatography is selected from aminopropyl bonded silica gel chromatographic column, is Refers to the aminopropyl group bonded to the silica gel matrix, and the detector used in the high-performance liquid chromatography is selected from a differential refractive index detector, an ultraviolet detector or a diode array detector; the detection wavelength is 180-280nm.
  8. 如权利要求5-7任一项所述乳制品中人乳寡糖的定性和/或定量检测方法，其特征在于，所述高效液相色谱法的流动相为水和有机溶剂，其体积比为10/90～90/10，使用等度洗脱或梯度洗脱；所述有机溶剂选自甲醇、乙醇、丙醇、异丙醇、丁醇、乙腈中的一种或多种；优选的，所述流动相为60-75％(v/v)的乙腈水溶液；进一步优选的，所述流动为65-70％(v/v)的 乙腈水溶液。The qualitative and/or quantitative detection method of human milk oligosaccharides in dairy products as claimed in any one of claims 5-7, is characterized in that, the mobile phase of described high performance liquid chromatography is water and organic solvent, and its volume ratio 10/90～90/10, using isocratic elution or gradient elution; the organic solvent is selected from one or more of methanol, ethanol, propanol, isopropanol, butanol, acetonitrile; preferred , the mobile phase is 60-75% (v/v) acetonitrile aqueous solution; further preferably, the mobile phase is 65-70% (v/v) acetonitrile aqueous solution.
  9. 如权利要求5-8任一项所述乳制品中人乳寡糖的定性和/或定量检测方法，其特征在于，所述高效液相色谱法的流动相流速为0.2-1mL/min，柱温为20-60℃；优选的，流动相流速为0.4-0.8mL/min；进一步优选的，流动相流速为0.5-0.6mL/min。The qualitative and/or quantitative detection method of human milk oligosaccharides in dairy products according to any one of claims 5-8, wherein the mobile phase flow rate of the high performance liquid chromatography is 0.2-1mL/min, and the column The temperature is 20-60° C.; preferably, the flow rate of the mobile phase is 0.4-0.8 mL/min; more preferably, the flow rate of the mobile phase is 0.5-0.6 mL/min.
  10. 如权利要求5所述乳制品中人乳寡糖的定性和/或定量检测方法，其特征在于，所述人乳寡糖为乳糖-N-四糖和/或乳糖-N-三糖。The qualitative and/or quantitative detection method of human milk oligosaccharides in dairy products according to claim 5, characterized in that the human milk oligosaccharides are lactose-N-tetrasaccharide and/or lactose-N-triose.
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