A method for detecting human milk oligosaccharides in dairy products

Abstract

The present invention relates to the technical field of dairy product detection, and provides a method for detecting human milk oligosaccharides in dairy products. The detection method comprises: a dairy product to be tested is pre-treated and mixed with an internal standard containing both laminaria triose and mannotriose to obtain a mixed solution; the mixed solution is enzymatically hydrolyzed and derivatized to obtain a derivative; the derivative is subjected to liquid chromatography detection; for the dairy product to be tested containing GOS, enzymatic hydrolysis is performed using amylotransglucosidase to test LNT and LNnT therein, and enzymatic hydrolysis is performed using a mixed enzyme of amylotransglucosidase and β-galactosidase to test 2'-FL, 3-FL, 3'-SL and 6'-SL therein; for the dairy product to be tested not containing GOS, enzymatic hydrolysis is performed using amylotransglucosidase to test 2'-FL, 3-FL, LNT, LNnT, 3'-SL and 6'-SL therein. The present invention has the technical characteristics of wide applicability, high sensitivity, high efficiency and strong promotion.

Description

Technical Field

[0001] The present invention relates to the technical field of dairy product detection, and in particular to a method for detecting human milk oligosaccharides in dairy products. Background Art

[0002] Human milk oligosaccharides (HMOs), as key nutritional elements for infant growth, play multiple physiological roles, including promoting the proliferation and colonization of probiotics, inhibiting the growth of harmful bacteria, preventing neonatal necrotizing colitis, regulating immune function, and promoting brain and neural development. Currently, seven HMOs have been approved for fortification internationally, including 2'-FL, 3-FL, LNT, LNnT, 3'-SL, 6'-SL, and DFL. The first six are widely used in various infant formula products. my country has approved two HMOs, 2'-FL and LNnT, as nutritional fortifiers in infant formula, and more HMOs are expected to be approved in the future.

[0003] However, due to the complex nature of dairy product matrices, the presence of milk fat, proteins, peptides, amino acids, lactose, and other oligosaccharides in the matrix can complicate the detection of HMOs. Studies have shown that existing detection methods fail to meet the multiple requirements of broad applicability, high sensitivity and accuracy, high efficiency, and strong scalability when detecting HMOs in infant formula and children's formula. Consequently, these methods struggle to support the research and development of these products. Summary of the Invention

[0004] As mentioned above, the existing dairy product matrices are complex, and the detection of human milk oligosaccharides in dairy products brings a lot of interference, especially when human milk oligosaccharides including LNT and / or LNnT in a GOS-containing matrix are to be detected. The present invention finds that the existing methods are difficult to accurately test the content of human milk oligosaccharides in this situation. Based on this, the present invention provides a method for detecting human milk oligosaccharides in dairy products. By containing double internal standards of laminaria triose and mannotriose, and designing enzymatic hydrolysis schemes according to different detection objects, and further using liquid chromatography detection for detection, the test accuracy of human milk oligosaccharides can be significantly improved. In addition, this method also has the technical characteristics of wide applicability, high sensitivity, high efficiency and strong promotion.

[0005] In a first aspect, the present invention provides a method for detecting human milk oligosaccharides in dairy products, wherein the human milk oligosaccharides include 2'-FL, 3-FL, LNT, LNnT, 3'-SL and 6'-SL, and the detection method comprises:

[0006] The dairy product to be tested is pre-treated and mixed with an internal standard containing both laminaria triose and mannotriose to obtain a mixed solution; the mixed solution is enzymatically hydrolyzed and derivatized to obtain a derivative; the derivative is detected by liquid chromatography;

[0007] For the dairy product containing GOS, the enzymatic hydrolysis is performed using amyloglucosidase to test LNT and LNnT therein, and the enzymatic hydrolysis is performed using a mixed enzyme of amyloglucosidase and β-galactosidase to test 2'-FL, 3-FL, 3'-SL and 6'-SL therein;

[0008] For the dairy product to be tested that does not contain GOS, amyloglucosidase is used to perform the enzymatic hydrolysis to test 2'-FL, 3-FL, LNT, LNnT, 3'-SL and 6'-SL therein.

[0009] According to the method for detecting human milk oligosaccharides in dairy products provided by the present invention, the column temperature during liquid chromatography detection is 50-55°C, preferably 52-53°C.

[0010] According to the method for detecting human milk oligosaccharides in dairy products provided by the present invention, the enzymatic hydrolysis includes: enzymatic hydrolysis at 50-70°C for 30-60 min, preferably enzymatic hydrolysis at 60°C for 30-60 min, and more preferably enzymatic hydrolysis at 60°C for 30 min.

[0011] According to the method for detecting human milk oligosaccharides in dairy products provided by the present invention, the derivatization includes: derivatizing at 50-80°C for 30-60 minutes, preferably derivatizing at 65°C for 30-60 minutes, and further preferably derivatizing at 65°C for 30 minutes.

[0012] According to the method for detecting human milk oligosaccharides in dairy products provided by the present invention, the derivatization reagents used in the derivatization include 2-aminobenzamide (2-AB) and 2-methylpyridine-N-borane.

[0013] According to the method for detecting human milk oligosaccharides in dairy products provided by the present invention, the gradient conditions of the liquid chromatography detection are: an initial proportion of 8-12% mobile phase B, maintained for 3-8 minutes; increasing the proportion of mobile phase B to 18-22% within 30-40 minutes; increasing the proportion of mobile phase B to 75-85% within 0.1-1 minute, maintained for 2-5 minutes; reducing the proportion of mobile phase B to the initial proportion within 0.1-1 minute, maintained for 5-10 minutes;

[0014] Preferably, the gradient conditions for the liquid chromatography detection are: an initial proportion of 9-10% mobile phase B, maintained for 4-5 minutes, increasing the proportion of mobile phase B to 19-20% within 33-35 minutes, increasing the proportion of mobile phase B to 79-81% within 0.1-0.5 minutes, maintaining for 3-4 minutes, reducing the proportion of mobile phase B to the initial proportion within 0.1-0.5 minutes, and maintaining for 5-10 minutes.

[0015] According to the method for detecting human milk oligosaccharides in dairy products provided by the present invention, the mobile phase for the liquid chromatography detection is: mobile phase A is acetonitrile, and mobile phase B is 50 mM ammonium formate solution with a pH of 4.4.

[0016] According to the method for detecting human milk oligosaccharides in dairy products provided by the present invention, during the liquid chromatography detection, the total concentration of human milk oligosaccharides in the injection solution is 0.5-200 μg / mL, preferably 2-200 μg / mL.

[0017] According to the method for detecting human milk oligosaccharides in dairy products provided by the present invention, the conditions for the liquid chromatography detection include:

[0018] The chromatographic column is a BEH Glycan column;

[0019] The flow rate is 0.3-0.6 mL / min, preferably 0.45-0.55 mL / min;

[0020] The injection volume is 2~5μL, preferably 4~5μL;

[0021] The excitation wavelength is 330 nm;

[0022] The emission wavelength is 420nm.

[0023] According to the method for detecting human milk oligosaccharides in dairy products provided by the present invention, the dairy products to be tested include one or a combination of two or more of infant formula cow's milk-based milk powder without GOS, partially hydrolyzed infant formula cow's milk-based milk powder, infant formula cow's milk-based milk powder containing GOS, children's formula cow's milk-based milk powder and infant formula soy-based milk powder.

[0024] The method of the present invention can achieve accurate detection under the conditions of 30 minutes of enzymatic hydrolysis and 30 minutes of derivatization, greatly improving the detection efficiency.

[0025] According to the method for detecting human milk oligosaccharides in dairy products provided by the present invention, the pretreatment comprises diluting the dairy product to be tested with water to obtain a sample aqueous solution with a mass concentration of 0.05-0.2 g / mL.

[0026] The dairy product to be tested includes a solid sample and a liquid sample.

[0027] The method for detecting human milk oligosaccharides in dairy products provided by the present invention comprises:

[0028] (1) Dissolve the dairy product to be tested in water to form a sample aqueous solution with a mass concentration of 10-30 g / 80-240 mL. Preferably, the mass concentration of the sample aqueous solution is 15-25 g / 180-220 mL.

[0029] (2) The aqueous solution of step (1) is further diluted 10 to 50 times with water to obtain a sample to be tested. Preferably, the water is diluted 40 to 50 times.

[0030] (3) The sample to be tested in step (2) is mixed with an internal standard in a volume ratio of 1 to 3:1, wherein the internal standard is a mixed aqueous solution of laminaria triose and mannotriose, and the concentrations of laminaria triose and mannotriose are both above 50 μg / ml.

[0031] (4) Enzymatic hydrolysis:

[0032] When the sample to be tested does not contain galacto-oligosaccharides (GOS) and the human milk oligosaccharides to be tested include one or a combination of two or more of 2'-FL, 3-FL, LNT, LNnT, 3'-SL and 6'-SL, a 50-100 U / mL amyloglucosidase solution is mixed with the mixed solution obtained in step (3), and enzymatic hydrolysis is performed at 50-70°C for 30-60 minutes, and then cooled to room temperature to obtain an enzymatic hydrolyzate. The volume ratio of the amyloglucosidase solution to the sample to be tested obtained in step (2) in the mixed solution is 0.5-1.5:1.

[0033] When the sample to be tested contains galacto-oligosaccharides (GOS), and the human milk oligosaccharides to be tested include one or a combination of two or more of 2'-FL, 3-FL, 3'-SL and 6'-SL, a mixed enzyme solution obtained by mixing a 50-100 U / mL amyloglucosidase solution and a 2000-6000 U / mL β-galactosidase solution in a volume ratio of 7-9:3-1 is mixed with the mixed solution obtained in step (3), and enzymatic hydrolysis is carried out at 50-70°C for 30-60 minutes, and then cooled to room temperature to obtain an enzymatic hydrolyzate. The volume ratio of the mixed enzyme solution to the sample to be tested obtained in step (2) in the mixed solution is 0.5-1.5:1.

[0034] When the sample to be tested contains galacto-oligosaccharides (GOS) and the human milk oligosaccharides to be tested are LNT and / or LNnT, a 50-100 U / mL amyloglucosidase solution is mixed with the mixed solution obtained in step (3), and enzymatic hydrolysis is performed at 60°C for 30-60 minutes, followed by cooling to room temperature to obtain an enzymatic hydrolyzate. The volume ratio of the amyloglucosidase solution to the sample to be tested obtained in step (2) in the mixed solution is 0.5-1.5:1.

[0035] (5) The enzymatic hydrolysate obtained in step (4) is mixed with a derivatization reagent in a volume ratio of 0.5 to 1.5:1, derivatized at 50 to 80°C for 30 to 60 minutes, cooled to room temperature, and centrifuged at 10,000 to 20,000 rpm for 5 minutes to obtain a derivative; wherein the derivatization reagent is obtained by dissolving 2-aminobenzamide (2-AB) and 2-methylpyridine-N-borane in a solvent, and the solvent is obtained by mixing DMSO and acetic acid in a volume ratio of 5 to 9:5 to 1, and the concentration of 2-AB in the derivatization reagent is 0.2 to 0.5 mol / L, and the concentration of 2-methylpyridine-N-borane is 0.5 to 1.5 mol / L.

[0036] (6) Liquid chromatography is used to detect the obtained derivatives and determine the content of human milk oligosaccharides in the dairy products to be tested.

[0037] The present invention provides a method for detecting human milk oligosaccharides in dairy products. By containing a double internal standard of laminaria triose and mannotriose, and designing an enzymatic hydrolysis scheme according to different detection objects, and further adopting liquid chromatography detection for detection, the test accuracy of human milk oligosaccharides can be significantly improved. In addition, the method also has the technical characteristics of wide applicability, high sensitivity, high efficiency and strong promotion. Among them, the double internal standard detection method of the present invention not only solves the problem that laminaria triose will be degraded by β-galactosidase, but also avoids the need to add different internal standards according to different enzymatic hydrolysis methods, increasing the complexity of pre-treatment and leading to the wrong addition of internal standards, etc., with wider applicability and more accurate quantitative analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0039] Figure 1 Schematic diagram of the testing method of the present invention.

[0040] Figure 2 This is the test result diagram of test case 1.

[0041] Figure 3 This is a test result diagram of Test Example 5 provided by the present invention, wherein the black line corresponds to Example 1, the blue line corresponds to Example 3, and the green line corresponds to Example 4.

[0042] Figure 4 This is a test result diagram of Test Example 6 provided by the present invention, wherein the black line corresponds to Example 1, the blue line corresponds to Example 5, and the green line corresponds to Example 6.

[0043] Figure 5 This is the test result chart of Test Example 7 using Nestlé Platinum NAN as the dairy product to be tested.

[0044] Figure 6 This is the test result chart for Test Example 7 using commercially available fresh milk and MySheng Youth Protein Drink as the tested dairy products.

[0045] Figure 7 This is the test result diagram of Test Case 8, where the blue line is Test 1 and the black line is Test 2.

[0046] Figure 8 This is the test result diagram of Test Case 9, where the blue line is Test 3 and the black line is Test 4.

[0047] Figure 9 This is the test result diagram of test case 10. DETAILED DESCRIPTION

[0048] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0049] The following combination Figures 1 to 7 The present invention describes a method for detecting human milk oligosaccharides in dairy products.

[0050] Where specific techniques or conditions are not specified in the examples, the experiments were carried out according to those described in literature in the field or according to the product specifications. Reagents or instruments used without manufacturer identification are conventional products available through regular channels. Some raw materials and their information are shown in the table below.

[0051] Table 1

[0052]

[0053] Derivatization reagent: 2-Aminobenzamide (2-AB) and 2-methylpyridine-N-borane were dissolved in a solvent prepared by mixing DMSO and acetic acid in a volume ratio of 7:3. The concentration of 2-AB in the derivatization reagent was 0.35 mol / L, and the concentration of 2-methylpyridine-N-borane was 1.0 mol / L.

[0054] Amylo-glucosidase solution: Amylo-glucosidase was dissolved in 0.2 M sodium acetate buffer at a pH of 4.5.

[0055] Mixed enzyme solution: A 70 U / mL amyloglucosidase solution and a 4000 U / mL β-galactosidase solution were mixed in a volume ratio of 8:2.

[0056] Example 1

[0057] A method for detecting HMOs in dairy products, comprising the following steps:

[0058] (1) Sample preparation: Dissolve the dairy product to be tested in water to form a solution with a mass concentration of 25g / 200g.

[0059] (2) The solution obtained in step (1) was further diluted 25 times with water to obtain a sample to be tested.

[0060] (3) The sample to be tested obtained in step (2) was mixed with the internal standard in a volume ratio of 2:1 to obtain a mixed solution, wherein the internal standard was a mixed aqueous solution of laminaria triose and mannotriose, and the concentrations of laminaria triose and mannotriose were both 100 μg / ml.

[0061] (4) Enzymatic hydrolysis: A 70 U / mL amylotransferase solution was mixed with the mixed solution obtained in step (3), and the mixture was hydrolyzed at 60°C for 30 min. The mixture was cooled to room temperature to obtain an enzymatic hydrolyzate. The volume ratio of the amylotransferase solution to the sample obtained in step (2) in the mixed solution was 1:1.

[0062] (5) The enzymatic hydrolysate obtained in step (4) was mixed with a derivatization reagent in a volume ratio of 1:1, derivatized at 65°C for 30 minutes, cooled to room temperature, and centrifuged at 15,000 rpm for 5 minutes to obtain the derivative.

[0063] (6) The derivative obtained in step (5) is detected by liquid chromatography. The specific conditions of liquid chromatography are:

[0064] Chromatographic column: BEH Glycan column;

[0065] Column temperature: 52°C;

[0066] Mobile phase: Mobile phase A is acetonitrile, mobile phase B is 50 mM ammonium formate solution (pH = 4.4);

[0067] Flow rate: 0.50 mL / min;

[0068] Injection volume: 5 μL;

[0069] Gradient conditions: initial proportion of mobile phase B was 10%, maintained for 4 min, the proportion of mobile phase B was increased to 19.9% within 34 min, the proportion of mobile phase B was increased to 80% within 0.5 min, maintained for 3 min, and the proportion of mobile phase B was reduced to the initial proportion within 0.5 min and maintained for 5 min.

[0070] Excitation wavelength: 330 nm;

[0071] Emission wavelength: 420nm.

[0072] Example 2

[0073] The method is basically the same as Example 1, except that the volume of the 70 U / mL amylotransferase solution in step (4) is replaced by the mixed enzyme solution.

[0074] Example 3

[0075] The method is basically the same as Example 1, except that the enzymatic hydrolysis time in step (4) is increased from 30 min to 45 min.

[0076] Example 4

[0077] The method is basically the same as Example 1, except that the enzymatic hydrolysis time in step (4) is increased from 30 min to 60 min.

[0078] Example 5

[0079] The method is basically the same as Example 1, except that the derivatization time in step (5) is increased from 30 min to 45 min.

[0080] Example 6

[0081] The method is basically the same as Example 1, except that the derivatization time in step (5) is increased from 30 min to 60 min.

[0082] Example 7

[0083] The process is basically the same as Example 1, except that the column temperature in step (6) is adjusted to 50°C.

[0084] Example 8

[0085] The process is basically the same as Example 1, except that the column temperature in step (6) is adjusted to 55°C.

[0086] Example 9

[0087] The method is basically the same as Example 1, except that the gradient condition in step (6) is:

[0088] The initial proportion was 8% mobile phase B, which was maintained for 3 min;

[0089] Increase the proportion of mobile phase B to 18% within 30 min;

[0090] Increase the proportion of mobile phase B to 75% within 0.1 min and maintain for 2 min;

[0091] Reduce the proportion of mobile phase B to the initial proportion within 0.1 min and maintain it for 5 min.

[0092] Example 10

[0093] The method is basically the same as Example 1, except that the gradient condition in step (6) is:

[0094] The initial proportion was 12% mobile phase B, which was maintained for 8 min;

[0095] Increase the proportion of mobile phase B to 22% within 40 min;

[0096] Increase the proportion of mobile phase B to 85% within 1 minute and maintain for 5 minutes;

[0097] Reduce the proportion of mobile phase B to the initial proportion within 1 min and maintain it for 10 min.

[0098] Example 11

[0099] The process is basically the same as Example 2, except that the column temperature in step (6) is adjusted to 50°C.

[0100] Example 12

[0101] The process is basically the same as Example 2, except that the column temperature in step (6) is adjusted to 55°C.

[0102] Example 13

[0103] The method is basically the same as Example 2, except that the gradient condition in step (6) is:

[0104] The initial proportion was 8% mobile phase B, which was maintained for 3 min;

[0105] Increase the proportion of mobile phase B to 18% within 30 min;

[0106] Increase the proportion of mobile phase B to 75% within 0.1 min and maintain for 2 min;

[0107] Reduce the proportion of mobile phase B to the initial proportion within 0.1 min and maintain it for 5 min.

[0108] Example 14

[0109] The method is basically the same as Example 2, except that the gradient condition in step (6) is:

[0110] The initial proportion was 12% mobile phase B, which was maintained for 8 min;

[0111] Increase the proportion of mobile phase B to 22% within 40 min;

[0112] Increase the proportion of mobile phase B to 85% within 1 minute and maintain for 5 minutes;

[0113] Reduce the proportion of mobile phase B to the initial proportion within 1 min and maintain it for 10 min.

[0114] Comparative Example 1

[0115] The process is basically the same as Example 1, except that the column temperature in step (6) is adjusted to 60°C.

[0116] Test Example 1 HPLC Test of 6 HMOs with Double Internal Standards

[0117] The method is basically the same as Example 1, except that standard substances of different concentrations are used as test samples and step (4) is omitted.

[0118] See the table below for standard product information.

[0119] Table 2

[0120]

[0121] The test results are as follows Figure 2 shown.

[0122] With the concentration of HMOs standard as the horizontal axis and the peak area ratio of HMOs response / internal standard response as the vertical axis, draw the linear regression equation of HMOs standard and determine R 2 The specific results are shown in the table below.

[0123] Table 3

[0124]

[0125] From the above test, we can conclude that for 2'-FL, its concentration is in the range of 2~200μg / mL, R 2 Greater than 0.995, which can meet the detection requirements; for other human milk oligosaccharides, in the range of 1~100μg / mL, R 2 Greater than 0.995, which can meet the detection requirements.

[0126] Test Example 2 Precision

[0127] The dairy products to be tested were cow's milk-based milk powder without GOS infant formula, cow's milk-based milk powder with GOS infant formula, partially hydrolyzed cow's milk-based milk powder, soy-based infant formula, and children's reconstituted cow's milk-based milk powder, respectively. The tests were conducted according to the methods in the above examples. The specific corresponding relationships are shown in the following table.

[0128] Table 4

[0129]

[0130] Each dairy product was tested in six replicates, with two replicates per day, over three days. The RSD values were calculated to assess the precision of the method. The results are shown in the table below.

[0131] Table 5

[0132]

[0133] As can be seen from the table above, the inter-day precision of the six HMOs in the five tested dairy products was RSD between 0.4 and 2.9%, which was less than 10%, meeting the requirements of the test method.

[0134] Test Case 3: Accuracy

[0135] HMOs standards were added to the dairy products to be tested, and the tests were performed according to the methods in the above examples. The specific corresponding relationships are shown in the following table.

[0136] Table 6

[0137]

[0138] Each dairy product was tested six times, with two replicates per day, over three days. The recovery rate was calculated to assess the accuracy of the method. The results are shown in the table below.

[0139] Table 7

[0140]

[0141] As can be seen from the table above, among the five dairy products tested, the matrix spike recoveries of the six HMOs ranged from 91.3% to 108%, meeting the accuracy requirement of 80% to 110%.

[0142] Test Example 4 Detection Limit and Quantification Limit

[0143] The method is essentially the same as Example 1, except that in step (2), the standard is diluted with water to the lowest point of linearity as the test solution (the concentration corresponding to 2'-FL is 2 mg / L, and the concentration corresponding to other human milk oligosaccharides is 1 mg / L). The signal-to-noise ratio (S / N) corresponding to the HMOs is calculated from the obtained spectrum. The detection limit concentration of the liquid phase method is determined by 3 times the baseline noise signal, and the quantification limit concentration of the liquid phase method is determined by 10 times the baseline noise signal. The dilution factor used in the previous treatment process is then multiplied, where the solid sample is 225 times and the liquid sample is 25 times, to obtain the final quantification limit and detection limit for the solid and liquid.

[0144] The detection limit (3 S / N) of 2'-FL and 3-FL in solid samples was 0.4 mg / 100 g, and the quantification limit (10 S / N) was 1.5 mg / 100 g.

[0145] The detection limit of LNnT and 6'-SL was 0.6 mg / 100 g, and the quantification limit was 2.0 mg / 100 g.

[0146] The detection limit of LNT and 3'-SL was 0.7 mg / 100 g, and the quantification limit was 2.5 mg / 100 g.

[0147] The detection limit (3S / N) of 2'-FL and 3-FL in liquid samples was 0.05 mg / 100 g, and the quantification limit (10S / N) was 0.17 mg / 100 g.

[0148] The detection limit of LNnT and 6'-SL was 0.07 mg / 100 g, and the quantification limit was 0.25 mg / 100 g.

[0149] The detection limit of LNT and 3'-SL was 0.08 mg / 100 g, and the quantification limit was 0.30 mg / 100 g.

[0150] Test Example 5 Effect of different enzymatic hydrolysis times on test results

[0151] The HMOs standard was used as the test solution. In the test solution, the concentration of 2'-FL was 50 mg / L, and the concentrations of other HMOs were all 25 mg / L. The test methods of Examples 1, 3, and 4 were used for testing.

[0152] The test results are as follows Figure 3 As shown, from Figure 3 It can be seen that the enzymatic hydrolysis times of Examples 1, 3 and 4 are different, namely 30 min, 45 min and 60 min, respectively. However, the test results show that the responses of the various substances are consistent, so it can be considered that the enzymatic hydrolysis time of 30 min can also meet the experimental requirements.

[0153] Test Example 6: Impact of Different Derivation Times on Test Results

[0154] The HMOs standard was used as the test solution. In the test solution, the concentration of 2'-FL was 50 mg / L, and the concentrations of other HMOs were all 25 mg / L. The test methods of Examples 1, 5, and 6 were used for testing.

[0155] The test results are as follows Figure 4 As shown, from Figure 4 It can be seen that the derivatization times used in Examples 1, 5 and 6 are different, namely 30 min, 45 min and 60 min, respectively. However, the test results show that the responses of the various substances are consistent, so it can be considered that the derivatization time of 30 min can also meet the experimental requirements.

[0156] Test Example 7: Effect of different column temperatures on test results

[0157] Nestle Platinum NAN was used as the dairy product to be tested, and the testing methods of Example 1 and Comparative Example 1 were used for testing.

[0158] The test results are as follows Figure 5 As shown, from Figure 5 As can be seen, using the method of Example 1, there is a miscellaneous peak next to the laminarin peak (in the red box). However, using the method of Comparative Example 1, there is only one peak at the laminarin peak location. This indicates that the testing method of Comparative Example 1 causes laminarin and impurities to co-emit. This results in an overly high peak area for laminarin, ultimately leading to inaccurate content data.

[0159] It is speculated that the impurities are caused by the raw milk raw material. Therefore, further verification was carried out: a standard of kelp trisaccharide, commercially available fresh milk, and Maisheng Youth Protein Drink (concentrated protein sample) were used as the dairy products to be tested, and the method of Comparative Example 1 was used for testing.

[0160] The test results are as follows Figure 6 As shown in the figure, the impurity in the samples of laminaria trisaccharide and milk has the same retention time, while there is no obvious impurity peak at this position in the concentrated protein sample, which proves that the impurity comes from raw milk.

[0161] Test Example 8: Verification test of degradation of laminaria trisaccharide internal standard caused by β-galactosidase

[0162] The present invention has found in experiments that β-galactosidase can cause the degradation of the laminaria triose internal standard. The experimental process is as follows:

[0163] Experiment 1: This procedure is essentially the same as Example 1, except that the dairy product to be tested is replaced with a solution of 100 mg / L prepared from a laminarin standard, and step (1) is omitted. Preparation of the laminarin standard: Weigh an appropriate amount of laminarin powder and dilute to a 10 mL volumetric flask with water. Draw a certain amount of the above solution and dilute to 100 mg / L with water.

[0164] Experiment 2: This is essentially the same as Example 2, except that the dairy product to be tested is replaced with a solution of 100 mg / L laminarin standard solution, and step (1) is omitted. Laminarin standard solution preparation: Same as Experiment 1.

[0165] The test results of experiments 1 and 2 are as follows Figure 7 As shown, the blue line is test 1, the black line is test 2, Figure 7 As can be seen, the response of the blue line is significantly higher than that of the black line, indicating that the use of β-galactosidase will lead to the degradation of the laminaria triose internal standard.

[0166] The experiment also found that if laminarin is added as an internal standard after enzymatic hydrolysis, the problem of degradation will also occur.

[0167] Test Example 9: Verification that β-galactosidase does not cause degradation of the mannotriose internal standard

[0168] The experimental process is as follows:

[0169] Experiment 3: This is essentially the same as Example 1, except that the dairy product to be tested is replaced with a solution of a 100 mg / L mannotriose standard solution, and step (1) is omitted. Mannotriose standard preparation: Weigh an appropriate amount of mannotriose powder and dilute to a 10 mL volumetric flask with water. Draw a certain amount of the above solution and dilute to 100 mg / L with water.

[0170] Experiment 4: This is essentially the same as Example 2, except that the dairy product to be tested is replaced with a solution of a mannotriose standard solution at a concentration of 100 mg / L, and step (1) is omitted. Mannotriose standard solution preparation: Same as Experiment 3.

[0171] The test results of experiments 3 and 4 are as follows Figure 8 As shown, the blue line is test 3, the black line is test 4, Figure 8 As can be seen in the figure, the blue line overlaps with the black line. This indicates that the two enzymes used do not cause degradation of mannotriose and can be accurately quantified.

[0172] Test Example 10

[0173] Mengniu Yimibaba was used as the dairy product to be tested, and the test method of Example 1 was used for testing.

[0174] The test results are as follows Figure 9 As shown, from Figure 9As can be seen, when spiked into a GOS-containing matrix, the mannotriose peak appears to be positioned right in the middle of the GOS-induced peak cluster, thus affecting the integration of the mannotriose internal standard and, consequently, quantitation. Therefore, when detecting LNT and LNnT in a GOS-containing matrix, only laminaria trisaccharide should be used as the internal standard, as mannotriose will interfere. However, when testing Mengniu Yimibaba as the dairy product using the test method described in Example 2, LNT and LNnT are degraded.

[0175] As demonstrated in Test Examples 7 to 9 above, when processing complex matrices containing GOS, β-galactosidase must be used for enzymatic hydrolysis of 2'-FL, 3-FL, 3'-SL, and 6'-SL, and mannotriose is used as the internal standard for quantitative analysis. For LNT and LNnT, when only amyloglucosidase is used for enzymatic hydrolysis, laminariose is used as the internal standard for quantitative analysis. This method ensures detection accuracy and reliability under different enzymatic hydrolysis conditions. Furthermore, adding a double internal standard before enzymatic hydrolysis greatly simplifies the entire pretreatment process and avoids the problem of incorrect internal standard addition. Furthermore, verification using the methods described in Examples 7 to 14 of the present invention revealed that Examples 7 to 10 achieved test results substantially equivalent to those of Example 1, and that Examples 11 to 14 achieved test results substantially equivalent to those of Example 2.

[0176] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for detecting human milk oligosaccharides in dairy products, characterized in that: The human milk oligosaccharides include 2'-FL, 3-FL, LNT, LNnT, 3'-SL and 6'-SL, and the detection method includes: The dairy product to be tested is pre-treated and mixed with an internal standard containing both laminaria triose and mannotriose to obtain a mixed solution; the mixed solution is enzymatically hydrolyzed and derivatized to obtain a derivative; the derivative is detected by liquid chromatography; For the dairy product containing GOS, the enzymatic hydrolysis is performed using amyloglucosidase to test LNT and LNnT therein, and the enzymatic hydrolysis is performed using a mixed enzyme of amyloglucosidase and β-galactosidase to test 2'-FL, 3-FL, 3'-SL and 6'-SL therein; For the dairy product to be tested that does not contain GOS, amyloglucosidase is used to perform the enzymatic hydrolysis to test 2'-FL, 3-FL, LNT, LNnT, 3'-SL and 6'-SL therein.

2. The method for detecting human milk oligosaccharides in dairy products according to claim 1, characterized in that: The column temperature during the liquid chromatography detection is 50-55°C.

3. The method for detecting human milk oligosaccharides in dairy products according to claim 1, characterized in that: The enzymatic hydrolysis comprises: enzymatic hydrolysis at 50-70° C. for 30-60 minutes.

4. The method for detecting human milk oligosaccharides in dairy products according to claim 1, characterized in that: The derivatization comprises: derivatizing at 50-80° C. for 30-60 minutes.

5. The method for detecting human milk oligosaccharides in dairy products according to claim 1, characterized in that: The derivatization reagents used in the derivatization include 2-aminobenzamide and 2-methylpyridine-N-borane.

6. The method for detecting human milk oligosaccharides in dairy products according to claim 1, characterized in that: The mobile phases for the liquid chromatography detection were: mobile phase A was acetonitrile, and mobile phase B was 50 mM ammonium formate solution with a pH of 4.

4.

7. The method for detecting human milk oligosaccharides in dairy products according to claim 6, characterized in that: The gradient conditions for the liquid chromatography detection are as follows: an initial proportion of 8-12% mobile phase B, maintained for 3-8 minutes; increasing the proportion of mobile phase B to 18-22% within 30-40 minutes; increasing the proportion of mobile phase B to 75-85% within 0.1-1 minute, maintained for 2-5 minutes; and reducing the proportion of mobile phase B to the initial proportion within 0.1-1 minute, maintained for 5-10 minutes.

8. The method for detecting human milk oligosaccharides in dairy products according to claim 1, characterized in that: During the liquid chromatography detection, the total concentration of human milk oligosaccharides in the injection solution is 0.5-200 μg / mL.

9. The method for detecting human milk oligosaccharides in dairy products according to claim 1, characterized in that: The conditions for the liquid chromatography detection include: The chromatographic column is a BEH Glycan column; Flow rate: 0.3~0.6mL / min; The injection volume is 2~5μL; The excitation wavelength is 330 nm; The emission wavelength is 420nm.

10. The method for detecting human milk oligosaccharides in dairy products according to any one of claims 1 to 9, characterized in that: The dairy product to be tested includes one or a combination of two or more of infant formula cow's milk-based milk powder without GOS, partially hydrolyzed infant formula cow's milk-based milk powder, infant formula cow's milk-based milk powder containing GOS, children's formulated cow's milk-based milk powder and infant formula soy-based milk powder.

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