Method for detecting various oligosaccharides in cow milk

Abstract

The invention relates to the technical field of detection, in particular to a method for detecting various oligosaccharides in cow milk. The invention provides a method for detecting various oligosaccharides in cow milk, which comprises the following steps: (1) centrifuging a cow milk sample, and discarding precipitate to obtain supernate; (2) mixing the supernate and absolute ethyl alcohol, standing for 12-16 hours, centrifuging, taking filtrate, and concentrating the filtrate to obtain a concentrated sample; (3) separating and purifying the concentrated sample to obtain a purified solution; (4) concentrating and freeze-drying the purified solution, and carrying out 2-AB derivatization to obtain a sample to be detected; and (5) quantifying by using an internal standard method, and determining the oligosaccharide content in the sample to be detected by using a high performance liquid chromatography. The method can be used for simply and efficiently detecting 10 oligosaccharides at one time.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and in particular to a method for detecting multiple oligosaccharides in milk. Background Art

[0002] As people's demand for dairy products continues to increase, dairy cow breeding has gradually become one of the important directions for the development of my country's animal husbandry. At present, some progress has been made in dairy cow breeding, such as the research and development of local breeds such as Chinese grassland black and white dairy cows, Sanhe cattle and Xinjiang brown cattle, as well as the introduction and adaptive breeding of foreign breeds such as Holstein cows, Jersey cows, Swiss brown cattle and Simmental cattle. The research on the nutritional components of human and cow milk is becoming increasingly in-depth at home and abroad, and nutrients such as prebiotics have also become a hot topic in scientific research. Oligosaccharides, also known as oligosaccharides, are a kind of prebiotics in milk. They are composed of 2 to 10 monosaccharides connected by glycosidic bonds. They are the fourth largest nutrient in breast milk and a new functional sugar source. They integrate nutrition, health care and diet therapy. They are widely used in food, health products, beverages, medicine, feed additives and other fields.

[0003] Lactose-N-neotetraose (LNnT), 2 ， -fucosyllactose (2'FL), 3 ， -sialyllactose (3'SL) and 6 ， -Sialyl lactose (6'SL) and other high content, mature milk 3'FL content is the most abundant. Nowadays, the proportion of insufficient breastfeeding has exceeded 20%, and infant formula milk powder is needed as a milk substitute. This product is generally made of cow's milk, but the content of bovine milk oligosaccharides (BMOs) in cow's milk is relatively low. BMOs and human milk oligosaccharides (HMOs) are similar in composition and structure but not the same, and the proportions of the two are very different. Oligosaccharides in cow's milk are composed of six monomers, including Glc, Gal, GlcNAc, Fuc, N-glycolylneuraminic acid and Neu5Ac. Most BMOs have a lactose core [Gal9 (β1-4) Glc]. By quantitatively detecting multiple oligosaccharides in milk from cows of different breeds and regions, analyzing the content differences of bovine milk oligosaccharides in different regions, breeds and types, and studying the laws of their changes, we can obtain the best milk source breed and the best milk source area that is closest to the composition of human milk oligosaccharides.

[0004] The material composition of human and animal milk is very complex, which has caused great interference to the qualitative and quantitative detection of oligosaccharides in milk. In order to study the changing patterns and trends of the types and contents of oligosaccharides in milk of humans or different species of animals during different lactation periods, a large number of samples need to be tested, and high-throughput detection of samples is still a headache for scientific researchers. In addition to the great difficulties in the detection methods and instruments of milk oligosaccharides, other substances such as protein and fat in milk can also cause serious interference to the accurate detection of oligosaccharides. The more difficult problem is the special structure of oligosaccharides and the fact that they do not contain strong ultraviolet and fluorescent absorption functional groups. So far, no unified high-performance liquid chromatography method has been established to analyze all oligosaccharides. Many separation parameters (elution form, analysis time, separation of complex compounds and column efficiency, column stability and column service life) restrict the chromatographic separation technology of oligosaccharides. According to the methods reported in previous studies, the separation and identification of oligosaccharides were carried out, but it was found in the separation process that due to the large number of target oligosaccharides detected and the sensitivity of the chromatographic column and detector, the separation and identification of all oligosaccharides could not be completed, so the method needs to be optimized.

[0005] Therefore, there is still a need in the art for an improved method for detecting multiple oligosaccharides in milk. Summary of the invention

[0006] The object of the present invention is to provide a method for detecting multiple oligosaccharides in milk, which can simply and efficiently detect 10 oligosaccharides at one time.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for detecting multiple oligosaccharides in milk, comprising the following steps:

[0009] (1) Centrifuging the milk sample, discarding the precipitate, and obtaining the supernatant;

[0010] (2) mixing the supernatant and anhydrous ethanol and letting it stand for 12 to 16 hours, centrifuging, taking the filtrate, and concentrating the filtrate to obtain a concentrated sample;

[0011] (3) separating and purifying the concentrated sample to obtain a purified solution;

[0012] (4) concentrating and freeze-drying the purified solution, and derivatizing with 2-AB to obtain a sample to be tested;

[0013] (5) quantification by internal standard method, using high performance liquid chromatography to determine the oligosaccharide content in the sample to be tested;

[0014] The chromatographic conditions are as follows: the chromatographic column is an XBridge BEH Amide Column, the mobile phase A is pure acetonitrile, the mobile phase B is an ammonium formate solution, the column temperature is 30-50°C, the injection volume is 15-25 μl, and the flow rate is 0.3-0.6 mL / min.

[0015] Preferably, in step (1), the centrifugal speed is 11000-13000 rpm; and the centrifugal time is 30-50 min.

[0016] Preferably, in step (2), the volume ratio of the supernatant and anhydrous ethanol is 1:1-3; in step (2), the centrifugal speed is 5000-6000 rpm, the centrifugal time is 40-50 min, and the centrifugal temperature is 1-5°C.

[0017] Preferably, in step (2), the concentration method is a water bath assisted nitrogen blowing concentration method; and the volume of the concentrated sample is 2 to 4 mL.

[0018] Preferably, in step (3), the separation and purification step comprises: SPE C18 column separation and purification, and graphite carbon column impurity removal.

[0019] Preferably, in step (4), the concentrated freeze-drying method is a water bath assisted nitrogen blowing concentration method.

[0020] Preferably, the internal standard substance used in the internal standard method for quantification is oligofructose.

[0021] Preferably, the initial concentration of the ammonium formate solution is 45-55 mmol / L.

[0022] Preferably, in step (5), the elution method used in the high performance liquid chromatography is gradient elution; the conditions of the gradient elution are: 0-2.3 min, 95% mobile phase A; 2.3-4.9 min, 95%-90% mobile phase A; 4.9-32 min, 90%-82% mobile phase A; 32-48 min, 82%-80% mobile phase A; 48-61 min, 80%-78% mobile phase A, 61-89 min, 78%-74.6% mobile phase A; 89-92 min, 74.6-30% mobile phase A; 92-98 min, 30%-90% mobile phase A; 98-99.5 min, 90%-95% mobile phase A.

[0023] Preferably, in step (5), the oligosaccharide comprises 2 ， -fucosyllactose, 3 ， -fucosyllactose, 3 ， -Sialyl lactose, lactoyl-N-fucopentaose I, 6 ，-sialyllactose, 6 ， -Sialyl-N-acetylated lactosamine, 3 ， -One or more of galactosyl lactose, disialactose, lactose-N-tetraose and lactose-N-neotetraose.

[0024] Beneficial effects of the present invention:

[0025] The present invention provides a method for detecting multiple oligosaccharides in milk. The method of the present invention can detect 10 oligosaccharides at one time by only using a liquid chromatography-tandem ultraviolet detector: ， -fucosyllactose, 3 ， -fucosyllactose, 3 ， -Sialyl lactose, lactoyl-N-fucopentaose I, 6 ， -sialyllactose, 6 ， -Sialyl-N-acetylated lactosamine, 3 ， -galactosyl lactose, disialactose, lactose-N-tetraose and lactose-N-neotetraose; the method of the present invention is simple and convenient; the method of the present invention uses internal standard calibration to ensure the accuracy of the detection result; and the detection limit is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the peak diagram of oligosaccharide standards;

[0027] Figure 2 This is the peak diagram of oligosaccharide standard and one of the samples of Beijing Holstein cattle;

[0028] Figure 3 The contents of 10 oligosaccharides in milk of different breeds of cattle, among which A is 2'FL, B is DSL, C is 3FL, D is LNFPI, E is 3'SL, F is LNT, G is 3'GL, H is LNnT, I is 6'SLN, and J is 6'SL; 1 is Beijing Holstein cattle, 2 is Hebei Holstein cattle, 3 is Jersey cattle, 4 is Shuxuanhua cattle, 5 is Sanhe cattle, 6 is Xinjiang brown cattle, and 7 is Simmental cattle;

[0029] Figure 4 The percentage of 10 oligosaccharides in milk of different breeds of cattle, A is Beijing Holstein cattle, B is Hebei Holstein cattle, C is Jersey cattle, D is Shuxuanhua cattle, E is Sanhe cattle, F is Xinjiang brown cattle, and G is Simmental cattle;

[0030] Figure 5 It is the test result of comparative example 1;

[0031] Figure 6 is the total oligosaccharide content in 7 kinds of milk in Comparative Example 1; DETAILED DESCRIPTION

[0032] The present invention provides a method for detecting multiple oligosaccharides in milk, comprising the following steps:

[0033] (1) Centrifuging the milk sample, discarding the precipitate, and obtaining the supernatant;

[0034] (2) mixing the supernatant and anhydrous ethanol and letting it stand for 12 to 16 hours, centrifuging, taking the filtrate, and concentrating the filtrate to obtain a concentrated sample;

[0035] (3) separating and purifying the concentrated sample to obtain a purified solution;

[0036] (4) concentrating and freeze-drying the purified solution, and derivatizing with 2-AB to obtain a sample to be tested;

[0037] (5) quantification by internal standard method, using high performance liquid chromatography to determine the oligosaccharide content in the sample to be tested;

[0038] The chromatographic conditions are as follows: the chromatographic column is an XBridge BEH Amide Column, the mobile phase A is acetonitrile, the mobile phase B is an ammonium formate solution, the column temperature is 30-50°C, the injection volume is 15-25 μl, and the flow rate is 0.3-0.6 mL / min.

[0039] In the present invention, in step (1), the centrifugal speed is 11000-13000 rpm, preferably 11500-12500 rpm, and more preferably 12000 rpm; the centrifugal time is 30-50 min, preferably 35-45 min, and more preferably 40 min.

[0040] In the present invention, in step (2), the volume ratio of the supernatant and anhydrous ethanol is 1:1-3, preferably 1:1.3-2.7, and more preferably 1:2;

[0041] The standing time is preferably 13 to 15 hours, more preferably 14 hours;

[0042] The standing temperature is 1 to 5°C, preferably 2 to 4°C, and more preferably 3°C;

[0043] During the standing process, it is necessary to shake it every 2 hours to ensure complete reaction;

[0044] In step (2), the centrifugal speed is 5000-6000 rpm, preferably 5300-5700 rpm, and more preferably 5500 rpm; the centrifugal time is 40-50 min, preferably 43-47 min, and more preferably 45 min; the centrifugal temperature is 1-5°C, preferably 2-4°C, and more preferably 3°C.

[0045] In the present invention, in step (2), the concentration method is a water bath assisted nitrogen blowing concentration method; the volume of the concentrated sample is 2 to 4 mL, preferably 3 mL;

[0046] During the concentration process, the temperature of the water bath is 40-50°C, preferably 43-47°C, and more preferably 45°C; the flow rate of nitrogen is 5-15 L / min, preferably 8-12 L / min, and more preferably 10 L / min.

[0047] In the present invention, in step (3), the separation and purification step includes: separation and purification by SPEC18 column, and impurity removal by graphite carbon column;

[0048] The specific steps of separation and purification are as follows: activating and balancing an SPE C18 column and a graphite carbon column; passing a concentrated sample through an SPE C18 column, eluting oligosaccharides with 20 mL of pure water to obtain an eluate; passing the eluate through a graphite carbon column, adsorbing oligosaccharides on the column, then desalting the column with 20 mL of pure water, and eluting the oligosaccharides with 25% acetonitrile.

[0049] In the present invention, in step (4), the concentrated freeze-drying method is a water bath assisted nitrogen blowing concentration method;

[0050] During the concentrated freeze-drying process, the temperature of the water bath is 40-50°C, preferably 43-47°C, and more preferably 45°C; the flow rate of nitrogen is 5-15 L / min, preferably 8-12 L / min, and more preferably 10 L / min; the nitrogen blowing time is 2.5-3.5 h, preferably 2.8-3.2 h, and more preferably 3 h.

[0051] In the present invention, in step (5), the internal standard substance used in the internal standard method for quantification is oligofructose.

[0052] In the present invention, in step (5), the specification of the chromatographic column is 4.6mm*250mm, 5μm; the initial concentration of the ammonium formate solution is 45-55mmol / L, preferably 48-52mmol / L, and more preferably 50mmol / L; the pH of the ammonium formate solution is 4-5, and more preferably 4.5;

[0053] The column temperature is preferably 35-45°C, more preferably 40°C; the injection volume is preferably 18-22μl, more preferably 20μl; the flow rate is 0.3-0.6mL / min, preferably 0.4-0.5mL / min, more preferably 0.5mL / min at 0-89min, and 0.4mL / min at 90-99.5min.

[0054] In the present invention, in step (5), the elution method used in the high performance liquid chromatography is gradient elution; the conditions of the gradient elution are: 0-2.3 min, 95% mobile phase A; 2.3-4.9 min, 95%-90% mobile phase A; 4.9-32 min, 90%-82% mobile phase A; 32-48 min, 82%-80% mobile phase A; 48-61 min, 80%-78% mobile phase A, 61-89 min, 78%-74.6% mobile phase A; 89-92 min, 74.6-30% mobile phase A; 92-98 min, 30%-90% mobile phase A; 98-99.5 min, 90%-95% mobile phase A.

[0055] In the present invention, in step (5), the oligosaccharide comprises 2 ， -fucosyllactose (2'FL), 3 ， -fucosyllactose (3'FL), 3 ， -sialyllactose (3'SL), lactoyl-N-fucopentaose I (LNFPI), 6 ， -sialyllactose (6'SL), 6 ， -Sialyl-N-acetylated lactosamine (6'SLN), 3 ， -One or more of galactosyl lactose (3'GL), disialactose (DSL), lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT).

[0056] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0057] In the embodiments and comparative examples of the present invention, the sources of the reagents used are shown in Table 1:

[0058] Table 1 Reagent names and their purchase sources

[0059]

[0060]

[0061] In the embodiments and comparative examples of the present invention, the methods for preparing the materials are as follows:

[0062] (1) 2-AB derivatization reagent: ① Use an electronic analytical balance to accurately weigh 262.1 mg of 2-AB into a 50 mL centrifuge tube, and then accurately weigh 345.6 mg of sodium cyanoborohydride and put it into a 50 mL centrifuge tube; ② Use a pipette to accurately pipette 1.65 mL of acetic acid into a 15 mL centrifuge tube, and pipette 3.85 mL of dimethyl sulfoxide into the acetic acid to prepare a 30% acetic acid solvent; Use a vortex to fully mix the solvent, and add the solvent in ② to the centrifuge tube mixed with 2-AB and sodium cyanoborohydride to dissolve. The solution will have precipitated particles, which are fully shaken using a vortex to disperse the particles to obtain a 2-AB derivatization reagent with a concentration of 0.3 mol / L;

[0063] (2) 2-AMAC derivatization reagent: Weigh 21.2 mg of 2-AMAC solid powder and place it in a clean centrifuge tube. Prepare a dimethyl sulfoxide solution containing 15% acetic acid. Pour the prepared solution into the centrifuge tube. After the powder is dissolved, dilute to 1.0 mL with water to obtain a 2-AMAC derivatization reagent with a concentration of 0.1 mol / L.

[0064] (3) Internal standard solution: 1 mg of oligofructose (internal standard, FOS) was accurately weighed using an electronic analytical balance and placed in a 2 mL centrifuge tube. 200 μL of the derivatization reagent was added. The tube was placed in a 60 °C water bath and heated for 2 h. After the reaction was completed, the tube was immediately transferred to a 4 °C refrigerator to terminate the derivatization reaction and obtain an internal standard solution.

[0065] (4) Ammonium formate solution: 3.152 g of ammonium formate was accurately weighed using an electronic analytical balance and placed in a 1000 mL conical flask. Water was added to make the volume 1 L and the pH was adjusted to 4.4 with formic acid. The bottle containing the mobile phase was first rinsed with 10 mL of ammonium formate, and then the ammonium formate was poured into the bottle. The solution was degassed in an ultrasonic degasser for 30 min to obtain an ammonium formate solution.

[0066] (5) Acetonitrile aqueous solution (70% V / V): Pipette 15 mL of ultrapure water, dilute with pure acetonitrile and make up to 50.0 mL to obtain an acetonitrile aqueous solution;

[0067] (6) Treatment of pure acetonitrile: Measure 1 L of pure acetonitrile into a reagent bottle and degas in an ultrasonic degasser for 30 min;

[0068] (7) 25% acetonitrile solution: Pipette 37.5 mL of ultrapure water, dilute with pure acetonitrile and make up to 50.0 mL to obtain a 25% acetonitrile solution.

[0069] Example 1

[0070] Milk samples were collected from Beijing Holstein, Hebei Holstein, Jersey, Sichuan Shuxuanhua, Inner Mongolia Sanhe, Xinjiang Brown and Simmental cattle, a total of 1,000 samples. After each collection, the samples were mixed and placed in a foam box filled with dry ice before being transported back to the laboratory and stored at -20°C.

[0071] (1) Take 8 mL of each milk sample and place it in four 2 mL centrifuge tubes. Centrifuge at 12000 rpm for 40 min to remove fat, discard the precipitate, and obtain the supernatant;

[0072] (2) Pour the supernatant into a 50 mL centrifuge tube, add two volumes of anhydrous ethanol to remove protein impurities, place in a refrigerator at 4°C, shake once every 2 hours to allow the reaction to be complete, and after standing for 14 hours, centrifuge the 50 mL centrifuge tube at 5400 rpm and 4°C for 45 minutes. The protein precipitates in the lower layer. Take the filtrate and concentrate it in a circular water bath nitrogen blower. Set the water bath temperature to 45°C and the nitrogen flow rate to 10 L / min. The nitrogen blow time is 3 hours to concentrate to 3 mL to obtain a concentrated sample;

[0073] (3) Separation and purification using an SPE C18 column and impurity removal using a graphite carbon column: activating and balancing an SPE C18 column and a graphite carbon column; passing the concentrated sample through an SPE C18 column, eluting the oligosaccharides with 20 mL of pure water to obtain an eluate; passing the eluate through a graphite carbon column, adsorbing the oligosaccharides on the column, and then desalting the column with 20 mL of pure water, and then eluting the oligosaccharides with a 25% acetonitrile solution to obtain a purified solution;

[0074] (4) Concentrating and freeze-drying the purified solution; concentrating and freeze-drying the purified solution in a circular water bath nitrogen blowing apparatus, setting the water bath temperature to 45° C., the nitrogen flow rate to 10 L / min, and the nitrogen blowing time to 3 h, to obtain a concentrated freeze-dried substance; using FOS as an internal standard and 2-AB derivatization reagent as a derivatization reagent to prepare an internal standard solution, and using the internal standard solution to perform 2-AB derivatization on the concentrated freeze-dried substance to obtain a sample to be tested;

[0075] (5) The internal standard method (with FOS as the internal standard) was used for quantification. The contents of 10 oligosaccharides, including 2'FL, 3'FL, 3'SL, LNFPI, 6'SL, 6'SLN, 3'GL, DSL, LNT and LNnT, in the milk of seven breeds of cattle, including Beijing Holstein, Hebei Holstein, Jersey, Shuxuanhua, Sanhe, Dengchuan, Xinjiang Brown and Simmental, were determined by HPLC, and their changes were analyzed. The peak diagrams of oligosaccharide standards and samples are shown in Figure 2. Figures 1-2 As shown;

[0076] The chromatographic conditions are as follows: the chromatographic column is an XBridge BEH Amide Column (4.6mm*250mm, 5μm), the mobile phase A is pure acetonitrile, the mobile phase B is 50mmol / L ammonium formate solution (pH 4.4), the column temperature is 40℃, the injection volume is 20μl; the flow rate is 0.5mL / min from 0 to 89min, and the flow rate is 0.4mL / min from 90 to 99.5min;

[0077] The conditions of gradient elution are: 0-2.3 min, 95% mobile phase A; 2.3-4.9 min, 95%-90% mobile phase A; 4.9-32 min, 90%-82% mobile phase A; 32-48 min, 82%-80% mobile phase A; 48-61 min, 80%-78% mobile phase A; 61-89 min, 78%-74.6% mobile phase A; 89-92 min, 74.6-30% mobile phase A; 92-98 min, 30%-90% mobile phase A; 98-99.5 min, 90%-95% mobile phase A;

[0078] The contents of various oligosaccharides were calculated based on the concentration ratio of the test sample and the internal standard, the ratio of their corresponding chromatographic peak areas and the relative correction factor. The detection and calculation results of the retention time, peak area and correction factor of FOS and 10 oligosaccharide standards are shown in Table 2. The calculation results of the contents of various oligosaccharides are shown in Table 3 and Table 4. Figures 3-4 As shown;

[0079] The calculation formula is:

[0080] Where ε is the correction factor, As is the peak area of ​​the internal standard (FOS), Ar is the peak area of ​​the oligosaccharide standard, Ms is the concentration of the internal standard, and As is the concentration of the oligosaccharide standard;

[0081] Where Mi is the content of oligosaccharides in the sample, ε is the correction factor, Ai is the peak area of ​​the oligosaccharide standard, As is the peak area of ​​the internal standard, and Mr is the concentration of the internal standard; if necessary, it is converted into a percentage of the labeled amount based on the dilution multiple, sampling volume and labeled amount, or converted into a percentage based on the dilution multiple and sampling volume.

[0082] Table 2 Retention time, peak area and correction factor of FOS and 10 oligosaccharide standards

[0083]

[0084]

[0085] Table 3 Results of 10 oligosaccharide contents in milk of 7 cattle breeds (unit: μg / mL)

[0086]

[0087] As can be seen from Table 2, 10 oligosaccharides can be detected at one time using this method.

[0088] Comparative Example 1

[0089] The difference from Example 1 is:

[0090] Chromatographic conditions: Waters BEHAmide column (150 mm*2.1 mm, 1.7 μm); column temperature: 40°C; injection volume: 5 μL; flow rate: 0.4 mL / min. Injection chamber temperature: 20°C; mobile phase A: 25 mmol / L ammonium formate solution. Mobile phase B: pure acetonitrile; other steps are the same as in Example 1;

[0091] This method was used to detect the content of 10 oligosaccharides and total oligosaccharides. Figure 1 The test results of the sample with the peak in the sample are as follows Figures 5-6 shown.

[0092] from Figure 5 As can be seen from the figure, due to the large number of target oligosaccharides that need to be detected, and due to reasons such as the chromatographic column and detector sensitivity, the separation and identification of all oligosaccharides cannot be completed. Therefore, this method cannot separate the 10 oligosaccharides well.

[0093] Comparative Example 2

[0094] The difference from Example 1 is:

[0095] The anhydrous ethanol in step (2) was replaced with an acetonitrile aqueous solution (70% V / V), and the other steps were the same as those in Example 2;

[0096] The results showed that the ethanol protein removal method can more effectively retain low-abundance and low-molecular-weight oligosaccharides in the detection of oligosaccharides in milk than the acetonitrile protein removal method, thereby improving the accuracy and efficiency of detection.

[0097] Comparative Example 3

[0098] The difference from Example 1 is:

[0099] The 2-AB derivatization in step (4) was replaced by 2-AMAC derivatization, and the other steps were the same as in Example 2;

[0100] The results showed that the 2-AMAC derivatization procedure was more complicated and that the 2-AB derivatization provided higher sensitivity and stronger signals.

[0101] Therefore, deproteinization plays an important role in oligosaccharide separation. If contaminants such as proteins are not removed from the sample during sample pretreatment, the performance of the chromatographic column will be reduced, which will ultimately affect the accuracy of the chromatographic analysis results. Comparison of Example 1 and Comparative Example 2 shows that ethanol is most suitable for the removal of milk protein;

[0102] In the embodiments and comparative examples, a liquid chromatography-tandem ultraviolet detector with lower sensitivity is used. By comparing Example 1 and Comparative Example 3, it can be seen that the effect of oligosaccharide derivatization using 2-AMAC in the early stage is not ideal, and the separation of the oligosaccharide absorption peaks is not good. The use of 2-AB derivatization can efficiently separate oligosaccharides and has less impact on the samples to be tested.

[0103] It can be seen from the above examples that the present invention provides a method for detecting multiple oligosaccharides in milk, which can simply and efficiently detect 10 oligosaccharides at one time.

[0104] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting multiple oligosaccharides in milk, characterized in that: The steps include: (1) Centrifuging the milk sample, discarding the precipitate, and obtaining the supernatant; (2) mixing the supernatant and anhydrous ethanol and letting it stand for 12 to 16 hours, centrifuging, taking the filtrate, and concentrating the filtrate to obtain a concentrated sample; (3) separating and purifying the concentrated sample to obtain a purified solution; (4) concentrating and freeze-drying the purified solution, and derivatizing with 2-AB to obtain a sample to be tested; (5) quantification by internal standard method, using high performance liquid chromatography to determine the oligosaccharide content in the sample to be tested; The chromatographic conditions are as follows: the chromatographic column is an XBridge BEH Amide Column, the mobile phase A is pure acetonitrile, the mobile phase B is an ammonium formate solution, the column temperature is 30-50°C, the injection volume is 15-25 μl, and the flow rate is 0.3-0.6 mL / min.

2. The method according to claim 1, characterized in that In step (1), the centrifugal speed is 11000-13000 rpm; the centrifugal time is 30-50 min.

3. The method according to claim 2, characterized in that In step (2), the volume ratio of the supernatant to anhydrous ethanol is 1:1-3; the centrifugal speed in step (2) is 5000-6000 rpm, and the centrifugal time is 40-50 min. ， The centrifugal temperature is 1-5°C.

4. The method according to claim 3, characterized in that In step (2), the concentration method is a water bath assisted nitrogen blowing concentration method; the volume of the concentrated sample is 2 to 4 mL.

5. The method according to claim 4, characterized in that In step (3), the separation and purification steps include: separation and purification using a SPEC18 column, and impurity removal using a graphite carbon column.

6. The method according to claim 5, characterized in that In step (4), the concentrated freeze-drying method is a water bath assisted nitrogen blowing concentration method.

7. The method according to claim 6, characterized in that In step (5), the internal standard substance used in the internal standard method for quantification is oligofructose.

8. The method according to claim 7, characterized in that The initial concentration of the ammonium formate solution is 45-55 mmol / L.

9. The method according to claim 8, characterized in that In step (5), the elution method used in the high performance liquid chromatography is gradient elution; the conditions of the gradient elution are: 0 to 2.3 min, 95% mobile phase A; 2.3 to 4.9 min, 95% to 90% mobile phase A; 4.9-32min, 90%-82% mobile phase A; 32-48min, 82%-80% mobile phase A; 48-61min, 80%-78% mobile phase A, 61-89min, 78%-74.6% mobile phase A; 89-92min, 74.6-30% mobile phase A; 92-98min, 30%-90% mobile phase A; 98-99.5min, 90%-95% mobile phase A.

10. The method according to claim 9, characterized in that In step (5), the oligosaccharide includes one or more of 2'-fucosyllactose, 3'-fucosyllactose, 3'-sialyllactose, lacto-N-fucopentaose I, 6'-sialyllactose, 6'-sialyllactose, 6'-sialyl-N-acetylated lactosamine, 3'-galactosyl lactose, disialactose, lactose-N-tetraose and lactose-N-neotetraose.

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