A method for determining milk fat globule membrane protein in food raw material powder

Abstract

The present invention proposes a method for determining milk fat globule membrane protein in food raw material powder, which belongs to the technical field of food detection. The method comprises: (1) sample pretreatment; (2) determining chromatographic conditions; (3) determining mass spectrometry conditions; (4) drawing a standard curve; and (5) detection, thereby obtaining the content of milk fat globule membrane protein in food raw material powder. The present invention can effectively determine the content of milk fat globule membrane protein in food raw material powder, with high accuracy, low detection limit, good linear relationship, good repeatability and stability, and provides a convenient and sensitive detection method for its detection.

Description

Technical Field

[0001] The invention relates to the technical field of food detection, and in particular to a method for determining milk fat globule membrane protein in food raw material powder. Background Art

[0002] Milk fat is one of the main components of milk, generally accounting for 3%-5% of milk components, and is dispersed in the milk in the form of fat globules. Under a microscope, it is spherical or ellipsoidal, and the surface is covered by a 10-20nm membrane, called milk fat globule membrane (MFGM). There are 25%-70% membrane proteins on the milk fat globule membrane, called milk fat globule membrane proteins (MFGMP). There are 120-170 types of proteins identified by proteomics methods. Milk fat globule membrane proteins have rich development and utilization value.

[0003] MFGM is mainly composed of a variety of active lipids and proteins, which come from the endoplasmic reticulum membrane, apical plasma membrane and cytoplasm compartments of mammary epithelial cells during lactation, accounting for 2%-6% of the mass of milk fat globules. The lipid components on MFGM are mainly divided into neutral lipids and polar lipids. Polar lipids account for 26%-40% of the lipid components of MFGM, including glycerophospholipids [phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol (PI)] and sphingolipids [especially sphingomyelin (sM)]. Among them, PE, PC, and SM are more abundant, while Ps and PI are less. The role of polar lipids is mainly to regulate lipid metabolism and participate in nervous system activities. Neutral lipids include triglycerides, sterols and sterol esters (mainly cholesterol), sphingolipids (cerebrosides and gangliosides), as well as diglycerides, monoglycerides, free fatty acids, etc. Among them, triglycerides account for 56%-62% of the lipid components of MFGM and are the non-membrane lipid components of MFGM during the separation and purification process. In addition, gangliosides have the function of regulating intestinal immunity and participating in the development of the nervous system.

[0004] Proteins on MFGM account for 25%-70% of MFGM, 1%-4% of the total protein content in milk, and 1% of the mass of milk fat globules. The main proteins of MFGM include 8 kinds, namely xanthine dehydrogenase / oxidase (Xo / XDH), butyric acid protein (BTN), lactadhesin (MFDE8 / PAS6 / 7), mucin 1 (MUCl), MUC15 / PASⅢ, differentiation antigen cluster CD36 (cD36 / PASⅣ), adipose differentiation-related protein (ADPH) and fat-associated key protein (FABP), etc. The last two proteins are non-glycosylated proteins. Among them, the content of butyric acid protein on MGFM in breast milk is higher than that in cow's milk, and the content of CD-36 and lactadhesin is lower than that in cow's milk, and there is no difference in the rest. MFGM minor proteins include the glycosylated enzyme carbonic anhydrase, milk alkaline phosphatase (AP), lactoferrin, osteopontin (OPN) and lysozyme, and the latter three are mainly present in whey and to a lesser extent in M3.

[0005] However, there is currently a lack of relatively accurate methods for determining milk fat globule membrane protein in food.

[0006] Chinese patent application CN103232991A discloses a method for synthesizing a dopamine-modified magnetic mesoporous silicon material and its application, wherein the magnetic mesoporous silicon material is dispersed in a dopamine aqueous solution, a sodium monohydrogen phosphate-disodium hydrogen phosphate buffer solution is added, the reaction is performed, and the washing is performed; the material is dispersed in a sodium monohydrogen phosphate-disodium hydrogen phosphate buffer solution, and glutaraldehyde is added, and the resulting solution is oscillated and reacted at room temperature, and washed; the material is dispersed in a trypsin-containing ammonium bicarbonate buffer solution, and the reaction is performed, and the washing is performed. The material uses magnetic microspheres as a skeleton, provides magnetism, is conducive to subsequent separation and analysis, has good pore size distribution and biocompatibility, has a simple synthesis method and low cost, and can fix enzymes inside the pores for size exclusion selective enzymolysis. However, the material does not have good compatibility with phospholipids, making it difficult to be used in the present invention for separation.

[0007] Chinese patent CN110252256B discloses a magnetic ionic liquid, its application, modified activated carbon and its preparation method. The preparation method of the modified activated carbon comprises the following steps: mixing the magnetic ionic liquid with activated carbon, reacting at 30-90°C for 2-24h, and obtaining modified activated carbon after separation treatment. The modified activated carbon provided by the present invention significantly increases the saturation amount of VOC waste gas adsorbed, improves the adsorption efficiency, increases the autoignition point, significantly increases the lower explosion limit, and significantly reduces the possibility of combustion and explosion. In addition, the modified activated carbon can effectively reduce the oxygen content in the activated carbon adsorption environment, and can further reduce the possibility of combustion and explosion of the gas-powder mixture. However, although the patent discloses a modified activated carbon with good adsorption performance, the activated carbon material is an inorganic structure and it is difficult to have good compatibility with the organic phospholipid structure, thus limiting its application in the field of the present invention. Summary of the invention

[0008] The purpose of the present invention is to propose a method for determining milk fat globule membrane protein in food raw material powder, which can effectively determine the content of milk fat globule membrane protein in food raw material powder, has high accuracy, low detection limit, good linear relationship, good repeatability and stability, and provides a convenient and sensitive detection method for its detection.

[0009] The technical solution of the present invention is achieved in this way:

[0010] The present invention provides a method for determining milk fat globule membrane protein in food raw material powder, comprising the following steps:

[0011] (1) Sample pretreatment: Weigh the sample and add it to water, add modified magnetic particles, stir and adsorb, separate by magnet, add the solid back to the ammonium bicarbonate solution, perform ultrasonic extraction, separate by magnet, make the liquid constant, add dithiosulfate solution, react at constant temperature, add iodoacetamide solution after cooling to room temperature, react at constant temperature in the dark, add trypsin solution after cooling to room temperature, perform enzymatic hydrolysis in a constant temperature water bath, add formic acid solution after cooling to room temperature, mix well and centrifuge, filter through a microporous filter membrane, and obtain the sample solution;

[0012] (2) Chromatographic conditions: The chromatographic column is a C18 column with an inner diameter of ,granularity ; In the mobile phase, phase A is acetonitrile, phase B is 0.1% formic acid solution, gradient elution; flow rate, 0.2-0.3 mL / min; column temperature: 35-45℃; injection volume: 5μL;

[0013] (3) Mass spectrometry conditions: The ion source is an electrospray ion source; the scanning mode is positive ion; the nebulizer gas, curtain gas, auxiliary heating gas, and collision gas are all high-purity nitrogen;

[0014] (4) Drawing of standard curve: Inject the mixed standard working solution separately, draw the standard curve with the peak area ratio of the analytical compound and the internal standard compound as the ordinate and the concentration ratio of the analytical compound as the abscissa. The response values ​​of the characteristic peptides in the standard working solution and the sample solution to be tested should be within the linear response range of the instrument;

[0015] (5) Detection: The test solution in step (1) is detected using the detection conditions in step (2) and step (3), and the detection results are quantitatively analyzed and calculated using the standard working curve in step (4), thereby obtaining the content of milk fat globule membrane protein in the food raw material powder.

[0016] As a further improvement of the present invention, the modified magnetic particles are magnetic activated carbon coated with surface alkyl-modified polydopamine, and the preparation method is as follows:

[0017] S1. Add activated carbon powder to water, add ferric chloride and ferrous chloride, add aqueous ammonia dropwise under inert gas protection, heat and stir to react, filter, calcine, wash, and dry to obtain magnetic activated carbon;

[0018] S2. adding magnetic activated carbon to water, adding dopamine hydrochloride and a catalyst, heating and stirring the reaction, separating with a magnet, washing, and drying to obtain magnetic particles;

[0019] S3. Add the magnetic particles into dichloromethane, add alkali and chloroalkane, stir to react at room temperature, separate with a magnet, wash, and dry to obtain modified magnetic particles.

[0020] As a further improvement of the present invention, the mass ratio of the activated carbon powder, ferric chloride, ferrous chloride and ammonia water in step S1 is 10:3.24:1.26:7-10, the temperature of the heating and stirring reaction is 80-90°C, the time is 2-4h, the temperature of the calcination is 400-500°C, and the time is 1-2h; the mass ratio of the magnetic activated carbon, dopamine hydrochloride and catalyst in step S2 is 10:3-5:0.1-0.5, the catalyst is a Tris-HCl solution with a pH of 8.5-9.5, the temperature of the heating and stirring reaction is 40-45°C, and the time is 1-3h; the mass ratio of the magnetic particles, the base and the chloroalkane in step S3 is 10:3-4:1-2, the chloroalkane is selected from at least one of 1-chlorododecane, 1-chlorotetradecane, 1-chlorohexadecane and 1-chlorooctadecane, the base is NaOH, KOH or triethylamine, and the time of the reaction is 2-3h.

[0021] As a further improvement of the present invention, the concentration of the ammonium bicarbonate solution in step (1) is 45-55 mmol / L, the power of the ultrasonic extraction is 1000-2000 W, the time is 20-40 min, the concentration of the dithiosulfate solution is 90-110 mmol / L, the temperature of the isothermal reaction is 55-65°C, and the time is 0.5-1 h; the concentration of the iodoacetamide solution is 90-110 mmol / L, the temperature of the light-proof isothermal reaction is 25-35°C, and the time is 20-40 min, the concentration of the formic acid solution is 3-7 wt%, the concentration of the trypsin solution is 0.5-1.5 mg / mL, the temperature of the isothermal water bath enzymatic hydrolysis is 35-40°C, and the time is 10-12 h, and the pore size of the microporous filter membrane is .

[0022] As a further improvement of the present invention, the preparation method of the mixed standard working solution in step (4) is as follows: the mixed standard solution is diluted with a 0.2% formic acid-5% acetonitrile mixed solution to a mixed standard dilution of 10 μg / mL and 1 μg / mL, and then 0 μL, 2.5 μL, 5 μL, 10 μL, 25 μL of the mixed standard dilution of 1 μg / mL and 5 μL, 10 μL, 25 μL, 50 μL, 100 μL of the mixed standard dilution of 10 μg / mL are respectively taken, and 50 μL of the mixed internal standard working solution is added to each, and the volume is adjusted to 1 mL with 0.2% formic acid-5% acetonitrile to prepare standard working solutions with a series of concentrations of 0, 2.5, 5, 10, 25, 50, 100, 250, 500, and 1000 ng / mL.

[0023] As a further improvement of the present invention, the preparation method of the mixed internal standard working solution is as follows: a mixed internal standard is prepared with a 0.2% formic acid-5% acetonitrile mixed solution, and each internal standard is diluted to 10 μg / mL; the mixed internal standard is isotope-labeled XDH-VRH, isotope-labeled MUC1-EKH, isotope-labeled ADPN-DRH, isotope-labeled BTN-TRH and isotope-labeled PAS6 / 7-QRH, wherein the peptide chain of the isotope-labeled XDH-VRH is: , the isotope-labeled peptide chain of MUC1-EKH is , the peptide chain of isotope-labeled ADPN-DRH is The peptide chain of isotope-labeled BTN-TRH is , the isotope-labeled peptide chain of PAS6 / 7-QRH is .

[0024] As a further improvement of the present invention, the preparation method of the mixed standard solution is as follows: weigh specific peptide standards and use 0.2% formic acid-5% acetonitrile mixed solution to prepare mixed standard solutions of 500 μg / mL, respectively. The specific peptide standards are XDH-VR, MUC1-EK, ADPN-DR, BTN-TR and PAS6 / 7-QR, the peptide chain of XDH-VR is: VSLSTTGFYR, the peptide chain of MUC1-EK is: EGTTAEWVK, the peptide chain of ADPN-DR is: DSVASTITGVVDR, the peptide chain of BTN-TR is: TPLPLAGPPR, and the peptide chain of PAS6 / 7-QR is: QFQFIQVAGR.

[0025] As a further improvement of the present invention, the retention times of the characteristic peptides MUC1-EK, XDH-VR, BTN-TR, ADPN-DR, and PAS6 / 7-QR are 5.74 min, 6.09 min, 6.07 min, 6.26 min, and 6.32 min, respectively.

[0026] As a further improvement of the present invention, the calculation method formula of the characteristic peptide content in the sample is as follows, and the calculation result deducts the blank value: ; Where: X——the content of each characteristic peptide in the sample (respectively X MUC1-EK , X BTN-TR , X ADPN-DR , X PAS6 / 7-QR , X XDH-VR ), the unit is milligram per gram (mg / g); ——The mass of the sample, in grams (g); ——The constant volume of the sample, in milliliters (mL); ——The concentration of each characteristic peptide in the sample solution, in nanograms per milliliter (ng / mL).

[0027] As a further improvement of the present invention, the calculation formula for the content of milk fat globule membrane protein in the food raw material powder is as follows: 总 =W 粘蛋白-1 +W 嗜乳脂蛋白 +W 亲脂素 +W 乳凝集素 +W 黄嘌呤脱氢酶

[0028] Where: W 总 ——Total content of milk fat globule membrane protein in the sample, in milligrams per gram (mg / g); W 粘蛋白-1 ——The content of mucin-1 in the sample, , the unit is milligram per gram (mg / g); W 嗜乳脂蛋白 - the content of lactophilin in the sample, , the unit is milligram per gram (mg / g); W 亲脂素 - the content of lipophilin in the sample, , the unit is milligram per gram (mg / g); W 乳凝集素 - the content of lactadhesin in the sample, , the unit is milligram per gram (mg / g); W 黄嘌呤脱氢酶 - the content of xanthine dehydrogenase in the sample, , the unit is milligram per gram (mg / g).

[0029] The present invention has the following beneficial effects:

[0030] In the pretreatment process of the present invention, modified magnetic particles are added. The modified magnetic particles are magnetic activated carbon coated with alkyl-modified polydopamine on the surface. The modified magnetic particles not only have a large specific surface area and can increase the adsorption amount of protein in the sample, but also, after the surface is modified by polydopamine, the surface is reacted with chloroalkane, thereby introducing long-chain alkyl groups. The long-chain alkyl groups can have good binding ability with phospholipids, so that the modified magnetic particles can have good specific adsorption to milk fat globule membrane proteins in the raw materials, thereby effectively separating the milk fat globule membrane proteins in the raw materials.

[0031] The separated solid is then added back to the ammonium bicarbonate solution, which is a mild denaturant that can change the charge state of the protein, thereby promoting the dissociation of the protein from the milk fat globule membrane. The pH value of the ammonium bicarbonate solution is close to the isoelectric point of the protein, which helps to reduce the interaction between proteins and makes the protein easier to extract. In addition, the ammonium bicarbonate solution can also help destroy the secondary and tertiary structures of the protein, making it easier to be hydrolyzed by enzymes such as trypsin.

[0032] The method comprises the following steps: ultrasonically extracting the protein with an ammonium bicarbonate solution, cooling the solution to room temperature and fixing the volume, adding an isotope internal standard, subsequently adding dithiothreitol (DTT) to open the disulfide bonds in or between proteins, protecting the solution with iodoacetamide, adding an excess of alkaline trypsin to perform enzymatic hydrolysis at 37°C, selecting specific peptides, performing reverse phase chromatography separation, performing quadrupole tandem mass spectrometry, and performing internal standard method quantitative determination, thereby effectively determining the content of milk fat globule membrane protein in food raw material powder, having high accuracy, low detection limit, good linear relationship, good repeatability and good stability, and providing a convenient and sensitive detection method for the detection thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0034] Figure 1 is the multiple reaction monitoring chromatogram of the standard solution; and are the chromatograms of MUC1-EK, MUC1-EKH, BTN-TR, BTN-TRH, ADPN-DR, ADPN-DRH, XDH-VR, XDH-VRH, PAS6 / 7-QR, and PAS6 / 7-QRH, respectively;

[0035] Figure 2 These are the test result graphs of goat milk detected by the method of Example 1 in Test Example 1 of the present invention, which are the test result graphs of MUC1-EK, MUC1-EKH, BTN-TR, BTN-TRH, ADPN-DR, ADPN-DRH, XDH-VR, XDH-VRH, PAS6 / 7-QR, and PAS6 / 7-QRH, respectively. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part 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 creative work are within the scope of protection of the present invention.

[0037] Preparation Example 1 Preparation of modified magnetic particles

[0038] Here’s how:

[0039] S1. Add 10g activated carbon powder to 500mL water, add 3.24g ferric chloride and 1.26g ferrous chloride, add 7g ammonia water dropwise under nitrogen protection, heat to 80℃, stir to react for 2h, filter, calcine at 400℃ for 1h, wash, and dry to obtain magnetic activated carbon;

[0040] S2. Add 10 g of magnetic activated carbon to 200 mL of water, add 3 g of dopamine hydrochloride and 0.1 g of catalyst, heat to 40°C, stir to react for 1 h, separate with a magnet, wash, and dry to obtain magnetic particles;

[0041] The catalyst is a Tris-HCl solution with a pH of 8.5;

[0042] S3. 10 g of magnetic particles were added to 200 mL of dichloromethane, 3 g of triethylamine and 1 g of 1-chlorooctadecane, and the mixture was stirred at room temperature for 2 h. The mixture was separated by a magnet, washed and dried to obtain modified magnetic particles.

[0043] Preparation Example 2 Preparation of modified magnetic particles

[0044] Here’s how:

[0045] S1. Add 10g activated carbon powder to 500mL water, add 3.24g ferric chloride and 1.26g ferrous chloride, add 10g ammonia water dropwise under nitrogen protection, heat to 90℃, stir to react for 4h, filter, calcine at 500℃ for 2h, wash, and dry to obtain magnetic activated carbon;

[0046] S2. Add 10 g of magnetic activated carbon to 200 mL of water, add 5 g of dopamine hydrochloride and 0.5 g of catalyst, heat to 45 ° C, stir and react for 3 h, separate with a magnet, wash, and dry to obtain magnetic particles;

[0047] The catalyst is a Tris-HCl solution with a pH of 9.5;

[0048] S3. 10 g of magnetic particles were added to 200 mL of dichloromethane, 4 g of triethylamine and 2 g of 1-chlorododecane, and the mixture was stirred at room temperature for 3 h. The mixture was separated by a magnet, washed and dried to obtain modified magnetic particles.

[0049] Preparation Example 3 Preparation of modified magnetic particles

[0050] Here’s how:

[0051] S1. Add 10g activated carbon powder to 500mL water, add 3.24g ferric chloride and 1.26g ferrous chloride, add 8g ammonia water dropwise under nitrogen protection, heat to 85℃, stir to react for 3h, filter, calcine at 450℃ for 1-2h, wash, and dry to obtain magnetic activated carbon;

[0052] S2. Add 10 g of magnetic activated carbon to 200 mL of water, add 4 g of dopamine hydrochloride and 0.3 g of catalyst, heat to 42 ° C, stir and react for 2 h, separate with a magnet, wash, and dry to obtain magnetic particles;

[0053] The catalyst is a Tris-HCl solution with a pH of 9;

[0054] S3. 10 g of magnetic particles were added to 200 mL of dichloromethane, 3.5 g of triethylamine and 1.5 g of 1-chlorohexadecane, and the mixture was stirred at room temperature for 2.5 h. The mixture was separated by a magnet, washed, and dried to obtain modified magnetic particles.

[0055] Comparative Preparation Example 1

[0056] Compared with Preparation Example 3, the difference is that step S3 is not performed.

[0057] Here’s how:

[0058] S1. Add 10g activated carbon powder to 500mL water, add 3.24g ferric chloride and 1.26g ferrous chloride, add 8g ammonia water dropwise under nitrogen protection, heat to 85℃, stir to react for 3h, filter, calcine at 450℃ for 1-2h, wash, and dry to obtain magnetic activated carbon;

[0059] S2. Add 10 g of magnetic activated carbon to 200 mL of water, add 4 g of dopamine hydrochloride and 0.3 g of catalyst, heat to 42 ° C, stir and react for 2 h, separate with a magnet, wash, and dry to obtain magnetic particles;

[0060] The catalyst is a Tris-HCl solution with a pH of 9. Example 1

[0061] 1. Reagents: Ammonium bicarbonate: high-grade; Acetonitrile: chromatographic grade; Formic acid: chromatographic grade. Dithiothreitol: purity 99.5%, purchased from Solarbio. Iodoacetamide: purchased from Solarbio. Bovine trypsin: purchased from Sigma, ≥10,000 BAEE units / mg protein

[0062] 2. Solution preparation

[0063] (1) 0.1% formic acid solution: Use a pipette to transfer 1.0 mL of formic acid into a 1000 mL volumetric flask, dilute with water and make up to volume.

[0064] (2) 0.2% formic acid-5% acetonitrile mixed solution: Use a pipette to transfer 0.2 mL of formic acid and 5 mL of acetonitrile into a 100 mL volumetric flask, dilute with water and make up to the mark.

[0065] (3) 5% formic acid solution: Pipette 5 mL of formic acid into a 100 mL volumetric flask, dilute with water and make up to volume.

[0066] (4) Ammonium bicarbonate solution (50 mmol): Weigh 1.976 g of ammonium bicarbonate into a 500 mL volumetric flask, dissolve it in water and make up to the mark.

[0067] (5) Dithiosulfate solution (100 mmol / L): Weigh 0.155 g of dithiosulfate in a 10 mL volumetric flask, dissolve it with ammonium bicarbonate solution and dilute to the mark. Store at 4°C.

[0068] (6) Iodoacetamide solution (100 mmol / L): Weigh 0.185 g of iodoacetamide into a 10 mL volumetric flask, dissolve it with ammonium bicarbonate solution and make up to the mark. Keep away from light and use it immediately.

[0069] (7) Bovine trypsin solution: Accurately weigh 5 mg of bovine trypsin into a 5 mL volumetric flask, dissolve it with ammonium bicarbonate solution and make up to the mark to prepare a 1.0 mg / mL trypsin solution. Transfer it to a plastic tube and store it at -20°C.

[0070] 3. Standard substances and standard solutions

[0071] (1) Characteristic peptide standards: XDH-VR (peptide chain is VSLSTTGFYR): purity is 97.37%, peptide content is 68.26%; MUC1-EK (peptide chain is EGTTAEWVK): purity is 95.91%, peptide content is 74.24%; ADPN-DR (peptide chain is DSVASTITGVVDR): purity is 95.48%, peptide content is 77.09%; BTN-TR (peptide chain is TPLPLAGPPR): purity is 97.99%, peptide content is 76.16%; PAS6 / 7-QR (peptide chain is QFQFIQVAGR): purity is 96.87%, peptide content is 77.30%. Specific peptides were purchased from biomatik.

[0072] (2) Internal standard: XDH-VRH (peptide chain ): Purity is 97.17%; MUC1-EKH (peptide chain is ): Purity is 96.17%; ADPN-DRH (peptide chain is ): Purity is 98.55%; BTN-TRH (peptide chain is ): Purity is 95.73%; PAS6 / 7-QRH (peptide chain is ): Purity: 95.73%. All were purchased from Biomatik Company.

[0073] (3) Preparation of standard stock solution: Weigh an appropriate amount of specific peptide standard and prepare a 500 μg / mL standard solution with a 0.2% formic acid-5% acetonitrile mixed solution. Transfer the solution to a plastic tube and store at -20°C.

[0074] (4) Preparation of internal standard stock solution: Weigh an appropriate amount of internal standard and prepare a 500 μg / mL internal standard solution using a 0.2% formic acid-5% acetonitrile mixed solution. Transfer the solution to a plastic tube and store at -20°C.

[0075] (5) Preparation of mixed standard intermediate solution: Take a certain volume of the standard stock solution and dilute it to 100 μg / mL with a 0.2% formic acid-5% acetonitrile mixed solution and store it at -20°C.

[0076] (6) Preparation of mixed internal standard intermediate solution: Take a certain volume of internal standard stock solution and dilute it to 10 μg / mL with 0.2% formic acid-5% acetonitrile mixed solution and store it at -20℃.

[0077] (7) Preparation of mixed internal standard working solution: dilute the mixed internal standard intermediate solution to 1.0 μg / mL with a 0.2% formic acid-5% acetonitrile mixed solution.

[0078] (8) Preparation of mixed standard working solution: First, dilute the mixed standard intermediate solution to 10 μg / mL and 1 μg / ml with 0.2% formic acid-5% acetonitrile mixed solution. Then, pipette 0 μL, 2.5 μL, 5 μL, 10 μL, 25 μL of the mixed standard dilution (1 μg / mL) and 5 μL, 10 μL, 25 μL, 50 μL, 100 μL of the mixed standard dilution (10 μg / mL), add 50 μL of internal standard working solution, and dilute to 1 mL with 0.2% formic acid-5% acetonitrile to prepare standard working solutions with concentrations of 0, 2.5, 5, 10, 25, 50, 100, 250, 500, 1000 ng / mL before use.

[0079] 5. Instruments and equipment

[0080] High performance liquid chromatography-tandem quadrupole mass spectrometer; ultrasonic cleaner; vortex mixer; electronic balance: with a sensitivity of 0.00001g and 0.0001g; constant temperature water bath oscillator; refrigerated high-speed centrifuge (maximum speed 12000 r / min); disposable microporous filter head: with 0.22μm water-based microporous filter membrane (polyether sulfoxide).

[0081] 6. Sample preparation and preservation

[0082] Take 500g of representative sample, mix well, put into a clean sample container, seal and mark. Prevent the sample from being contaminated or the analyte content from changing.

[0083] 7. Determination steps

[0084] (1) Sample pretreatment

[0085] Weigh 0.1g of the mixed sample (accurate to 0.0001 g) in a 50 mL centrifuge tube, add 30 mL of water, add 1g of the modified magnetic particles prepared in Preparation Example 3, stir for adsorption, separate with a magnet, add the solid back to 30 mL of ammonium bicarbonate solution, mix on a vortex mixer for 1 min, place on an ultrasonic cleaner for ultrasonic extraction for 30 min, separate the solid with a magnet, transfer the liquid to a 50 mL volumetric flask, wash the solid with ammonium bicarbonate solution, add the washing liquid to the volumetric flask, and dilute to the mark with ammonium bicarbonate solution. Transfer 200 μL of the sample solution to a 1.5 mL centrifuge tube, add 50 μL of the mixed internal standard working solution, add 100 μL of dithiothreitol solution, react at 60°C for 1 h, cool to room temperature, add 200 μL of iodoacetamide solution, react at 30°C in the dark for 30 min, cool to room temperature, add 10 μL of trypsin solution, and hydrolyze overnight in a constant temperature water bath at 37°C. After cooling to room temperature, add 440 μL of formic acid solution, mix well, centrifuge at 10000 r / min for 5 min, and then filter through a 0.22 μm water-based microporous filter membrane to obtain the sample test solution.

[0086] (2) Determination

[0087] 1) Liquid chromatography conditions

[0088] a) Chromatographic column: C18 column, (inner diameter), particle size or equivalent;

[0089] b) Mobile phase: Gradient elution program see Table 1;

[0090] c) Flow rate: 0.25 mL / min;

[0091] d) Column temperature: 40°C;

[0092] e) Injection volume: 5μL.

[0093] Table 1 Mobile phase gradient elution program

[0094]

[0095] 2) Mass spectrometry conditions

[0096] a) Ion source: electrospray ion source;

[0097] b) Scanning mode: positive ion;

[0098] c) Detection method: multiple reaction monitoring (MRM);

[0099] d) Nebulizer gas, curtain gas, auxiliary heating gas and collision gas are all high-purity nitrogen; the flow rate of each gas should be adjusted before use so that the mass spectrometer sensitivity meets the detection requirements;

[0100] e) Parameters such as spray voltage, declustering voltage, collision energy, etc. should be optimized to achieve optimal sensitivity. The reference conditions are as follows:

[0101] Scanning mode: positive ion scanning; detection mode: multiple reaction monitoring (MRM); curtain gas (CUR): 20 Psi; nebulizer gas (GS1): 50 Psi; auxiliary heating gas (GS2): 55 Psi; collision gas (CAD): 7 Psi; electrospray voltage (IS): 5000 V; ion source temperature (TEM): 450 °C; qualitative ion pairs, quantitative ion pairs, collision energy, declustering voltage, collision cell entrance and exit voltages, and collision cell exit voltages are shown in Table 2.

[0102] Table 2 Multiple reaction monitoring conditions

[0103]

[0104] Note:" ” is the quantitative ion.

[0105] (3) Liquid chromatography-mass spectrometry / mass spectrometry detection

[0106] 1) Quantitative determination

[0107] For quantification by the internal standard method, the mixed standard working solution is injected separately, and the standard curve is drawn with the peak area ratio of the analytical compound and the internal standard compound as the vertical axis and the concentration ratio of the analytical compound as the horizontal axis. The sample is quantified using the standard working curve. The response values ​​of the characteristic peptides in the standard working solution and the sample solution to be tested should be within the linear response range of the instrument. If the content exceeds the range of the standard curve, it should be diluted to an appropriate concentration before analysis. Under the above chromatographic conditions, the reference retention times of the characteristic peptides MUC1-EK, XDH-VR, BTN-TR, ADPN-DR, and PAS6 / 7-QR are 5.74min, 6.09min, 6.07min, 6.26min, and 6.32min, respectively. The multiple reaction monitoring chromatogram of the standard solution is shown in Figure 1 .

[0108] 2) Qualitative determination

[0109] Under the same experimental conditions, the retention time of the chromatographic peak of the analyte in the sample solution is the same as that of the standard working solution, and the selected ion pairs all appear in the sample solution spectrum after background subtraction. When the relative abundance of each qualifier ion is compared with the relative abundance of the standard ion, the deviation does not exceed the range specified in Table 2, then it can be determined that the corresponding analyte exists in the sample.

[0110] Table 2 Maximum allowable deviation of relative ion abundance for qualitative confirmation

[0111]

[0112] 3) Blank test

[0113] Except that no sample is added, the above measurement steps are followed.

[0114] 4) Calculation and presentation of results

[0115] Calculate the content of characteristic peptides in the sample using a chromatography data processor or according to formula (1), and the blank value must be deducted from the calculation result.

[0116]

[0117] Where: X——the content of each characteristic peptide in the sample (respectively X MUC1-EK , X BTN-TR , X ADPN-DR , X PAS6 / 7-QR , X XDH-VR ), the unit is milligram per gram (mg / g); ——The mass of the sample, in grams (g); ——The constant volume of the sample, in milliliters (mL); ——The concentration of each characteristic peptide in the sample solution, in nanograms per milliliter (ng / mL).

[0118] Carry out two parallel measurements at the same time and take the arithmetic mean as the measurement result.

[0119] The total content of milk fat globule membrane protein in the sample is calculated according to formula (2):

[0120]

[0121] Where: W 总 ——Total content of milk fat globule membrane protein in the sample, in milligrams per gram (mg / g); W 粘蛋白-1 ——The content of mucin-1 in the sample, , the unit is milligram per gram (mg / g); W 嗜乳脂蛋白 - the content of lactophilin in the sample, , the unit is milligram per gram (mg / g); W 亲脂素 - the content of lipophilin in the sample, , the unit is milligram per gram (mg / g); W 乳凝集素 - the content of lactadhesin in the sample, , the unit is milligram per gram (mg / g); W 黄嘌呤脱氢酶 - the content of xanthine dehydrogenase in the sample, , the unit is milligram per gram (mg / g).

[0122] 8. Method validation

[0123] The results are shown in Table 3.

[0124] Table 3

[0125]

[0126] It can be seen from the above table that the method of the present invention has low detection limit and quantification limit, high linear correlation coefficient, small RSD and wide linear range.

[0127] Comparative Example 1

[0128] Compared with Example 1, the difference is that the sample pretreatment method is different.

[0129] The details are as follows: weigh 0.1g of the mixed sample (accurate to 0.0001 g) into a 50 mL centrifuge tube, add 30 mL of ammonium bicarbonate solution, mix well on a vortex mixer for 1 min, place it on an ultrasonic cleaner for ultrasonic extraction for 30 min, transfer it to a 50 mL volumetric flask, and dilute it to the mark with ammonium bicarbonate solution. Pipette 200 μL of the sample solution into a 1.5 mL centrifuge tube, add 50 μL of the mixed internal standard working solution, add 100 μL of dithiothreitol solution, react at 60°C for 1 h, cool to room temperature, add 200 μL of iodoacetamide solution, react at 30°C in the dark for 30 min, cool to room temperature, add 10 μL of trypsin solution, and enzymolyze overnight in a constant temperature water bath at 37°C. After cooling to room temperature, add 440 μL of formic acid solution, mix well, centrifuge at 10000 r / min for 5 min, and then filter through a 0.22 μm water system microporous filter membrane to obtain the sample test solution.

[0130] Comparative Example 2

[0131] Compared with Example 1, the difference is that the magnetic particles prepared in Comparative Preparation Example 1 replace the modified magnetic particles.

[0132] Test Example 1 Method Verification

[0133] The same batch of raw milk samples (Seperex TM The results of the determination of the content of milk fat globule membrane proteins (including mucin-1, butyrophilin, lipophilin, lactadhesin, and xanthine dehydrogenase) in MULPROTECTOR (wherein the content of MFGM-milk fat globule membrane proteins is 50-60 mg / g) (detected by the methods in Example 1, Comparative Example 1 or 2, respectively), and the average recovery and precision of the spiked detection results at two levels of 5 mg / g (mucin-1, butyrophilin, lipophilin, lactadhesin, and xanthine dehydrogenase were all 1 mg / g) and 10 mg / g (mucin-1, butyrophilin, lipophilin, lactadhesin, and xanthine dehydrogenase were all 2 mg / g) are shown in Table 4.

[0134] Table 4

[0135]

[0136] As can be seen from the table above, the content of milk fat globule membrane protein in the raw milk detected by Example 1 of the present invention is 55.6 mg / g, and the detection result is shown in the figure below: Figure 2 ; The average recovery rate was 93.1-94.9%, and the precision was 3.1-3.7%.

[0137] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining milk fat globule membrane protein in food raw material powder, characterized in that: The following steps are involved: (1) Sample pretreatment: weigh the sample and add it to water, add modified magnetic particles, stir and adsorb, separate by magnet, add the solid back into ammonium bicarbonate solution, perform ultrasonic extraction, separate by magnet, adjust the liquid to volume, add dithiosulfate solution, react at constant temperature, add iodoacetamide solution after cooling to room temperature, react at constant temperature in the dark, add trypsin solution after cooling to room temperature, perform enzymatic hydrolysis in a constant temperature water bath, add formic acid solution after cooling to room temperature, mix well and centrifuge, filter through a microporous filter membrane to obtain the sample test solution; the modified magnetic particles are magnetic activated carbon coated with alkyl-modified polydopamine on the surface; (2) Chromatographic conditions: The chromatographic column is a C18 column; the mobile phase is acetonitrile, the mobile phase is 0.1% formic acid solution, and the gradient elution is performed; the flow rate is 0.2-0.3 mL / min; (3) Mass spectrometry conditions: the ion source is an electrospray ion source; the scanning mode is positive ion; (4) Drawing of standard curve: Inject the mixed standard working solution separately, draw the standard curve with the peak area ratio of the analytical compound and the internal standard compound as the ordinate and the concentration ratio of the analytical compound as the abscissa. The response values ​​of the characteristic peptides in the standard working solution and the sample solution to be tested should be within the linear response range of the instrument; (5) Detection: the test solution in step (1) is detected using the detection conditions in step (2) and step (3), and the detection results are quantitatively analyzed and calculated using the standard working curve in step (4), thereby obtaining the content of milk fat globule membrane protein in the food raw material powder; The preparation method of the modified magnetic particles is as follows: S1. Add activated carbon powder to water, add ferric chloride and ferrous chloride, add aqueous ammonia dropwise under inert gas protection, heat and stir to react, filter, calcine, wash, and dry to obtain magnetic activated carbon; S2. adding magnetic activated carbon to water, adding dopamine hydrochloride and a catalyst, heating and stirring the reaction, separating with a magnet, washing, and drying to obtain magnetic particles; S3. The magnetic particles are added to dichloromethane, a base and an alkyl chloride are added, the reaction is stirred at room temperature, separated by a magnet, washed, and dried to obtain modified magnetic particles; The mass ratio of activated carbon powder, ferric chloride, ferrous chloride and ammonia water in step S1 is 10:3.24:1.26:7-10, the temperature of the heating and stirring reaction is 80-90°C, the time is 2-4h, and the temperature of the calcination is 400-500°C, and the time is 1-2h; the mass ratio of magnetic activated carbon, dopamine hydrochloride and catalyst in step S2 is 10:3-5:0.1-0.5, the catalyst is Tris-HCl solution with pH=8.5-9.5, the temperature of the heating and stirring reaction is 40-45°C, and the time is 1-3h; the mass ratio of magnetic particles, base and chloroalkane in step S3 is 10:3-4:1-2, the chloroalkane is selected from at least one of 1-chlorododecane, 1-chlorotetradecane, 1-chlorohexadecane and 1-chlorooctadecane, the base is NaOH, KOH or triethylamine, and the time of the stirring reaction at room temperature is 2-3h; The substances tested are listed in the following table: 。 2. The measuring method according to claim 1, characterized in that In step (1), the concentration of the ammonium bicarbonate solution is 45-55 mmol / L, the power of the ultrasonic extraction is 1000-2000 W, the time is 20-40 min, the concentration of the dithiocarbamide solution is 90-110 mmol / L, the temperature of the isothermal reaction is 55-65 ° C, and the time is 0.5-1 h; the concentration of the iodoacetamide solution is 90-110 mmol / L, the temperature of the light-proof isothermal reaction is 25-35 ° C, and the time is 20-40 min. The concentration of the formic acid solution is 3-7wt%, the concentration of the trypsin solution is 0.5-1.5 mg / mL, the temperature of the constant temperature water bath enzymatic hydrolysis is 35-40 ° C, and the time is 10-12 h. The pore size of the microporous filter membrane is 0.22 m.

3. The measuring method according to claim 1, characterized in that The preparation method of the mixed standard working solution in step (4) is as follows: dilute the mixed standard solution with a 0.2% formic acid-5% acetonitrile mixed solution to a mixed standard dilution of 10 μg / mL and 1 μg / mL, then respectively take 0 μL, 2.5 μL, 5 μL, 10 μL, 25 μL of the 1 μg / mL mixed standard dilution and 5 μL, 10 μL, 25 μL, 50 μL, 100 μL of the 10 μg / mL mixed standard dilution, add 50 μL of the mixed internal standard working solution, dilute to 1 mL with 0.2% formic acid-5% acetonitrile, and prepare standard working solutions with a series of concentrations of 0, 2.5, 5, 10, 25, 50, 100, 250, 500, and 1000 ng / mL.

4. The measuring method according to claim 3, characterized in that The preparation method of the mixed internal standard working solution is as follows: a mixed internal standard is prepared with a 0.2% formic acid-5% acetonitrile mixed solution, and each internal standard is diluted to 10 μg / mL; the mixed internal standard is isotope-labeled XDH-VRH, isotope-labeled MUC1-EKH, isotope-labeled ADPN-DRH, isotope-labeled BTN-TRH and isotope-labeled PAS6 / 7-QRH.

5. The measuring method according to claim 3, characterized in that The preparation method of the mixed standard solution is as follows: weigh specific peptide standards and use 0.2% formic acid-5% acetonitrile mixed solution to prepare mixed standard solutions of 500 μg / mL, respectively. The specific peptide standards are XDH-VR, MUC1-EK, ADPN-DR, BTN-TR and PAS6 / 7-QR.

6. The measuring method according to claim 5, characterized in that The retention times of the characteristic peptides MUC1-EK, XDH-VR, BTN-TR, ADPN-DR, and PAS6 / 7-QR are 5.74 min, 6.09 min, 6.07 min, 6.26 min, and 6.32 min, respectively.

7. The measuring method according to claim 6, characterized in that The calculation method formula of the characteristic peptide content in the sample is as follows, and the blank value is deducted from the calculation result: ； Where: X——the content of each characteristic peptide in the sample, which are X MUC1-EK , X BTN-TR , X ADPN-DR , X PAS6 / 7-QR , X XDH-VR , in milligrams per gram; ——The mass of the sample, in grams; ——The constant volume of the sample, in milliliters; ——The concentration of each characteristic peptide in the sample solution, in nanograms per milliliter.

8. The measuring method according to claim 7, characterized in that The calculation formula of the content of milk fat globule membrane protein in the food raw material powder is as follows: IN 总 =In 粘蛋白-1 +W 嗜乳脂蛋白 +W 亲脂素 +W 乳凝集素 +W 黄嘌呤脱氢酶 ; Where: W 总 ——Total content of milk fat globule membrane protein in the sample, in milligrams per gram; W 粘蛋白-1 ——The content of mucin-1 in the sample, W 粘蛋白-1 =X MUC1-EK ×58091.82 / 1020.50, in milligrams per gram; W 嗜乳脂蛋白 ——The content of lactophilin in the sample, W 嗜乳脂蛋白 =X BTN-TR ×59276.71 / 1018.60, in milligrams per gram; W 亲脂素 ——The content of lipophilin in the sample, W 亲脂素 =X ADPN-DR ×49368.08 / 1319.6801, in milligrams per gram; W 乳凝集素 ——The content of lactadhesin in the sample, W 乳凝集素 =X PAS6 / 7-QR ×47410.83 / 1193.6425, in milligrams per gram; W 黄嘌呤脱氢酶 ——The content of xanthine dehydrogenase in the sample, W 黄嘌呤脱氢酶 =X XDH-VR ×146790.47 / 1130.584, unit is milligrams per gram.

Images