Method for measuring content of lactoferrin in dairy product

Abstract

The invention relates to a method for determining the content of lactoferrin in a dairy product. According to the method, through aptamer enrichment and composite enzymolysis treatment, matrix interference can be effectively reduced, and the content of lactoferrin in the dairy product can be more accurately and rapidly detected.

Description

Technical Field

[0001] This invention relates generally to the food industry, and specifically to a method for determining the lactoferrin content in dairy products. Background Technology

[0002] Lactoferrin is a multifunctional glycoprotein naturally found in the milk (especially colostrum) and other secretions of mammals. As a core functional active ingredient in dairy products, it possesses a variety of important physiological functions, including broad-spectrum antibacterial and antiviral activity, immune regulation, iron absorption promotion, gut microbiota regulation, and cell growth promotion. These unique biological characteristics make lactoferrin a key indicator for evaluating the nutritional and functional value of dairy products, especially infant formula, functional liquid milk, and health-promoting dairy products, and have also garnered significant attention in the high-end food, nutritional supplement, and even biopharmaceutical fields. With consumers' increasing demand for the nutritional and health benefits of dairy products, and the gradual improvement of relevant national standards and industry regulations, the accurate addition and content monitoring of lactoferrin in dairy products has become a crucial aspect of product quality control, market regulation, and consumer rights protection.

[0003] However, the complex composition of dairy products, rich in casein, whey protein, fat, lactose, and minerals, can severely interfere with the analysis of the target protein (lactoferrin) during detection. This places extremely high demands on the specificity, sensitivity, and accuracy of detection methods. Currently, commonly used methods in the industry mainly include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS / MS), but each of these mainstream technical routes has its own limitations.

[0004] Specifically, while ELISA is relatively simple to operate, it relies on the specific reaction between antigen and antibody, making it susceptible to cross-reaction interference in practical applications. Furthermore, batch variations, stability, and cost of the antibodies themselves directly affect the repeatability and reliability of the test results. HPLC primarily uses a UV detector for quantitative analysis. Since many coexisting proteins in dairy products absorb in the UV range, peak overlap and insufficient resolution can easily occur, leading to significant quantitative errors. LC-MS / MS, with its high specificity and sensitivity, theoretically has significant advantages. However, its pretreatment process is complex and lengthy, typically requiring time-consuming enzymatic digestion and stringent sample purification steps. More importantly, existing sample pretreatment techniques lack the specific enrichment capacity for lactoferrin, making it difficult to efficiently and selectively capture the target protein and remove interfering components in complex matrices. This results in overall low efficiency, making it unsuitable for large-scale, rapid, and accurate screening applications.

[0005] In summary, given the important nutritional value of lactoferrin and increasingly stringent market regulations, existing detection technologies still have significant shortcomings in effectively purifying the complex matrices of dairy products, achieving highly selective enrichment of lactoferrin, and balancing accuracy, specificity, throughput, and cost. Therefore, there is an urgent need in this field to develop a new method for the determination of lactoferrin that is faster, more robust, and provides accurate and reliable results. Summary of the Invention

[0006] This invention was made in view of the aforementioned problems existing in the prior art.

[0007] Specifically, the present invention is achieved by the following:

[0008] 1. A method for determining the lactoferrin content in dairy products, the method comprising the following steps:

[0009] (1) Provide a sample of the dairy product to be tested, wherein the dairy product is in the form of liquid, particulate solid or block solid;

[0010] (2) Pretreatment: The sample is pretreated to obtain a pretreated aqueous solution, the pretreatment comprising:

[0011] The steps of diluting or dissolving the dairy product sample to obtain a homogeneous solution; and

[0012] The homogenized solution is subjected to a step of removing impurities such as proteins and fats to obtain a pretreated sample aqueous solution;

[0013] (3) Lactoferrin-specific enrichment: Magnetic nanomaterials with surface-modified lactoferrin aptamers are contacted with a pretreated sample aqueous solution to adsorb and separate lactoferrin. Then, the magnetic nanomaterials are separated from the solution, and the bound lactoferrin is eluted from the magnetic nanomaterials to obtain a lactoferrin enrichment solution.

[0014] (4) Enzymatic hydrolysis: After optionally adjusting the pH of the lactoferrin enrichment solution, the lactoferrin is enzymatically hydrolyzed by a complex enzyme system of trypsin and proteinase K to degrade the lactoferrin into characteristic peptides, thereby obtaining a characteristic peptide solution.

[0015] (5) Peptide purification: The characteristic peptide solution is purified by solid phase extraction column, the solvent is removed from the eluent and the resulting solid is dissolved to obtain the characteristic peptide purified solution.

[0016] (6) Chromatographic and mass spectrometric detection: Using an ultra-high performance liquid chromatography-tandem mass spectrometry system, characteristic ion pairs of characteristic peptides were detected in multiple reaction monitoring mode, and the chromatographic peak area of ​​lactoferrin characteristic peptides was determined; and

[0017] (7) Calculate lactoferrin content: Calculate the lactoferrin content in the dairy product sample based on the peak area.

[0018] 2. The method as described in Project 1, wherein the dairy product is liquid milk such as whole milk, yogurt, colostrum, milk powder such as infant formula, maternal milk powder, adult milk powder, and milk powder for the middle-aged and elderly, protein powder, cheese such as natural cheese, processed cheese / functional cheese, milk tablets, or dairy beverages.

[0019] 3. The method described in any one of items 1-2, wherein...

[0020] When the dairy product sample is a liquid, it is diluted with phosphate buffer to obtain a homogeneous solution.

[0021] When the dairy product sample is a particulate solid, it is dissolved in phosphate buffer to obtain a homogeneous solution.

[0022] When the dairy product sample is a blocky solid, it is crushed and then dissolved in phosphate buffer to obtain a homogeneous solution.

[0023] The conjugate acid and base in the phosphate buffer solution are sodium dihydrogen phosphate and disodium hydrogen phosphate, or potassium dihydrogen phosphate and dipotassium hydrogen phosphate; the concentration of the phosphate buffer solution is 0.005-0.05 mol / L; the concentration refers to the total concentration of the conjugate acid and base in the buffer solution; the pH value of the phosphate buffer solution is 5.8-7.8, preferably 6.5-7.5;

[0024] When the dairy product is liquid, phosphate buffer should be used at a rate of 1-5 mL per 1 mL of dairy product sample. When the dairy product is solid, such as particulate or blocky solid, phosphate buffer should be used at a rate of 1-30 mL per 1 g of dairy product sample.

[0025] 4. The method described in any one of items 1-3, wherein:

[0026] Protein removal is performed by precipitation; and

[0027] Fat removal is carried out by centrifugation and / or extraction with organic solvents, such as nonpolar solvents like n-hexane, diethyl ether, and / or petroleum ether, preferably n-hexane.

[0028] 5. The method of any one of items 1-4, wherein the pretreatment comprises: adding an aqueous solution of trichloroacetic acid to the obtained homogeneous solution, mixing thoroughly and allowing it to stand, then centrifuging, taking the supernatant, and obtaining the pretreated sample aqueous solution.

[0029] 6. The method of any one of items 1-5, wherein the magnetic nanomaterial is iron(II,III) oxide (Fe3O4), neodymium iron boron magnet, or a mixture thereof, such as iron(II,III) oxide (Fe3O4).

[0030] 7. The method of any one of items 1-6, wherein the magnetic nanomaterial has a particle size of 1-500 nm, for example 10-100 nm.

[0031] 8. The method of any one of items 1-7, wherein the lactoferrin aptamer is a DNA aptamer or an RNA aptamer, preferably a DNA aptamer, and preferably, the DNA aptamer has the following nucleotide sequence: 5'-AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT-3'.

[0032] 9. The method of any one of items 1-8, wherein the magnetic nanomaterial with surface-modified lactoferrin aptamer is prepared by:

[0033] Magnetic nanoparticles are surface-treated with coupling agents such as silane coupling agents (e.g., 3-aminopropyltriethoxysilane) to obtain surface-aminated magnetic nanoparticles; and

[0034] The surface amino groups of the surface-amino-treated magnetic nanoparticles are chemically linked to the carboxyl groups of the lactoferrin aptamer.

[0035] The connection is carried out, for example, in a medium in the presence of a carbodiimide crosslinking agent (e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and an activating enhancer (e.g., N-hydroxysuccinimide), the medium being, for example, 2-(N-morpholino)ethanesulfonic acid (MES) buffer.

[0036] 10. The method as described in any one of items 1-9, wherein in step (3),

[0037] The contact is performed at a temperature of 25-40°C, for example 35-37°C, for 5-60 minutes, for example 20-30 minutes;

[0038] The separation of magnetic nanomaterials from solution is achieved by the action of an external magnetic field.

[0039] The lactoferrin bound to the magnetic nanomaterial is eluted using a tris(hydroxymethyl)aminomethane (Tris)-HCl buffer solution with a pH of, for example, 7.0-9.0, or 8.5, and containing 0.3-0.8 mol / L NaCl.

[0040] 11. The method as described in any one of items 1-10, wherein in step (4),

[0041] Optionally, the pH of the lactoferrin enrichment solution is adjusted to 7.8-8.5 before enzymatic hydrolysis;

[0042] The mass ratio of trypsin to proteinase K is 0.1:1-10:1;

[0043] The total concentration of added enzymes was 0.1-10 mg / mL;

[0044] The mass ratio of enzyme to lactoferrin substrate is 1:10 to 1:100;

[0045] The enzymatic hydrolysis reaction is carried out at a temperature of 30-60℃;

[0046] The enzymatic hydrolysis reaction is carried out for 1-24 hours, after which a terminator such as formic acid is added to terminate the enzymatic hydrolysis reaction.

[0047] 12. The method as described in any one of items 1-11, wherein in step (5),

[0048] The solid-phase extraction column is a C18 solid-phase extraction column;

[0049] The purification is performed by gradient elution using acetonitrile-water solutions of different concentrations. The gradient elution can be performed, for example, as follows: first elute with 5% v / v acetonitrile-water solution; then elute with 80% v / v acetonitrile-water solution.

[0050] Before elution, the solid-phase extraction column was activated sequentially with methanol and ultrapure water;

[0051] After elution, the eluent is collected and the solvent is removed from the collected eluent; the solvent can be removed by blowing it dry with nitrogen.

[0052] The dissolution was performed using an aqueous formic acid solution.

[0053] 13. The method as described in any of items 1-12, wherein in step (6),

[0054] The ultra-high performance liquid chromatography uses a C18 column;

[0055] Mobile phase A is a 0.1% v / v formic acid aqueous solution, and mobile phase B is a 0.1% v / v formic acid acetonitrile solution;

[0056] The gradient elution program is as follows: 0-5 min 5%-20% B, 5-10 min 20%-40% B, 10-12 min 40%-90% B, 12-15 min 90% B, 15-16 min 90%-5% B, 16-20 min 5% B;

[0057] The characteristic ion pair is a parent ion with m / z 835.4 and daughter ions with m / z 926.5 and m / z 789.4;

[0058] The mass spectrometry parameters of the multi-reaction monitoring mode may include, for example, an electrospray ionization source, positive ion mode, capillary voltage of 3.2 kV, ion source temperature of 110 °C, desolvation gas temperature of 380 °C, desolvation gas flow rate of 900 L / h, collision gas as argon, and collision energies of 25 eV and 22 eV, respectively.

[0059] 14. The method described in any one of items 1-13, in step (7) using a calibration curve established by lactoferrin standards, calculates the content of lactoferrin in the dairy product sample based on the chromatographic peak area of ​​the characteristic peptide;

[0060] The calibration curve is established using the formula y=k×x+b, where y is the chromatographic peak area of ​​the characteristic peptide, x is the concentration of lactoferrin standard, k is the slope, and b is the intercept.

[0061] The lactoferrin content in dairy product samples is calculated using the formula X=(x×V×D) / m, where X is the lactoferrin content in the sample, V is the volume of the lactoferrin enrichment solution, D is the dilution factor, and m is the sample mass.

[0062] The method of this invention can effectively reduce matrix interference and more accurately and quickly detect the content of lactoferrin in dairy products. Its detection accuracy and repeatability are significantly better than traditional methods. Attached Figure Description

[0063] Figure 1 A flowchart illustrating a method according to an embodiment of the present invention is shown schematically.

[0064] Figure 2 The multireaction monitoring chromatogram of the characteristic peptides in Example 2 is shown. Detailed Implementation

[0065] Unless otherwise specified, the technical terms in this specification have the same meaning as those generally understood by those skilled in the art; however, in case of any conflict, the definitions in this specification shall prevail.

[0066] Furthermore, in the context of this invention, the terms "comprising" or "including" do not exclude other possible elements. The compositions of this invention (including the various embodiments described herein) may comprise, consist of, or consist substantially of the following elements: the essential elements and necessary limitations of the invention as described herein, and any other or optional ingredients, components, or limitations as described herein or as otherwise desired.

[0067] In this application, unless otherwise stated, when referring to the percentage composition of the eluent, it refers to the volume percentage; when referring to other percentage compositions, it refers to the mass composition.

[0068] In this application, unless otherwise stated, when referring to the concentration of a solid solute in a solution of a liquid solvent, it refers to the mass concentration; when referring to the concentration of a liquid solute in a solution of a liquid solvent, it refers to the volume concentration.

[0069] Unless otherwise specified, temperature in this application refers to room temperature (25°C).

[0070] It should be noted that the various aspects, features, implementation methods, experimental examples, and advantages described in this application are compatible and / or can be combined together.

[0071] This invention relates to a method for determining the lactoferrin content in dairy products, the method comprising the following steps:

[0072] (1) Provide a sample of the dairy product to be tested, wherein the dairy product is in the form of liquid, particulate solid or block solid;

[0073] (2) Pretreatment: The sample is pretreated to obtain a pretreated aqueous solution, the pretreatment comprising:

[0074] The steps of diluting or dissolving the dairy product sample to obtain a homogeneous solution; and

[0075] The homogenized solution is subjected to a step of removing impurities such as proteins and fats to obtain a pretreated sample aqueous solution;

[0076] (3) Lactoferrin-specific enrichment: Magnetic nanomaterials with surface-modified lactoferrin aptamers are contacted with a pretreated sample aqueous solution to adsorb and separate lactoferrin. Then, the magnetic nanomaterials are separated from the solution, and the bound lactoferrin is eluted from the magnetic nanomaterials to obtain a lactoferrin enrichment solution.

[0077] (4) Enzymatic hydrolysis: After optionally adjusting the pH of the lactoferrin enrichment solution, the lactoferrin is enzymatically hydrolyzed by a complex enzyme system of trypsin and proteinase K to degrade the lactoferrin into characteristic peptides, thereby obtaining a characteristic peptide solution.

[0078] (5) Peptide purification: The characteristic peptide solution is purified by solid phase extraction column, the solvent is removed from the eluent and the resulting solid is dissolved to obtain the characteristic peptide purified solution.

[0079] (6) Chromatographic and mass spectrometric detection: Using an ultra-high performance liquid chromatography-tandem mass spectrometry system, characteristic ion pairs of characteristic peptides were detected in multiple reaction monitoring mode, and the chromatographic peak area of ​​lactoferrin characteristic peptides was determined; and

[0080] (7) Calculate lactoferrin content: Calculate the lactoferrin content in the dairy product sample based on the peak area.

[0081] Figure 1 A flowchart illustrating the method according to the present invention is shown schematically. Reference will be made below. Figure 1 The method will be described in detail.

[0082] Step (1): Provide the dairy product sample to be tested.

[0083] In step (1), a sample of dairy products to be tested is provided.

[0084] The type of dairy product is not particularly limited and refers to dairy products commonly used in the art, which may be in the form of liquid, granular solid or block solid; for example, the dairy product may be liquid milk such as whole milk, yogurt, colostrum, milk powder such as infant formula, maternal milk powder, adult milk powder, middle-aged and elderly milk powder, protein powder, cheese (e.g. natural cheese, processed cheese / functional cheese), milk tablets, or dairy beverages, etc.

[0085] Step (2): Preprocessing

[0086] In step (2), the sample is pretreated to obtain a pretreated sample aqueous solution.

[0087] The pretreatment includes: diluting or dissolving the dairy product sample to obtain a homogenized solution; and removing impurities and fats from the homogenized solution to obtain a pretreated aqueous sample solution.

[0088] In some embodiments, when the dairy product sample is a liquid, it is diluted with phosphate buffer to obtain a homogeneous solution; when the dairy product sample is a particulate solid, it is dissolved with phosphate buffer to obtain a homogeneous solution; when the dairy product sample is a blocky solid, it is pulverized and then dissolved with phosphate buffer to obtain a homogeneous solution.

[0089] The pulverization can be carried out by any suitable means known in the art (e.g., using a high-speed homogenizer).

[0090] The conjugate acid and base in the phosphate buffer solution may be sodium dihydrogen phosphate and disodium hydrogen phosphate, or potassium dihydrogen phosphate and dipotassium hydrogen phosphate; the concentration of the phosphate buffer solution may be, for example, 0.005-0.05 mol / L, such as 0.005, 0.010, 0.015, 0.02, 0.03, 0.04, 0.05 mol / L, or within the range defined by any two thereof; more preferably 0.01 mol / L; wherein the concentration refers to the total concentration of the conjugate acid and base in the buffer solution.

[0091] The pH value of the phosphate buffer solution may be, for example, 5.8-7.8, preferably 6.5-7.5, such as 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or within the range defined by any two thereof; more preferably 7.4.

[0092] There are no particular restrictions on the amount of phosphate buffer used for dilution or dissolution, and it can be appropriately selected depending on whether the dairy product is in liquid or solid form. For example:

[0093] - When the dairy product is in liquid form, phosphate buffer may be used, for example, in the following amounts per 1 mL of dairy product sample: 1-5 mL, for example 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 mL, or within the range defined by any two thereof, for example 2 mL;

[0094] - When the dairy product is a solid, such as a particulate or lumpy solid, the phosphate buffer should be used at a temperature that promotes dissolution and in an amount sufficient to fully dissolve the dairy product, for example, 1-30 mL per 1 g dairy product sample, such as 1, 2, 5, 10, 15, 20, 25, 30 mL, or within the range defined by any two thereof, preferably 5-10 mL.

[0095] The dilution and dissolution, as well as all other operations / steps in this application, are performed at temperatures below the denaturation temperature of lactoferrin; for example, at 0-65°C (e.g., 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65°C, or within the range defined by any two thereof).

[0096] The dilution and dissolution can be carried out under stirring or shaking to promote homogenization.

[0097] The removal of contaminating proteins and fats can be carried out by any means known in the art.

[0098] In this application, the term "miscellaneous protein" refers to proteins other than lactoferrin, which is the target protein, including casein, etc., as will be understood by those skilled in the art.

[0099] Removing extraneous proteins (while retaining lactoferrin) involves selectively separating lactoferrin from major extraneous proteins (especially casein) based on differences in properties such as isoelectric point, molecular size, charge, and affinity specificity.

[0100] In this implementation, the removal of contaminating proteins can be carried out, for example, by one or more of the following methods:

[0101] - Precipitation. Casein has an isoelectric point of about 4.6, while lactoferrin has an isoelectric point of about 8.0-8.5. By adjusting the pH of the solution to at or near the isoelectric point of casein (e.g., 4.5-4.7), casein precipitates, while lactoferrin remains in solution (dissolved in the supernatant). This precipitation (isoelectric point precipitation) can be carried out, for example, at a temperature below room temperature (e.g., 0-10°C, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C, or any range defined therein) to reduce co-precipitation. The precipitation can be carried out, for example, in the presence of trichloroacetic acid. The presence of trichloroacetic acid enhances the precipitation effect.

[0102] - Salting out. By adding a high concentration of neutral salt (such as ammonium sulfate), the protein hydration layer is contested, causing some of the impurities to precipitate.

[0103] In this implementation, fat removal can be carried out, for example, by one or more of the following methods:

[0104] Centrifugation. The centrifugation may be performed, for example, at a temperature below room temperature (e.g., 0-10°C, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C, or any range defined therein) to reduce the solubility of fat, thereby achieving better separation of fat. The centrifugation may be performed, for example, at a speed of 3000-20000 rpm (e.g., 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000 rpm, or any range defined therein). The centrifugation can be performed for, for example, 5-60 minutes (e.g., 5, 10, 15, 20, 25, 30, 40, 45, 50, 60 minutes, or a range defined by any two thereof). Fat, with its relatively low density, will rise to the surface under centrifugation, forming a fat layer (cream layer). The upper fat layer is discarded, while the lower aqueous phase is retained, thereby achieving partial or complete removal of the fat.

[0105] - Organic solvent extraction. The extraction may be carried out, for example, using nonpolar solvents such as n-hexane, diethyl ether, and / or petroleum ether, preferably n-hexane. The extraction may be carried out, for example, at a temperature below room temperature (e.g., 0-10°C, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C, or a range defined by any two thereof). The solution is mixed with the solvent, shaken, allowed to stand to separate into layers, and the lower aqueous phase is collected while the upper oil phase is discarded, thereby achieving partial or complete removal of the fat.

[0106] - Acidification / heating combined with centrifugation. By adjusting the solution pH to the isoelectric point of casein (4.6) and heating moderately (e.g., at about 40°C), casein and fat globules are co-precipitated.

[0107] - Filtration. Fat globules are physically trapped by using a filter membrane or glass fiber filter paper with a pore size smaller than the diameter of the fat globules (e.g., 0.45 μm).

[0108] In some embodiments, the removal of fat can be carried out by a combination of centrifugation and extraction with an organic solvent (e.g., n-hexane).

[0109] In some implementations, the removal of contaminating proteins and the removal of fat can be performed in any order (e.g., removing fat first and then removing contaminating proteins; or removing contaminating proteins first and then removing fat), or partially or completely simultaneously (e.g., performing the step of removing fat first, during which contaminating proteins are also partially removed, and then performing the step of removing the remaining contaminating proteins; or performing the step of removing contaminating proteins first, during which fat is also partially removed, and then performing the step of removing the remaining fat).

[0110] In some embodiments, the pretreatment includes: adding an aqueous trichloroacetic acid solution (e.g., 10% by mass) to the resulting homogeneous solution, mixing thoroughly, allowing it to stand (e.g., for 15-20 minutes), then centrifuging (e.g., at 8000-10000 r / min for 20-30 minutes at 0-10°C), and taking the supernatant to obtain the pretreated sample aqueous solution.

[0111] In some embodiments, the pretreatment includes: adding (e.g., 10% by mass) an aqueous trichloroacetic acid solution to the resulting homogeneous solution, mixing thoroughly, allowing it to stand (e.g., for 15-20 minutes), then centrifuging (e.g., at 8000-10000 rpm for 20-30 minutes at 0-10°C), taking the supernatant, adding n-hexane to it, mixing thoroughly (e.g., by vortexing at 2000-3000 rpm for 2-5 minutes), then centrifuging (e.g., at 5000-6000 rpm for 8-10 minutes at 0-10°C) to remove the upper n-hexane phase, obtaining a pretreated aqueous sample solution.

[0112] In some embodiments, the pretreatment includes: placing 10 mL of liquid sample in a centrifuge tube, adding 20 mL-25 mL of pH 7.4 phosphate buffer, vortexing at 3000 r / min-3200 r / min for 2-3 minutes, adding 5-7 mL of 10% trichloroacetic acid solution, letting stand for 15-20 minutes, centrifuging at 8000 r / min-10000 r / min for 20-30 minutes at 0-10℃, and taking the supernatant to obtain the pretreated sample solution; the concentration of the phosphate buffer is 0.01 mol / L.

[0113] The pretreatment effectively removes fats and proteins, reducing their interference with the chromatographic column, tubing, and detection signal.

[0114] (3) Specific enrichment of lactoferrin

[0115] In step (3), the magnetic nanomaterials with surface-modified lactoferrin aptamers are contacted with the pretreated sample aqueous solution to adsorb and separate lactoferrin. Then, the magnetic nanomaterials are separated from the solution, and the bound lactoferrin is eluted from the magnetic nanomaterials to obtain a lactoferrin enrichment solution.

[0116] The type of magnetic nanomaterial is not particularly limited and may be, for example, iron(II,III) oxide (Fe3O4), neodymium iron boron magnets, or mixtures thereof, such as iron(II,III) oxide (Fe3O4).

[0117] The particle size of the magnetic nanomaterial is not particularly limited and may be, for example, 1-500 nm, 10-100 nm, or, for example, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 nm, or a range defined by any two of these. In this application, particle size refers to the average particle size, which is measured, for example, by laser diffraction.

[0118] The type of lactoferrin aptamer is not particularly limited and may be, for example, a DNA aptamer or an RNA aptamer, preferably a DNA aptamer. Preferably, the DNA aptamer has the following nucleotide sequence: 5'-AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT-3'.

[0119] Magnetic nanomaterials with surface-modified lactoferrin aptamers can have the lactoferrin aptamers attached to the surface of the magnetic nanoparticles by any suitable means known in the art. For example, in some embodiments, the magnetic nanomaterials with surface-modified lactoferrin aptamers can be prepared as follows:

[0120] Magnetic nanoparticles are surface-treated with coupling agents such as silane coupling agents (e.g., amino-functionalized silane coupling agents such as 3-aminopropyltriethoxysilane) to obtain surface-aminated magnetic nanoparticles; and

[0121] The surface amino groups of the surface-amino-coated magnetic nanoparticles are chemically linked to the carboxyl groups of the lactoferrin aptamer.

[0122] The connection can be carried out, for example, in a medium in the presence of a carbodiimide crosslinking agent (e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and an activating enhancer (e.g., N-hydroxysuccinimide). This connects the magnetic nanoparticles to the aptamer via amide bonds. The medium can be, for example, a 2-(N-morpholino)ethanesulfonic acid (MES) buffer.

[0123] In some embodiments, the magnetic nanomaterials with surface-modified lactoferrin aptamers can be prepared, for example, by dispersing magnetic nanomaterials, such as Fe3O4 nanoparticles, in ultrapure water, adding a silane coupling agent, such as 3-aminopropyltriethoxysilane, for silanization modification, centrifuging and washing, and then dispersing in a medium, such as MES (2-(N-morpholino)ethanesulfonic acid) buffer, adding a carbodiimide crosslinking agent, such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and an activation enhancer, such as N-hydroxysuccinimide, for activation, then adding lactoferrin aptamers and reacting at room temperature for 1-2 hours, and obtaining the magnetic nanomaterials with surface-modified lactoferrin aptamers after magnetic separation; the MES buffer concentration is 0.05 mol / L and the pH is 6.0.

[0124] The contact may be carried out, for example, at temperatures of 25-40°C, such as 35-37°C, such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C, or within the range defined by any two of these.

[0125] The contact may last for, for example, 5 to 60 minutes, such as 5, 10, 15, 20, 25, 30, 40, 45, 50, 60 minutes, or a range defined by any two of these, such as 20 to 30 minutes.

[0126] By contacting magnetic nanomaterials with surface-modified lactoferrin aptamers with pretreated aqueous sample solutions, specific enrichment of lactoferrin on the surface of the magnetic nanomaterials was achieved, thereby realizing targeted adsorption and rapid separation of lactoferrin.

[0127] Subsequently, the magnetic nanomaterials were separated from the solution.

[0128] The separation can be performed by any suitable means known in the art. Preferably, the separation can be performed by the action of an external magnetic field. The strength of the magnetic field is not particularly limited and can be, for example, 0.2-0.5 Tesla, such as 0.2, 0.3, 0.4, 0.5 Tesla, or within the range defined by any two thereof. By employing an external magnetic field, the solution can be clarified rapidly (typically within 30 seconds to 5 minutes); and the effect on the target complex is extremely mild, with only the magnetic beads being immobilized, without generating shear forces or pressures, protecting the aptamer-lactoferrin complex; and it is highly selective, attracting only magnetic particles, while non-magnetic impurities remain in the solution.

[0129] After separating the magnetic nanomaterials from the solution, the bound lactoferrin was eluted from the magnetic nanomaterials, thereby obtaining a lactoferrin-enriched solution.

[0130] The elution can be performed using an eluent. The type of eluent is not particularly limited and can be, for example, an acidic eluent such as a tris(hydroxymethyl)aminomethane (Tris)-HCl buffer. The pH of the acidic eluent, such as the tris(hydroxymethyl)aminomethane (Tris)-HCl buffer, is not particularly limited and can be appropriately selected depending on its type. For example, the pH of the tris(hydroxymethyl)aminomethane (Tris)-HCl buffer can be, for example, 7.0-9.0, such as 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or within a range defined by any two thereof, such as 8.5.

[0131] In embodiments, the eluent, such as tris(hydroxymethyl)aminomethane (Tris)-HCl buffer, may contain NaCl to reduce or even avoid the elution of contaminating proteins that may be nonspecifically adsorbed onto the surface of the magnetic nanomaterial. Preferably, the concentration of NaCl may be, for example, 0.3-0.8 mol / L, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mol / L, or within the range defined by any two thereof. When the NaCl concentration is within this range, it can effectively break the specific binding of the aptamer to lactoferrin while reducing interference from nonspecific adsorption of contaminating proteins. This range can balance elution efficiency and protein stability; below 0.3 mol / L, the elution efficiency is insufficient, and above 0.8 mol / L, it may cause lactoferrin denaturation.

[0132] Through this (3) lactoferrin-specific enrichment step, efficient and specific separation of lactoferrin from solution was achieved.

[0133] (4) Enzymatic hydrolysis

[0134] In step (4), after optionally adjusting the pH of the lactoferrin enrichment solution, it is enzymatically hydrolyzed by a complex enzyme system of trypsin and proteinase K to degrade lactoferrin into characteristic peptides, thereby obtaining a characteristic peptide solution.

[0135] Prior to enzymatic hydrolysis, the pH of the lactoferrin enrichment solution may optionally be adjusted, for example to 7.8-8.5, such as 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or within a range defined by any two thereof, such as 8.0.

[0136] The mass ratio of trypsin to proteinase K is not particularly limited and can be, for example, 0.1:1 to 10:1, such as 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or within the range defined by any two of them, such as 3:1.

[0137] In this embodiment, there is no particular limitation on the amount of the complex enzyme system added to the lactoferrin enrichment solution and the total enzyme concentration added (in the resulting solution) is 0.1-10 mg / mL, for example 0.1, 0.2, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / mL, or within the range defined by any two of them, for example 1 mg / mL.

[0138] In the embodiments, the mass ratio of enzyme to lactoferrin substrate is not particularly limited and may be, for example, 1:10 to 1:100, such as 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, or within the range defined by any two of them, such as 1:50.

[0139] The enzymatic hydrolysis reaction is carried out, for example, at a temperature favorable to enzyme activity. The temperature may be, for example, 30-60°C, such as 30, 35, 37, 40, 45, 50, 55, 60°C, or within the range defined by any two thereof; preferably 37°C.

[0140] The enzymatic hydrolysis reaction may, for example, be carried out for 1 to 24 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 24 hours, or within the range defined by any two of them, such as 4 hours.

[0141] Then, a terminator is added to terminate the enzymatic hydrolysis reaction. The type of terminator is not particularly limited and can be, for example, an acid such as an organic acid like formic acid or acetic acid.

[0142] In some embodiments, the mass ratio of trypsin to proteinase K in the complex enzyme system is 3:1, and the total enzyme concentration is 1 mg / mL; the enzymatic hydrolysis treatment includes: adjusting the pH of the lactoferrin enrichment solution to 8.0, adding the complex enzyme system to make the enzyme-to-substrate mass ratio 1:50, enzymatic hydrolysis in a 37°C water bath with shaking for 4 hours, and adding 0.1% v / v formic acid aqueous solution to terminate the enzymatic hydrolysis reaction.

[0143] Lactoferrin is broken down into characteristic peptides through an enzymatic reaction, thereby obtaining a solution of the characteristic peptides.

[0144] (5) Peptide purification

[0145] In step (5), the characteristic peptide solution is purified using a solid-phase extraction column. The solvent is removed from the collected eluent and the resulting solid is dissolved to obtain the purified characteristic peptide solution.

[0146] The solid phase extraction column can be a C18 solid phase extraction column.

[0147] The purification can be carried out by gradient elution using acetonitrile-water solutions of different concentrations.

[0148] Before elution, the solid-phase extraction column can be activated. For example, the solid-phase extraction column can be activated sequentially with methanol and water (e.g., ultrapure water).

[0149] The gradient elution can be performed, for example, by rinsing with a 5% v / v acetonitrile aqueous solution and then eluting with an 80% v / v acetonitrile aqueous solution.

[0150] The eluent is then collected and the solvent is removed from it. The solvent can be removed by blowing it dry with nitrogen gas.

[0151] The dissolution can be carried out using an acid solution, such as an organic acid like an aqueous solution of formic acid.

[0152] In some embodiments, the solid-phase extraction column is a C18 solid-phase extraction column, and the purification of the characteristic peptide includes: activating the solid-phase extraction column sequentially with (e.g., 5 mL-7 mL) methanol and (e.g., 5 mL-7 mL) ultrapure water; loading the characteristic peptide solution onto the column (at a flow rate of, for example, 0.5 mL / min); rinsing with (e.g., 5 mL-7 mL) 5% v / v acetonitrile aqueous solution; eluting with (e.g., 3 mL-5 mL) 80% v / v acetonitrile aqueous solution, collecting the eluent, drying (e.g., blowing with nitrogen), and redissolving with (e.g., 0.1% v / v) formic acid aqueous solution to obtain the purified characteristic peptide solution.

[0153] (6) Chromatography-mass spectrometry detection

[0154] In step (6), an ultra-high performance liquid chromatography-tandem mass spectrometry system is used to detect the characteristic ion pairs of the characteristic peptides in multiple reaction monitoring mode, and the chromatographic peak area of ​​the lactoferrin characteristic peptides is determined.

[0155] Characteristic ion pairs of characteristic peptides can be detected using multiple reaction monitoring (MRM) mode to determine the chromatographic peak area of ​​lactoferrin characteristic peptides.

[0156] The ultra-high performance liquid chromatography (UHPLC) can use a C18 column. Its mobile phase A can be a 0.1% v / v formic acid aqueous solution, and its mobile phase B can be a 0.1% v / v formic acid acetonitrile solution.

[0157] The gradient elution program can be as follows: 0-5 min 5%-20% B, 5-10 min 20%-40% B, 10-12 min 40%-90% B, 12-15 min 90% B, 15-16 min 90%-5% B, 16-20 min 5% B.

[0158] In some embodiments, the characteristic ion pair is a parent ion with m / z 835.4 and daughter ions with m / z 926.5 and m / z 789.4. Those skilled in the art will understand that the characteristic ion pair corresponds to the characteristic peptide.

[0159] In some embodiments, the mass spectrometry parameters of the multi-reaction monitoring mode include: an electrospray ion source, positive ion mode, capillary voltage of 3.2 kV, ion source temperature of 110 °C, desolvation gas temperature of 380 °C, desolvation gas flow rate of 900 L / h, collision gas as argon, and collision energies of 25 eV and 22 eV, respectively.

[0160] (7) Calculate lactoferrin content

[0161] In step (7), the content of lactoferrin in the dairy product sample is calculated based on the peak area.

[0162] The calculation can be performed by any means known in the art.

[0163] For example, the content of lactoferrin in a dairy product sample can be calculated based on the chromatographic peak area of ​​the characteristic peptide using a calibration curve established from lactoferrin standards.

[0164] The calibration curve is established using the formula y=k×x+b, where y is the chromatographic peak area of ​​the characteristic peptide, x is the concentration of lactoferrin standard, k is the slope, and b is the intercept.

[0165] The lactoferrin content in dairy product samples is calculated using the formula X=(x×V×D) / m, where X is the lactoferrin content in the sample, V is the volume of the lactoferrin enrichment solution, D is the dilution factor, and m is the sample mass.

[0166] The concentration series of lactoferrin standards may be, for example, 0.05, 0.1, 0.5, 1, 5, 10 mg / L.

[0167] The measurement method of the present invention has the following advantages:

[0168] 1. High versatility and wide coverage: A general technical framework applicable to different forms of dairy products has been established. Differentiated pretreatment effectively removes matrix interference and enables accurate determination of a wide content range from 0.05 mg / 100g to 100 mg / 100g. It can meet the quality control needs of enterprise production lines and is also suitable for arbitration testing by regulatory agencies.

[0169] 2. High efficiency and specificity in pretreatment: Functionalized magnetic nanomaterials are used to specifically enrich lactoferrin. Combined with the high selectivity of aptamers and the convenience of magnetic separation, the capture efficiency of target proteins in complex matrices is significantly improved. Interfering components such as casein and fat are effectively removed, especially improving the detection signal-to-noise ratio of low-content samples (such as humanized milk powder).

[0170] 3. Rapid and highly accurate detection: The method shortens enzymatic digestion time through a complex enzyme system and achieves rapid separation and specific detection using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). The single-sample detection cycle can be controlled within 20 minutes. Quantitative methods based on characteristic peptides and calibration curves ensure the sensitivity and accuracy of the method, meeting the needs of trace analysis and batch detection.

[0171] 4. Adjustable conditions and flexible application: To address the differences in lactoferrin content in different samples, precise elution can be achieved by dynamically adjusting the elution conditions, further enhancing the applicability and reliability of the method.

[0172] In particular, compared with existing methods, the method of the present invention, through aptamer enrichment and complex enzymatic hydrolysis, can effectively reduce matrix interference and more accurately and rapidly detect the content of lactoferrin in dairy products. The detection accuracy and repeatability are significantly better than traditional methods.

[0173] Experimental Example

[0174] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to experimental examples. It should be understood that the specific experimental examples described herein are merely illustrative and not intended to limit the invention. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional in the art.

[0175] Preparation Example 1: Preparation of Functionalized Magnetic Nanomaterials (Magnetic Nanomaterials with Surface Modified Lactoferrin Aptamers)

[0176] 100 mg of Fe3O4 nanoparticles (50 nm in diameter) were dispersed in 50 mL of ultrapure water and sonicated for 15 minutes until uniformly dispersed. 5 mL of 3-aminopropyltriethoxysilane was added, and the mixture was stirred at 60 °C for 4 hours to perform silanization modification. After the reaction, the mixture was centrifuged at 10000 r / min for 10 minutes, the precipitate was collected, and washed three times with ultrapure water to remove unreacted reagents. The modified particles were dispersed in 50 mL of 0.05 mol / L, pH 6.0 MES buffer, and 20 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 15 mg of... N-hydroxysuccinimide was activated by shaking at room temperature for 1 hour; then 50 nmol of lactoferrin aptamer (purchased from Sangon Biotech (Shanghai) Co., Ltd., nucleotide sequence 5'-AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT-3', HPLC purity ≥98% by mass) was added, and the reaction was carried out at room temperature in the dark for 2 hours. The particles were separated by an external magnetic field of 0.5 Tesla and washed twice with 0.01 mol / L, pH 7.4 phosphate buffer (conjugate acid-base pair of sodium dihydrogen phosphate-disodium hydrogen phosphate) to obtain magnetic nanomaterials with surface-modified lactoferrin aptamer, which were stored at 4℃ for later use.

[0177] Example 1: Determination of lactoferrin in liquid milk

[0178] Take 10 mL of whole milk (Inner Mongolia Mengniu Dairy Group Co., Ltd., main components and contents as follows (per 100 g): water 87.2 g, protein 3.0 g, fat 3.7 g, carbohydrates 4.8 g, calcium 100 mg, sodium 62 mg) and place it in a 50 mL centrifuge tube. Add 20 mL of 0.01 mol / L phosphate buffer (pH 7.4, conjugate acid-base pair is sodium dihydrogen phosphate-disodium hydrogen phosphate), and vortex at 3000 r / min for 2 minutes. Add 5 mL of 10% trichloroacetic acid solution, let stand at room temperature for 15 minutes, and centrifuge at 8000 r / min for 20 minutes at 4℃. Collect the supernatant. Add 3 mL of n-hexane to the supernatant, vortex at 2000 r / min for 2 minutes, and centrifuge at 5000 r / min for 10 minutes at 4℃ to remove the upper n-hexane phase. Repeat the operation twice to obtain the pretreated sample solution. Preliminary estimation indicated that the lactoferrin content M in the sample solution was 50 mg / L. Based on the empirical formula C = 0.1 + 0.02 × lg(M), the sodium chloride concentration C was calculated to be 0.134 mol / L. NaCl was added to the sample solution to adjust the NaCl concentration to 0.3 mol / L. The estimation was achieved using rapid qualitative detection combined with empirical range derivation. The sample was initially screened using colloidal gold immunochromatographic strips to determine the approximate lactoferrin content range. Then, considering the typical content range of similar dairy products (the natural lactoferrin content in liquid milk is mostly 1-10 mg / L, while added products typically do not exceed 50 mg / L), the estimated content M was determined. The following examples follow the same procedure.

[0179] Add 5 mg of the functionalized magnetic nanomaterial prepared in Preparation Example 1 to the obtained sample solution and incubate with shaking at 37°C for 30 minutes; separate the material by an external magnetic field of 0.5 Tesla strength, discard the supernatant, add 5 mL of 0.05 mol / L Tris-HCl buffer containing 0.3 mol / L sodium chloride, pH 8.5 for elution, and collect the eluent to obtain lactoferrin enrichment solution. The pH of the enrichment solution was adjusted to 8.0 with 0.1 mol / L NaOH. A complex enzyme system (total enzyme concentration 1 mg / mL) of trypsin (Sangon Biotech (Shanghai) Co., Ltd., catalog number A003702) and proteinase K (Beijing Solarbio Science & Technology Co., Ltd., catalog number P9461) at a mass ratio of 3:1 was added to make the mass ratio of enzyme to lactoferrin substrate 1:50 (the mass of lactoferrin substrate was determined by calculating the estimated mass of lactoferrin based on the estimated content M and the volume of the sample solution, and then correcting it with the empirical value of the recovery rate of the enrichment step (about 85%-90%) to finally determine the substrate mass; the same applies below). The enzyme was hydrolyzed in a water bath at 37°C with shaking for 4 hours. The enzymatic reaction was terminated by adding 1 mL of 0.1% formic acid solution to obtain the characteristic peptide solution.

[0180] Characteristic peptides were purified using a C18 solid-phase extraction column (Nanjing Yizhiyuan Testing Technology Co., Ltd., KEPNO, Y1018): The column was activated sequentially with 5 mL of methanol and 5 mL of ultrapure water. The characteristic peptide solution was loaded at a flow rate of 0.5 mL / min. The column was then rinsed with 5 mL of 5% v / v acetonitrile aqueous solution and the eluent was discarded. The column was then eluted with 3 mL of 80% v / v acetonitrile aqueous solution, and the eluent was collected and dried under nitrogen at 40 °C. The eluent was then reconstituted with 1 mL of 0.1% v / v formic acid aqueous solution to obtain the purified characteristic peptide solution.

[0181] The purified solution was injected into an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry system (Agilent Technologies, 1290 Infinity II-Agilent 6470B). The chromatographic conditions were as follows: C18 column (2.1 mm × 100 mm, 1.7 μm), mobile phase A was 0.1% v / v formic acid aqueous solution, mobile phase B was 0.1% v / v formic acid acetonitrile solution, gradient elution program was 0–5 min 5%–20% B, 5–10 min 20%–40% B, 10–12 min 40%–90% B, 12–15 min 90% B, 15–16 min 90%–5% B, 16–20 min 5% B, flow rate 0.3 mL / min, column temperature 40 °C, and injection volume 5 μL. The mass spectrometry conditions were as follows: electrospray ionization source, positive ion mode, capillary voltage 3.2 kV, ion source temperature 110 °C, desolvation gas temperature 380 °C, desolvation gas flow rate 900 L / h, collision gas was argon, and multiple reaction monitoring mode was used to detect characteristic ion pairs (mother ion m / z 835.4, daughter ions m / z 926.5, 789.4) (corresponding to characteristic peptides), with collision energies of 25 eV and 22 eV, respectively.

[0182] Calibration curves were established using lactoferrin standard concentrations (0.05, 0.1, 0.5, 1, 5, 10 mg / L), yielding a regression equation of y = 12568x + 42.3 and a correlation coefficient R² = 0.9995. The sample content was calculated using the formula X = (x × V × D) / m, where x is the concentration read from the calibration curve (2.35 mg / L), V is the enrichment liquid volume (0.005 L), D is the dilution factor (3), and m is the sample mass (10 g). The lactoferrin content in the liquid milk was found to be 3.52 mg / 100 g. The results showed that the spiked recovery rate was 94.2%, and the intra-day precision RSD was 2.1%. This verifies the accuracy and repeatability of the detection method of this invention in liquid dairy products, providing a practical operational example for the detection of lactoferrin in liquid dairy products.

[0183] Example 2: Determination of lactoferrin in infant formula

[0184] Take 2g of infant formula milk powder sample (Heilongjiang Feihe Dairy Co., Ltd., main components and contents as follows (per 100g): protein 12.3g, fat 24.2g, carbohydrates 58g, DHA 45mg, vitamin A 600μg, calcium 385mg) and place it in a 50mL centrifuge tube. Add 20mL of 0.01mol / L, pH 7.4 phosphate buffer (conjugate acid-base pair is sodium dihydrogen phosphate-disodium hydrogen phosphate), shake in a 50℃ water bath for 10 minutes until completely dissolved, and vortex at 3000r / min for 2 minutes; add 5mL of... A 10% trichloroacetic acid solution was allowed to stand at room temperature for 15 minutes, then centrifuged at 8000 r / min for 20 minutes at 4°C. The supernatant was collected to obtain the pretreated sample solution. The estimated lactoferrin content M was 150 mg / L. The sodium chloride concentration C was calculated to be 0.144 mol / L according to the formula given in Example 1. NaCl was added to the sample solution to adjust the NaCl concentration in the solution to 0.4 mol / L.

[0185] Lactoferrin enrichment, enzymatic digestion, purification of characteristic peptides, and chromatographic and mass spectrometric detection were performed using the same method as in Example 1, except that the elution buffer contained 0.4 mol / L sodium chloride during the enrichment process.

[0186] The established calibration curve regression equation was y = 12610x + 38.7, with R² = 0.9993. The concentration x = 6.82 mg / L read from the calibration curve, combined with V = 0.005 L, D = 10, and m = 2 g, calculated the lactoferrin content in the sample to be 170.5 mg / 100 g. The spiked recovery rate of this detection result was 96.8%, and the inter-day precision RSD was 2.8%. The multiple reaction monitoring chromatogram of the characteristic peptide is shown below. Figure 2 As shown, the characteristic ion pairs exhibit symmetrical response peaks with no obvious interference peaks and a resolution of 1.8, indicating good resolution. This demonstrates that the method of the present invention can accurately detect lactoferrin in high-value-added powdered dairy products, meeting the accuracy and stability requirements for quality control of high-end dairy products.

[0187] Example 3: Determination of lactoferrin in hard cheese

[0188] Take 5g of hard cheese sample, homogenize it at 10000r / min for 30 seconds using a high-speed homogenizer, and place it in a 50mL centrifuge tube. Add 25mL of 0.01mol / L phosphate buffer (pH 7.4, conjugate acid-base pair: sodium dihydrogen phosphate-disodium hydrogen phosphate), and incubate at 60℃ for 20 minutes with vortexing once every 5 minutes. Add 5mL of 10% trichloroacetic acid solution, let stand at room temperature for 15 minutes, and centrifuge at 8000r / min for 20 minutes at 4℃. Collect the supernatant. Add 3mL of n-hexane to the supernatant and repeat the fat removal operation twice to obtain the pretreated sample solution. The estimated lactoferrin content M is 30mg / L, and the calculated sodium chloride concentration C is 0.130mol / L. Add NaCl to the sample solution to adjust the NaCl concentration to 0.3mol / L.

[0189] Lactoferrin enrichment, enzymatic digestion, purification of characteristic peptides, and chromatographic and mass spectrometric detection were performed using the same methods as in Example 1.

[0190] The calibration curve regression equation was y = 12585x + 40.2, with R² = 0.9994. The calculated lactoferrin content in the sample was 8.24 mg / 100g, with a spiked recovery rate of 93.5% and RSD = 2.5%. This example verifies the applicability of the method to complex solid dairy products, overcoming the limitations of traditional methods in the detection of solid high-fat samples.

[0191] The peak areas of characteristic peptides of standards at different concentrations are shown in Table 1. It can be seen that the linear relationship is good in the range of 0.05-10 mg / L.

[0192] Table 1. Relationship between standard concentration and characteristic peptide peak area

[0193]

[0194] Example 4: Method Performance Verification Experiment

[0195] The linear range and limit of detection were determined by preparing lactoferrin standard solutions with concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 20 mg / L, and detecting the lactoferrin using the method described in Example 1. A calibration curve was plotted with concentration on the x-axis and peak area on the y-axis, yielding the regression equation y = 12572x + 41.5, with a correlation coefficient R² = 0.9996. The linear range was 0.05–10 mg / L. The limit of detection, calculated using a signal-to-noise ratio of 3, was 0.02 mg / 100g, and the limit of quantitation, calculated using a signal-to-noise ratio of 10, was 0.07 mg / 100g.

[0196] Samples of liquid milk, milk powder, and cheese with known content were taken. The liquid milk was from Inner Mongolia Mengniu Dairy Group Co., Ltd., with the following main components and content (per 100g): water 87.2g, protein 3.0g, fat 3.7g, carbohydrates 4.8g, calcium 100mg, sodium 62mg; the milk powder was from Heilongjiang Feihe Dairy Co., Ltd., with the following main components and content (per 100g): protein 12.3g, fat 24.2g, carbohydrates 58g, DHA 45mg, vitamin A 600μg, calcium 385mg; the cheese was from Shanghai Miaokelanduo Food Technology Co., Ltd., with the following main components and content (per 100g): protein 23g, fat 23.4g, carbohydrates 6.7g, calcium 510mg, sodium 510mg. Lactoferrin standards at low, medium, and high concentrations (mg) were added to the samples, and each concentration level was measured six times in parallel. The spiked recoveries and relative standard deviations (RSDs) were calculated. The results are shown in Table 2. The spiked recoveries of the three samples ranged from 92.3% to 104.8%, and the RSDs were all ≤3.0%, indicating that the method has good accuracy and repeatability.

[0197] Table 2. Spike recovery and precision results for different samples.

[0198]

[0199] Weigh an appropriate amount of cation exchange resin (A764221, Macklin, Shanghai Macklin Biochemical Technology Co., Ltd.), and activate the resin by sequentially circulating it with 1 mol / L NaCl, 0.1 mol / L NaOH, and 0.1 mol / L HCl solutions. Finally, wash with deionized water until neutral. Then, equilibrate the resin with 0.02 mol / L phosphate buffer (pH 5.2, with a conjugate acid-base pair of sodium dihydrogen phosphate and disodium hydrogen phosphate). Mix the equilibrated resin with whole milk (Inner Mongolia Mengniu Dairy Group Co., Ltd., with the following main components and contents per 100g: water 87.2g, protein 3.0g, fat 3.7g, carbohydrates 4.8g, calcium 100mg, sodium 62mg) at a volume ratio of 1:15. Accurately adjust the pH of the mixture to 5.17 with 0.1 mol / L HCl or NaOH aqueous solution, and allow it to adsorb at room temperature for 3 hours. Under these conditions, positively charged lactoferrin in whey is specifically adsorbed onto the resin. After adsorption, the resin is allowed to settle completely, and the supernatant is collected. The supernatant is then filtered through a 0.45 μm filter membrane to obtain a liquid blank whey matrix free of lactoferrin. (All other reagents mentioned above were purchased from Beijing Solarbio Science & Technology Co., Ltd.)

[0200] Blank milk matrix without lactoferrin was taken, and lactoferrin standards of the same concentration as shown in Table 2 were added. The results were compared between this method and the traditional ELISA method. The results showed that the relative error between the measured value and the theoretical value by this method was 2.3%, while the relative error of the ELISA method was 14.6%. This indicates that this method, through aptamer enrichment and complex enzymatic digestion, can effectively reduce matrix interference and its detection accuracy is significantly better than that of the traditional method.

[0201] The above description is merely an exemplary embodiment of the present invention. It should be noted that those skilled in the art can make improvements to the present invention without departing from the inventive concept, and all such improvements fall within the scope of protection of the present invention.

Claims

1. A method for determining the lactoferrin content in dairy products, the method comprising the following steps: (1) Provide a sample of the dairy product to be tested, wherein the dairy product is in the form of liquid, particulate solid or block solid; (2) Pretreatment: The sample is pretreated to obtain a pretreated aqueous solution, the pretreatment comprising: The steps of diluting or dissolving the dairy product sample to obtain a homogeneous solution; and The homogenized solution is subjected to a step of removing impurities such as proteins and fats to obtain a pretreated sample aqueous solution; (3) Lactoferrin-specific enrichment: Magnetic nanomaterials with surface-modified lactoferrin aptamers are contacted with a pretreated sample aqueous solution to adsorb and separate lactoferrin. Then, the magnetic nanomaterials are separated from the solution, and the bound lactoferrin is eluted from the magnetic nanomaterials to obtain a lactoferrin enrichment solution. (4) Enzymatic hydrolysis: After optionally adjusting the pH of the lactoferrin enrichment solution, the lactoferrin is enzymatically hydrolyzed by a complex enzyme system of trypsin and proteinase K to degrade the lactoferrin into characteristic peptides, thereby obtaining a characteristic peptide solution. (5) Peptide purification: The characteristic peptide solution is purified by solid phase extraction column, the solvent is removed from the eluent and the resulting solid is dissolved to obtain the characteristic peptide purified solution. (6) Chromatographic and mass spectrometric detection: Using an ultra-high performance liquid chromatography-tandem mass spectrometry system, characteristic ion pairs of characteristic peptides were detected in multiple reaction monitoring mode, and the chromatographic peak area of ​​lactoferrin characteristic peptides was determined; and (7) Calculate lactoferrin content: Calculate the lactoferrin content in the dairy product sample based on the peak area.

2. The method of claim 1, wherein the dairy product is liquid milk such as whole milk, yogurt, colostrum, milk powder such as infant formula, maternal milk powder, adult milk powder, and milk powder for the elderly, protein powder, cheese such as natural cheese, processed cheese / functional cheese, milk tablets, or dairy beverages.

3. The method as described in any one of claims 1-2, wherein: When the dairy product sample is a liquid, it is diluted with phosphate buffer to obtain a homogeneous solution. When the dairy product sample is a particulate solid, it is dissolved in phosphate buffer to obtain a homogeneous solution. When the dairy product sample is a blocky solid, it is crushed and then dissolved in phosphate buffer to obtain a homogeneous solution. The conjugate acid and base in the phosphate buffer solution are sodium dihydrogen phosphate and disodium hydrogen phosphate, or potassium dihydrogen phosphate and dipotassium hydrogen phosphate; the concentration of the phosphate buffer solution is 0.005-0.05 mol / L; the concentration refers to the total concentration of the conjugate acid and base in the buffer solution; the pH value of the phosphate buffer solution is 5.8-7.8, preferably 6.5-7.5; When the dairy product is liquid, phosphate buffer should be used at a rate of 1-5 mL per 1 mL of dairy product sample. When the dairy product is solid, such as particulate or blocky solid, phosphate buffer should be used at a rate of 1-30 mL per 1 g of dairy product sample.

4. The method according to any one of claims 1-3, wherein: Protein removal is performed by precipitation; and Fat removal is carried out by centrifugation and / or extraction with organic solvents, such as nonpolar solvents like n-hexane, diethyl ether, and / or petroleum ether, preferably n-hexane.

5. The method of any one of claims 1-4, wherein the preprocessing comprises: Add trichloroacetic acid aqueous solution to the obtained homogeneous solution, mix well and let stand, then centrifuge, take the supernatant to obtain the pretreated sample aqueous solution.

6. The method according to any one of claims 1-5, wherein the magnetic nanomaterial is iron(II,III) oxide, neodymium iron boron magnet, or a mixture thereof.

7. The method according to any one of claims 1-6, wherein the particle size of the magnetic nanomaterial is 1-500 nm, for example 10-100 nm.

8. The method according to any one of claims 1-7, wherein the lactoferrin aptamer is a DNA aptamer or an RNA aptamer, preferably a DNA aptamer, and preferably, the DNA aptamer has the following nucleotide sequence: 5'-AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT-3'.

9. The method according to any one of claims 1-8, wherein the magnetic nanomaterial with surface-modified lactoferrin aptamer is prepared by: Magnetic nanoparticles are surface-treated with coupling agents such as silane coupling agents (e.g., 3-aminopropyltriethoxysilane) to obtain surface-aminated magnetic nanoparticles; and The surface amino groups of the surface-amino-treated magnetic nanoparticles are chemically linked to the carboxyl groups of the lactoferrin aptamer. The connection is carried out, for example, in a medium in the presence of a carbodiimide crosslinking agent (e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and an activating enhancer (e.g., N-hydroxysuccinimide), the medium being, for example, 2-(N-morpholino)ethanesulfonic acid (MES) buffer.

10. The method according to any one of claims 1-9, wherein in step (3), The contact is performed at a temperature of 25-40°C, for example 35-37°C, for 5-60 minutes, for example 20-30 minutes; The separation of magnetic nanomaterials from solution is achieved by the action of an external magnetic field. The lactoferrin bound to the magnetic nanomaterial is eluted using a tris(hydroxymethyl)aminomethane (Tris)-HCl buffer solution with a pH of, for example, 7.0-9.0, or 8.5, and containing 0.3-0.8 mol / L NaCl.

11. The method according to any one of claims 1-10, wherein in step (4), Optionally, the pH of the lactoferrin enrichment solution is adjusted to 7.8-8.5 before enzymatic hydrolysis; The mass ratio of trypsin to proteinase K is 0.1:1-10:1; The total concentration of added enzymes was 0.1-10 mg / mL; The mass ratio of enzyme to lactoferrin substrate is 1:10 to 1:100; The enzymatic hydrolysis reaction is carried out at a temperature of 30-60℃; The enzymatic hydrolysis reaction is carried out for 1-24 hours, after which a terminator such as formic acid is added to terminate the enzymatic hydrolysis reaction.

12. The method according to any one of claims 1-11, wherein in step (5), The solid-phase extraction column is a C18 solid-phase extraction column; The purification is performed by gradient elution using acetonitrile-water solutions of different concentrations. The gradient elution can be performed, for example, as follows: first elute with 5% v / v acetonitrile-water solution; then elute with 80% v / v acetonitrile-water solution. Before elution, the solid-phase extraction column was activated sequentially with methanol and ultrapure water; After elution, the eluent is collected and the solvent is removed from the collected eluent; the solvent can be removed by blowing it dry with nitrogen. The dissolution was performed using an aqueous formic acid solution.

13. The method according to any one of claims 1-12, wherein in step (6), The ultra-high performance liquid chromatography uses a C18 column; Mobile phase A is a 0.1% v / v formic acid aqueous solution, and mobile phase B is a 0.1% v / v formic acid acetonitrile solution; The gradient elution program is as follows: 0-5 min 5%-20% B, 5-10 min 20%-40% B, 10-12 min 40%-90% B, 12-15 min 90% B, 15-16 min 90%-5% B, 16-20 min 5% B; The characteristic ion pair is a parent ion with m / z 835.4 and daughter ions with m / z 926.5 and m / z 789.4; The mass spectrometry parameters of the multiple reaction monitoring mode may include, for example: The electrospray ionization source is in positive ion mode, with a capillary voltage of 3.2 kV, an ion source temperature of 110℃, a desolvation gas temperature of 380℃, a desolvation gas flow rate of 900 L / h, and argon as the collision gas, with collision energies of 25 eV and 22 eV respectively.

14. The method according to any one of claims 1-13, wherein in step (7), a calibration curve established by lactoferrin standards is used to calculate the content of lactoferrin in the dairy product sample based on the chromatographic peak area of ​​the characteristic peptide; The calibration curve is established using the formula y=k×x+b, where y is the chromatographic peak area of ​​the characteristic peptide, x is the concentration of lactoferrin standard, k is the slope, and b is the intercept. The lactoferrin content in dairy product samples is calculated using the formula X=(x×V×D) / m, where X is the lactoferrin content in the sample, V is the volume of the lactoferrin enrichment solution, D is the dilution factor, and m is the sample mass.

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