Method for enhancing solubility of fat-soluble vitamins in bovine serum albumin

Abstract

The invention discloses a method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin. The method comprises the following steps: acquiring a bovine serum albumin raw material and adding a PBS buffer solution; adding an antioxidant into the bovine serum albumin raw material added with the PBS buffer solution to form a 5%-10% BSA solution; adding 0.2%-2.0% of a surfactant into the BSA solution to form an enhanced base solution; adding a fat-soluble vitamin solution into the enhanced base solution to form a vitamin matrix solution; taking 50-200 [mu] L of a vitamin matrix solution, adding 10-50 [mu] L of a zinc sulfate solution, carrying out primary vortex mixing, adding 100-500 [mu] L of an internal standard solution, and carrying out secondary vortex mixing; and carrying out centrifugal treatment on the solution subjected to secondary vortex uniform mixing, taking supernate, and carrying out concentration detection on the fat-soluble vitamins by adopting an LC-MS (Liquid Chromatography-Mass Spectrometer) method. The method has the advantages of low cost, high reliability, high detection result accuracy and good product stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical detection, and particularly relates to a method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin. Background Art

[0002] Vitamins are a class of small-molecule organic compounds necessary for maintaining the normal physiological functions of the body and specific metabolic reactions within cells, and are important and indispensable substances for maintaining and regulating the normal metabolism of the body. Currently, there are 13 vitamins in total, which can be divided into two categories according to their solubility: fat-soluble and water-soluble. Fat-soluble vitamins are insoluble in water, soluble in fat and organic solvents, coexist with lipids in food, and are absorbed together with lipids. Their types include vitamin A, vitamin D, vitamin E, vitamin K, etc. Fat-soluble vitamins are important and indispensable substances for maintaining and regulating the normal metabolism of the body, and different vitamins play different roles in the human body. Vitamin A ensures the normal function of the retina, promotes growth and development, and maintains the immune system function; vitamin D has the functions of regulating calcium and phosphorus metabolism, promoting calcium and phosphorus absorption and bone formation, regulating immunity, and preventing the occurrence of autoimmune diseases; vitamin E has antioxidant effects and affects lipid metabolism, etc.; vitamin K can participate in blood coagulation as a coenzyme and synthesize specific proteins related to bone metabolism.

[0003] Currently, mass spectrometry has been widely used in the detection of fat-soluble vitamins in serum, which faces the problem of matrix selection for establishing a standard curve. Due to the presence of a certain concentration of endogenous fat-soluble vitamins in normal serum, it interferes with the detection method and cannot accurately quantify within the detection range. Therefore, it is impossible to directly use animal serum or human serum to prepare calibration products and quality control products with accurate concentrations. It is necessary to pretreat to remove endogenous fat-soluble vitamins or find a matrix that is the same as or similar to the matrix of the sample to be tested.

[0004] In the prior art, one method is to prepare negative serum by removing fat-soluble vitamins from serum as the matrix of calibration products and quality control products for fat-soluble vitamins (for example, Application No. 202210665695.X, "A Method and Kit for Preparing Negative Serum for Fat-Soluble Vitamins" discloses that fat-soluble vitamins in serum are removed by extraction with an extractant followed by ultraviolet lamp irradiation for 6 to 24 hours. However, this method of removing endogenous vitamins from serum has a diverse operation process, involves complex reagents, and takes a long time. Moreover, commercial negative serum is expensive. In addition, a certain amount of fat-soluble vitamins still remain in the serum treated by the above method, which will cause interference with the detection results.

[0005] Another method is to use a matrix similar to the test sample (e.g., bovine serum albumin (BSA)) to prepare calibration standards and quality control samples (e.g., Patent Application No. 202110538061.3 discloses "A rapid mass spectrometry detection kit and method for fat-soluble vitamins in serum" and Patent Application No. 201910862188.3 discloses "A method for high-throughput determination of multiple fat-soluble vitamins by ultra-high performance liquid chromatography-mass spectrometry"). BSA is a globulin in bovine serum, mainly playing roles in maintaining osmotic pressure, pH buffering, acting as a carrier, and providing nutrition. It is widely used in life science disciplines such as cell culture, in vitro diagnosis, human and veterinary drugs, molecular biology, serology, and general research. However, currently commercially available BSA or other alternative matrices are all water-soluble products, and the solubility of fat-soluble vitamins in water-soluble liquids is extremely low. The calibration standards or quality control samples prepared by existing technologies need to be accurately assigned by a detection system, and in the BSA solution, the samples are extremely prone to degradation due to the lack of proteins that can specifically bind to fat-soluble vitamins, affecting the accuracy of the detection results. Summary of the Invention

[0006] The object of the present invention is to propose a method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin in view of the above problems. This method has low cost, high reliability, high accuracy of detection results, and good product stability.

[0007] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0008] A method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin proposed by the present invention includes the following steps:

[0009] S1. Obtain bovine serum albumin raw material and add PBS buffer solution;

[0010] S2. Add an antioxidant to the bovine serum albumin raw material added with PBS buffer solution to form a 5% - 10% BSA solution;

[0011] S3. Add 0.2% - 2.0% surfactant to the BSA solution to form an enhanced base solution;

[0012] S4. Add a fat-soluble vitamin solution to the enhanced base solution to form a vitamin matrix solution;

[0013] S5. Take 50 μL - 200 μL of the vitamin matrix solution, add 10 μL - 50 μL of zinc sulfate solution, vortex mix once, and then add 100 μL - 500 μL of internal standard solution for secondary vortex mixing;

[0014] S6. Centrifuge the solution after mixing the secondary vortex, and take at least 100 μL of the supernatant for detecting the concentration of fat-soluble vitamins by LC-MS method.

[0015] Preferably, the content of the antioxidant is 1% - 5%, and it includes a first reagent and a second reagent. The first reagent is vitamin C or tea polyphenols, and the second reagent is at least one of 2,6-di-tert-butyl-p-cresol and butylated hydroxyanisole.

[0016] Preferably, the antioxidant is vitamin C at 5 mg / mL: 2,6-di-tert-butyl-p-cresol at 5 mg / mL = 1:1 - 2:1.

[0017] Preferably, the surfactant is Triton X-100 or Tween 20, and Triton X-100 is 0.5% - 1%, and Tween 20 is 0.2% - 2%.

[0018] Preferably, the fat-soluble vitamin solution includes vitamin A, vitamin D, vitamin E, and vitamin K. Vitamin D includes 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, vitamin K is vitamin K1, and the concentration of vitamin A is 20 ng / mL - 3000 ng / mL, the concentration of 25-hydroxyvitamin D2 is 1 ng / mL - 200 ng / mL, the concentration of 25-hydroxyvitamin D3 is 1 ng / mL - 200 ng / mL, the concentration of vitamin E is 0.2 μg / mL - 30.0 μg / mL, and the concentration of vitamin K1 is 0.1 ng / mL - 12 ng / mL.

[0019] Preferably, the ratio of the volume of the internal standard solution to the volume of the vitamin matrix solution is 1:5, and the concentration of the zinc sulfate solution is 0.4 mol / L - 4 mol / L.

[0020] Preferably, when forming the vitamin matrix solution by adding the fat-soluble vitamin solution to the enhanced base solution, the volume of the fat-soluble vitamin solution is less than or equal to 10% of the volume of the BSA solution, and it is added in a manner of stirring while adding at a uniform speed, and the stirring rate is not less than 500 rpm / min, and the stirring duration is not less than 10 min.

[0021] Preferably, the rotation speed of the first vortex mixing and the second vortex mixing is 400 rpm - 800 rpm, the duration is 30 min - 60 min, and the temperature is 18°C - 30°C; the rotation speed of the centrifugation is 4000 rpm - 5000 rpm, and the time is 5 min - 10 min.

[0022] Preferably, the LC-MS method is specifically as follows:

[0023] 1) Chromatographic conditions:

[0024] Mobile phase A: water containing 0.1% formic acid, mobile phase B: methanol containing 0.1% formic acid; chromatographic column: C18 column; column temperature: 40 °C; flow rate: 0.6 mL / min; injection volume: 25 μL; gradient elution is as follows:

[0025] Time / min Phase A / % Phase B / % 0 45 55 0.4 45 55 1.0 15 85 1.6 15 85 1.8 10 90 2.8 10 90 3.2 5 95 5.0 5 95 5.2 2 98 6.5 2 98 7.0 45 55 8.0 45 55

[0026] 2) Mass spectrometry conditions:

[0027] Positive ion detection mode; ion source temperature: 450 °C; nebulizing gas: 55 psi; curtain gas: 40 psi; multiple reaction monitoring scanning mode is adopted, and the monitored ion pairs are: vitamin A: 269.3>119.0; 25-hydroxyvitamin D2: 395.3>269.2; 25-hydroxyvitamin D3: 383.4>365.3; vitamin E: 431.3>137.1; vitamin K1: 451.4>187.2; vitamin A-d6: 275.2>122.0; 25-hydroxyvitamin D2-d3: 398.4>272.1; 25-hydroxyvitamin D3-d6: 389.6>371.0; vitamin E-d6: 437.2>143.1; vitamin K1-d7: 458.2>194.1.

[0028] Preferably, the method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin further includes:

[0029] S7. Freeze-drying the vitamin matrix solution to obtain a freeze-dried product, and the freeze-drying process is as follows:

[0030] S71. Pre-freeze the vitamin matrix solution, the cooling rate of pre-freezing is 0.5 °C / min to 1.5 °C / min, the temperature is -60 °C to -35 °C, the maintenance duration is 6 h to 10 h, and the vacuum degree is 0 mbar;

[0031] S72. Perform primary sublimation on the pre-frozen vitamin matrix solution, the cooling rate of primary sublimation is 0.2 °C / min to 0.5 °C / min, the temperature is -20 °C to -5 °C, the maintenance duration is 6 h to 10 h, and the vacuum degree is 0.15 mbar to 0.3 mbar;

[0032] S73. Perform secondary sublimation on the vitamin matrix solution after primary sublimation, the cooling rate of secondary sublimation is 0.3 °C / min to 0.8 °C / min, the temperature is -5 °C to 10 °C, the maintenance duration is 4 h to 8 h, and the vacuum degree is 0.2 mbar;

[0033] S74. Perform analytical drying on the vitamin matrix solution after secondary sublimation. The cooling rate of the analytical drying is 0.5°C / min to 1.5°C / min, the temperature is 20°C to 40°C, the duration is 8 hours to 12 hours, and the vacuum degree is 0.1 mbar.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] By adding antioxidants to the bovine serum albumin raw material with PBS buffer and adding surfactants to the BSA solution, this method reduces the surface tension of the BSA solution, enhances the solubility of fat-soluble vitamins in the BSA solution, makes the obtained calibrator index closer to the actual content, and can prepare a more stable fat-soluble vitamin product, improving the accuracy of detection and the stability of the product; the surfactant also has good environmental compatibility, is not easy to accumulate, causes little harm to water sources, animals, etc., and adding surfactants to the BSA solution can also reduce the use of other chemical reagents in addition to enhancing the solubility of fat-soluble vitamins, further reducing the impact on the environment, making it low-cost, highly reliable, and environmentally friendly, and suitable for detecting the concentration of fat-soluble vitamins in serum. Description of the Drawings

[0036] Figure 1 It is a flowchart of the method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin of the present invention;

[0037] Figure 2 It is a detection chromatogram of fat-soluble vitamins in bovine serum albumin of the present invention. Detailed Embodiments

[0038] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0039] It should be noted that unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field of the present application. The terms used in the specification of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0040] As Figure 1 - 2 shown, a method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin includes the following steps:

[0041] S1. Obtain bovine serum albumin raw material and add PBS buffer;

[0042] S2. Add an antioxidant to the bovine serum albumin raw material with PBS buffer to form a 5% - 10% BSA solution;

[0043] S3. Add 0.2% - 2.0% surfactant to the BSA solution to form an enhanced base solution;

[0044] S4. Add a fat-soluble vitamin solution to the enhanced base solution to form a vitamin matrix solution;

[0045] S5. Take 50 μL - 200 μL of the vitamin matrix solution, add 10 μL - 50 μL of zinc sulfate solution, vortex mix once, and then add 100 μL - 500 μL of the internal standard solution for secondary vortex mixing;

[0046] S6. Centrifuge the solution after secondary vortex mixing, and take at least 100 μL of the supernatant to detect the concentration of fat-soluble vitamins by LC-MS method.

[0047] Among them, this method enhances the solubility of fat-soluble vitamins in it and improves stability by adding a surfactant in a suitable proportion to the BSA solution. Specifically, the molecules of the surfactant usually contain two different types of groups: one end is a long-chain non-polar hydrophobic group (such as hydrocarbons), and the other end is a hydrophilic polar group (such as carboxylic acids, sulfonic acids, etc.), so that the surfactant can reduce the surface tension of the liquid in the solution and improve its surface activity ability. At a specific concentration, the surfactant can dissolve some substances insoluble in water and increase their solubility in water. At the same time, the emulsifying property of the surfactant forms a stable emulsion mixture between the fat-soluble vitamins and the BSA solution. On the other hand, to a certain extent without destroying the matrix, it weakens the influence of other interfering substances, thereby enhancing the stability of the product. Using the LC-MS method to verify the concentration of fat-soluble vitamins has high sensitivity and can detect lower concentrations of various fat-soluble vitamins. And the surfactant also has good environmental compatibility, is not easy to accumulate, causes little harm to water sources, animals, etc., and adding the surfactant to the BSA solution can also reduce the use of other chemical reagents, further reducing the impact on the environment, making it low-cost, highly reliable, and environmentally friendly.

[0048] In one embodiment, the content of the antioxidant is 1% - 5%, and it includes a first reagent and a second reagent. The first reagent is vitamin C or tea polyphenols, and the second reagent is at least one of 2,6 - di-tert-butyl-p-cresol and butylated hydroxyanisole.

[0049] In one embodiment, the antioxidant is vitamin C at 5 mg / mL: 2,6 - di-tert-butyl-p-cresol at 5 mg / mL = 1:1 - 2:1.

[0050] Among them, the preferred antioxidants are vitamin C at 5 mg / mL: 2,6-di-tert-butyl-p-cresol at 5 mg / mL = 1:1, and its concentration added to the BSA solution is 5%, which can further enhance the stability of the solution.

[0051] In one embodiment, the surfactant is Triton X-100 or Tween 20, and Triton X-100 is 0.5% - 1%, and Tween 20 is 0.2% - 2%.

[0052] In one embodiment, the fat-soluble vitamin solution includes vitamin A, vitamin D, vitamin E, and vitamin K. Vitamin D includes 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. Vitamin K is vitamin K1, and the concentration of vitamin A is 20 ng / mL to 3000 ng / mL, the concentration of 25-hydroxyvitamin D2 is 1 ng / mL to 200 ng / mL, the concentration of 25-hydroxyvitamin D3 is 1 ng / mL to 200 ng / mL, the concentration of vitamin E is 0.2 μg / mL to 30.0 μg / mL, and the concentration of vitamin K1 is 0.1 ng / mL to 12 ng / mL. It is easy to understand that the types and concentrations of each vitamin A, vitamin D, vitamin E, and vitamin K can also be selected according to actual needs.

[0053] In one embodiment, the ratio of the volume of the internal standard solution to the volume of the vitamin matrix solution is 1:5, and the concentration of the zinc sulfate solution is 0.4 mol / L to 4 mol / L.

[0054] In one embodiment, when forming the vitamin matrix solution by adding the fat-soluble vitamin solution to the enhanced base solution, the volume of the fat-soluble vitamin solution is less than or equal to 10% of the volume of the BSA solution, and it is added in a uniform speed while stirring, and the stirring rate is not less than 500 rpm / min, and the stirring duration is not less than 10 min.

[0055] In one embodiment, the rotation speeds of the first vortex mixing and the second vortex mixing are 400 rpm to 800 rpm, the duration is 30 min to 60 min, and the temperature is 18°C to 30°C; the rotation speed of the centrifugation treatment is 4000 rpm to 5000 rpm, and the time is 5 min to 10 min.

[0056] In one embodiment, the LC-MS method is specifically as follows:

[0057] 1) Chromatographic conditions:

[0058] Mobile phase A: water containing 0.1% formic acid, mobile phase B: methanol containing 0.1% formic acid; chromatographic column: C18 column; column temperature: 40°C; flow rate: 0.6 mL / min; injection volume: 25 μL; gradient elution is as follows in the table:

[0059]

[0060]

[0061] 2) Mass spectrometry conditions:

[0062] Positive ion detection mode; ion source temperature: 450 °C; nebulizing gas: 55 psi; curtain gas: 40 psi; multi-reaction monitoring scanning mode is adopted, and the monitored ion pairs are: Vitamin A: 269.3>119.0; 25-hydroxyvitamin D2: 395.3>269.2; 25-hydroxyvitamin D3: 383.4>365.3; Vitamin E: 431.3>137.1; Vitamin K1: 451.4>187.2; Vitamin A-d6: 275.2>122.0; 25-hydroxyvitamin D2-d3: 398.4>272.1; 25-hydroxyvitamin D3-d6: 389.6>371.0; Vitamin E-d6: 437.2>143.1; Vitamin K1-d7: 458.2>194.1.

[0063] Among them, for the monitored analyte ion pairs: the parent ion mass-to-charge ratio of Vitamin A is 269.3, and the corresponding daughter ion mass-to-charge ratio is 119.0; the parent ion mass-to-charge ratio of 25-hydroxyvitamin D2 is 395.3, and the corresponding daughter ion mass-to-charge ratio is 269.2; the parent ion mass-to-charge ratio of 25-hydroxyvitamin D3 is 383.4, and the corresponding daughter ion mass-to-charge ratio is 365.3; the parent ion mass-to-charge ratio of Vitamin E is 431.3, and the corresponding daughter ion mass-to-charge ratio is 137.1; the parent ion mass-to-charge ratio of Vitamin K1 is 451.4, and the corresponding daughter ion mass-to-charge ratio is 187.2; the parent ion mass-to-charge ratio of the Vitamin A-d6 isotope deuterated internal standard is 275.2, and the corresponding daughter ion mass-to-charge ratio is 122.0; the parent ion mass-to-charge ratio of the 25-hydroxyvitamin D2-d3 isotope deuterated internal standard is 398.4, and the corresponding daughter ion mass-to-charge ratio is 272.1; the parent ion mass-to-charge ratio of the 25-hydroxyvitamin D3-d6 isotope deuterated internal standard is 389.6, and the corresponding daughter ion mass-to-charge ratio is 371.0; the parent ion mass-to-charge ratio of the Vitamin E-d6 isotope deuterated internal standard is 437.2, and the corresponding daughter ion mass-to-charge ratio is 143.1; the parent ion mass-to-charge ratio of the Vitamin K1-d7 isotope deuterated internal standard is 458.2, and the corresponding daughter ion mass-to-charge ratio is 194.1.

[0064] Specifically, in LC-MS detection, the peak areas of the analytes vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, and vitamin K1 and the peak areas of the corresponding internal standards are recorded, and the ratios of the peak areas of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, and vitamin K1 to the peak areas of the corresponding internal standards are calculated. The peak areas are determined according to the corresponding mass-to-charge ratios. Using the standard curve plotted with the ratio (y) of the target concentration (x) of each vitamin in the calibrator to the corresponding peak area, and then substituting the ratio of the peak area of each vitamin in the sample to be tested to the peak area of the internal standard into the fitted standard curve equation, the concentration of each vitamin in the sample to be tested can be calculated, which is a well-known technique in the art and will not be elaborated here. The concentration detection results of the vitamin matrix solution are as Figure 2 shown, where the ordinate corresponding to each vitamin is the mass spectrometry response value (CPS), and the abscissa is time (Time), with the unit of min.

[0065] In one embodiment, the method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin further includes:

[0066] S7. Freeze-drying the vitamin matrix solution to obtain a freeze-dried product, and the freeze-drying process is as follows:

[0067] S71. Pre-freeze the vitamin matrix solution, with a pre-freezing cooling rate of 0.5 °C / min to 1.5 °C / min, a temperature of -60 °C to -35 °C, a holding duration of 6 h to 10 h, and a vacuum degree of 0 mbar;

[0068] S72. Perform primary sublimation on the pre-frozen vitamin matrix solution, with a primary sublimation cooling rate of 0.2 °C / min to 0.5 °C / min, a temperature of -20 °C to -5 °C, a holding duration of 6 h to 10 h, and a vacuum degree of 0.15 mbar to 0.3 mbar;

[0069] S73. Perform secondary sublimation on the vitamin matrix solution after primary sublimation, with a secondary sublimation cooling rate of 0.3 °C / min to 0.8 °C / min, a temperature of -5 °C to 10 °C, a holding duration of 4 h to 8 h, and a vacuum degree of 0.2 mbar;

[0070] S74. Perform analytical drying on the vitamin matrix solution after secondary sublimation, with an analytical drying cooling rate of 0.5 °C / min to 1.5 °C / min, a temperature of 20 °C to 40 °C, a holding duration of 8 h to 12 h, and a vacuum degree of 0.1 mbar.

[0071] Among them, after freeze-drying the vitamin matrix solution, the concentration of fat-soluble vitamins in the freeze-dried product can also be determined by LC-MS (liquid chromatography tandem mass spectrometry). It can be found through detection that the solubility of fat-soluble vitamins in bovine serum albumin by this method is significantly enhanced and the stability is greatly improved.

[0072] The following is verified by specific experimental data:

[0073] 1) The internal standard solution of fat-soluble vitamins in this example, and the corresponding fat-soluble vitamin solution also includes vitamin A, vitamin D, vitamin E and vitamin K. Vitamin D includes 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, and vitamin K is vitamin K1, and the concentrations are shown in Table 1.

[0074] Table 1

[0075]

[0076] 2) Screening of the type and concentration of surfactants, as shown in Table 2, different types and different concentrations of activators are added to the BSA solution.

[0077] Table 2

[0078]

[0079]

[0080]

[0081] According to the data in Table 2 above, it can be seen that choosing 0.5% - 1% of Triton X100 can improve the solubility of fat-soluble vitamins in the BSA solution.

[0082] 2) The freeze-drying process of the vitamin matrix solution in this example is specifically shown in Table 3.

[0083] Table 3

[0084] Step Cooling rate (℃ / min) Temperature (℃) Duration maintained (h) Vacuum degree (mbar) Remarks 1 1 -45 8 off Preliminary freezing 2 0.3 -10 8 0.2 Primary sublimation 3 0.4 0 6 0.2 Secondary sublimation 4 1 30 10 0.1 Analysis drying

[0085] 3) The LC-MS detection results are shown in Tables 4 and 5.

[0086] (1) Comparison of the concentration detection of fat-soluble vitamins in two matrices, the two matrices are BSA solution and enhanced base solution (BSA solution + surfactant), as shown in Table 4.

[0087] Table 4

[0088]

[0089]

[0090] When no surfactant was added to the BSA solution, the deviation between the concentration of fat-soluble vitamins and the theoretical concentration was as high as 40% - 60% (especially for vitamin E and vitamin K1). However, after adding the surfactant, the deviation between the concentration of fat-soluble vitamins and the theoretical concentration was within 15%, indicating that the surfactant enhanced the solubility of fat-soluble vitamins in the BSA solution.

[0091] (2) The real-time stability of different product types is shown in Table 5.

[0092] Table 5

[0093]

[0094]

[0095]

[0096] As can be seen from Table 5, in the BSA solution, two enhanced base liquids with and without surfactant were respectively prepared, and the same concentration of fat-soluble vitamin solution was added to these two enhanced base liquids. The solubility of fat-soluble vitamins in the BSA solution enhanced by the surfactant was verified by detecting the concentration of fat-soluble vitamins in these two vitamin matrix solutions by LC-MS method. That is, under the same conditions, the concentration deviation of fat-soluble vitamins in the vitamin matrix solution without surfactant exceeded 15% after being stored for 3 months under freezing conditions, while the vitamin matrix solution with surfactant could be stably stored for 3 months under freezing conditions; and the concentration deviation of fat-soluble vitamins in the freeze-dried product without surfactant exceeded 15% after being stored for 6 months under freezing conditions, while the freeze-dried product with surfactant could be stably stored for 12 months under freezing conditions, indicating that the surfactant can enhance the stability of the product. The concentration unit for each vitamin detection is ng / mL.

[0097] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0098] The above-described embodiments only represent the embodiments of the present application that are described in more specific and detailed ways, but should not be construed as a limitation on the scope of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin, characterized in that: The method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin comprises the following steps: S1. Obtain bovine serum albumin raw material and add PBS buffer solution; S2. Add an antioxidant to the bovine serum albumin raw material added with PBS buffer solution to form a 5%-10% BSA solution; S3. Add 0.2%-2.0% surfactant to the BSA solution to form an enhanced base solution; S4. Add a fat-soluble vitamin solution to the enhanced base solution to form a vitamin matrix solution; S5. Take 50 μL - 200 μL of the vitamin matrix solution, add 10 μL - 50 μL of zinc sulfate solution, vortex mix once and then add 100 μL - 500 μL of internal standard solution for secondary vortex mixing; S6. Centrifuge the solution after secondary vortex mixing, and take at least 100 μL of the supernatant for concentration detection of fat-soluble vitamins by LC-MS method.

2. The method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin according to claim 1, characterized in that: The content of the antioxidant is 1%-5%, and it includes a first reagent and a second reagent. The first reagent is vitamin C or tea polyphenols, and the second reagent is at least one of 2,6-ditert-butyl-p-cresol and butylated hydroxyanisole.

3. The method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin according to claim 2, characterized in that: The antioxidant is vitamin C at 5 mg / mL: 2,6-ditert-butyl-p-cresol at 5 mg / mL = 1:1 - 2:

1.

4. The method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin according to claim 1, characterized in that: The surfactant is Triton X100 or Tween 20, and the Triton X100 is 0.5%-1%, and the Tween 20 is 0.2%-2%.

5. The method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin according to claim 1, wherein: The fat-soluble vitamin solution includes vitamin A, vitamin D, vitamin E and vitamin K. The vitamin D includes 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. The vitamin K is vitamin K1. The concentration of vitamin A is 20 ng / mL - 3000 ng / mL, the concentration of 25-hydroxyvitamin D2 is 1 ng / mL - 200 ng / mL, the concentration of 25-hydroxyvitamin D3 is 1 ng / mL - 200 ng / mL, the concentration of vitamin E is 0.2 μg / mL - 30.0 μg / mL, and the concentration of vitamin K1 is 0.1 ng / mL - 12 ng / mL.

6. The method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin according to claim 1, characterized in that: The ratio of the volume of the internal standard solution to the volume of the vitamin matrix solution is 1:5, and the concentration of the zinc sulfate solution is 0.4 mol / L - 4 mol / L.

7. The method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin according to claim 1, characterized in that: When adding the fat-soluble vitamin solution to the enhanced base solution to form a vitamin matrix solution, the volume of the fat-soluble vitamin solution is less than or equal to 10% of the volume of the BSA solution, and it is added in a manner of stirring while adding at a constant speed, and the stirring rate is not less than 500 rpm / min, and the stirring duration is not less than 10 min.

8. The method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin according to claim 1, characterized in that: The rotation speed of the primary vortex mixing and the secondary vortex mixing is 400 rpm - 800 rpm, the duration is 30 min - 60 min, and the temperature is 18°C - 30°C; the rotation speed of the centrifugation treatment is 4000 rpm - 5000 rpm, and the time is 5 min - 10 min.

9. The method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin according to claim 1, wherein: The LC-MS method is specifically as follows: 1) Chromatographic conditions: Mobile phase A: water containing 0.1% formic acid, mobile phase B: methanol containing 0.1% formic acid; chromatographic column: C18 column; column temperature: 40 °C; flow rate: 0.6 mL / min; injection volume: 25 μL; gradient elution is as follows in the table: 2) Mass spectrometry conditions: Positive ion detection mode; ion source temperature: 450 °C; nebulizing gas: 55 psi; curtain gas: 40 psi; multi-reaction monitoring scanning mode is adopted, and the monitored ion pairs are: vitamin A: 269.3>119.0; 25-hydroxyvitamin D2: 395.3>269.2; 25-hydroxyvitamin D3: 383.4>365.3; vitamin E: 431.3>137.1; vitamin K1: 451.4>187.2; vitamin A-d6: 275.2>122.0; 25-hydroxyvitamin D2-d3: 398.4>272.1; 25-hydroxyvitamin D3-d6: 389.6>371.0; vitamin E-d6: 437.2>143.1; vitamin K1-d7: 458.2>194.

1.

10. The method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin according to claim 1, wherein: The method for enhancing the solubility of fat-soluble vitamins in bovine serum albumin further includes: S7. Freeze-drying the vitamin matrix solution to obtain a freeze-dried product, and the freeze-drying is specifically as follows: S71. Pre-freeze the vitamin matrix solution, the cooling rate of the pre-freezing is 0.5 °C / min to 1.5 °C / min, the temperature is -60 °C to -35 °C, the maintenance duration is 6 h to 10 h, and the vacuum degree is 0 mbar; S72. Perform primary sublimation on the pre-frozen vitamin matrix solution, the cooling rate of the primary sublimation is 0.2 °C / min to 0.5 °C / min, the temperature is -20 °C to -5 °C, the maintenance duration is 6 h to 10 h, and the vacuum degree is 0.15 mbar to 0.3 mbar; S73. Perform secondary sublimation on the vitamin matrix solution after primary sublimation, the cooling rate of the secondary sublimation is 0.3 °C / min to 0.8 °C / min, the temperature is -5 °C to 10 °C, the maintenance duration is 4 h to 8 h, and the vacuum degree is 0.2 mbar; S74. Perform analytical drying on the vitamin matrix solution after secondary sublimation, the cooling rate of the analytical drying is 0.5 °C / min to 1.5 °C / min, the temperature is 20 °C to 40 °C, the maintenance duration is 8 h to 12 h, and the vacuum degree is 0.1 mbar.

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