Method for measuring lactose content in protein component formula food for special medical purpose

Abstract

The invention discloses a method for determining lactose in protein component formula food for special medical purposes. The method comprises the following steps: adding a trichloroacetic acid solution, precipitating acetonitrile to remove protein in a sample, detecting by using a high performance liquid chromatograph with a differential refraction detector, and quantifying by using an external standard method. A chromatographic column used in the method is an amino chromatographic column (4.6 mm * 250 mm, the particle size is 5 [mu] m, and amino silane bonded silica gel is used as a filler), a good separation effect can be achieved, and accurate quantification is carried out. The method is good in reproducibility, high in sensitivity, simple to operate and low in detection cost, effectively eliminates the influence of incomplete protein precipitation on a chromatographic column when a sample with high protein content is measured, and is suitable for quantitative analysis of the lactose content in protein component formula food for special medical purposes.

Description

Technical Field

[0001] The invention belongs to the technical field of medical food detection, and in particular relates to a method for determining lactose in a protein component formula food for special medical purposes. Background Art

[0002] Lactose is a carbohydrate unique to the milk of humans and other mammals. It exists in milk and most animal dairy products. It is a disaccharide composed of glucose and galactose, with a molecular formula of C12H22O11. During the growth and development of infants and young children, lactose not only provides energy, but also participates in the development of the brain. During the digestion process, lactose is broken down by lactase in the body to form galactose and glucose that can be absorbed by the small intestine. However, according to statistics, about 70% of adults worldwide are affected by lactose intolerance or lactose malabsorption, and lactose-intolerant patients can consume foods containing lactose according to their own lactose tolerance. Therefore, it is particularly important to accurately measure the lactose content in food.

[0003] Special medical purpose protein component formula foods are non-complete nutritional formula foods specially processed and prepared for people who need protein supplementation due to specific diseases or medical conditions. The protein source can be one or more amino acids, protein hydrolysates, peptides or high-quality whole proteins, and the protein content of powder products is generally ≥75%. Common protein sources such as whey protein powder, concentrated whey protein powder, casein, etc. all contain lactose, but in the study of lactose content detection methods, it was found that when the standard method was used for detection, the protein precipitation was incomplete due to the high protein content of the sample. When the amino chromatographic column was used for determination, the pressure gradually increased and damaged the chromatographic column, resulting in time-consuming detection and high cost. Therefore, it is particularly important to develop a new detection method to eliminate the influence of incomplete protein precipitation on the chromatographic column and accurately determine the lactose content in special medical purpose protein component formula foods. Summary of the invention

[0004] The invention provides a method for determining the lactose content in a protein component formula food for special medical purposes. The method has the characteristics of good reproducibility, high sensitivity, simple operation, low inspection cost, and effectively eliminates the influence of incomplete protein precipitation on the chromatographic column.

[0005] The method for determining the lactose content in the protein component formula food for special medical purposes is: S1: After the sample is weighed, trichloroacetic acid solution and acetonitrile are added to precipitate the protein; S2: After ultrasonic extraction, the volume was fixed with trichloroacetic acid solution, centrifuged, and filtered through a membrane to obtain an injection solution; S3: Preparation of standard solution: weigh lactose standard to prepare lactose standard solution; S4: Determination by high performance liquid chromatography with a differential refractometer. Mobile phase: a mixture of acetonitrile and water in a volume ratio of 70:30.

[0006] The protein component formula food for special medical purposes is a powder containing concentrated whey protein and isolated soy protein.

[0007] In the S1, the concentration of trichloroacetic acid added is 1 wt %.

[0008] In S2, the ultrasonic extraction time is 10 min, the centrifugal speed is 8000-10000 rpm / min, and the centrifugal time is 10 min.

[0009] In the above-mentioned S2, the filter membrane is a 0.22 μm organic filter membrane.

[0010] The steps for preparing the lactose standard solution in S3 are as follows: (1) 20.0 mg / mL lactose standard stock solution: weigh 0.5 g of lactose standard, dissolve it in water and transfer it to a 25 mL volumetric flask, add 1.25 mL of acetonitrile, and dilute to the mark with water; (2) standard series working solution: pipette 0.100 mL, 0.50 mL, 1.00 mL, 1.50 mL and 2.00 mL of lactose standard stock solution into a 10.0 mL volumetric flask, dilute to the mark with water, and obtain a standard series working solution with a lactose mass concentration of 0.200 mg / mL, 1.00 mg / mL, 2.00 mg / mL, 3.00 mg / mL and 4.00 mg / mL.

[0011] The chromatographic column in S4 is an amino chromatographic column, 4.6 mm×250 mm, with a particle size of 5 μm, and aminosilane bonded silica gel is used as a filler.

[0012] The flow rate of the mobile phase in S4 is 1.0 mL / min; the injection volume is 10 μL; the column temperature is 40° C., and the detection condition of the differential refractometer is 40° C.

[0013] Compared with the prior art, this method has the following beneficial effects: 1. Compared with the standard method GB 5009.8-2023, the first method, high performance liquid chromatography, the biggest feature of the present invention is that it effectively eliminates the effect of incomplete protein precipitation on the chromatographic column. When using the conventional standard method to detect lactose in special medical protein component formula foods with high protein content, the protein precipitation of the machine test liquid after the filter membrane is incomplete, and protein still precipitates after adding the mobile phase (containing 70% acetonitrile), which leads to an increase in the column pressure of the chromatographic column and damages the chromatographic column. The conventional standard method is not suitable for the detection of lactose in special medical protein component formula foods with high protein content. The present invention adds trichloroacetic acid solution and acetonitrile to the sample, precipitates and removes the protein in the sample, detects with a high performance liquid chromatograph with a differential refractometer, and quantifies with the external standard method. After the machine test liquid after the filter membrane is added to the mobile phase, no protein precipitates, which effectively eliminates the effect of incomplete protein precipitation on the chromatographic column.

[0014] 2. Compared with the standard method, the present invention has the characteristics of good reproducibility, high sensitivity, simple operation, and low inspection cost. The present invention modifies the steps of adding water to dissolve and then adding zinc acetate and potassium ferrocyanide solution to precipitate protein in the conventional standard method, and uses trichloroacetic acid solution and acetonitrile to precipitate protein, shortening the ultrasonic extraction time from 30 min to 10 min, saving some reagent preparation and operation time, and reducing the detection cost. The pretreatment is simple and easy to operate, which can meet the requirements of lactose detection in protein component formula foods for special medical purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Example 1 Lactose standard curve diagram.

[0016] Figure 2 Example 1 High performance liquid chromatogram of lactose standard solution.

[0017] Figure 3 Example 1 High performance liquid chromatogram of lactose in protein component formula food for special medical purposes.

[0018] Figure 4 Example 2 Lactose standard curve diagram.

[0019] Figure 5 Example 2 High performance liquid chromatogram of lactose standard solution.

[0020] Figure 6 Example 2 High performance liquid chromatogram of lactose in protein component formula food for special medical purposes. DETAILED DESCRIPTION

[0021] The present invention will be further described and illustrated by way of examples and drawings below. Example 1

[0022] The lactose content of multiple batches of protein component formula foods (powders) for special medical purposes was determined. The specific implementation steps are as follows: S1: Weigh 2.0 g of the sample protein component formula food for special medical purposes (powder), add 25 mL of 1% trichloroacetic acid solution and 10 mL of acetonitrile.

[0023] S2: Vortex mix, ultrasonically extract for 10 min, dilute to 50 mL with 1% trichloroacetic acid solution, mix, centrifuge at 9000 rpm / min for 10 min, and take the supernatant to filter through a 0.22 μm organic filter membrane.

[0024] S3: Preparation of standard solutions: (1) Lactose standard stock solution (19.94 mg / mL): Weigh 0.5 g of lactose standard, dissolve it in water and transfer it to a 25 mL volumetric flask, add 1.25 mL of acetonitrile and dilute to the mark with water; (2) Standard series working solutions: Pipette 0.100 mL, 0.50 mL, 1.00 mL, 1.50 mL and 2.00 mL of lactose standard stock solution into a 10.0 mL volumetric flask and dilute to the mark with water to obtain a standard series working solution with lactose mass concentrations of 0.1994 mg / mL, 0.9970 mg / mL, 1.9940 mg / mL, 2.9910 mg / mL and 3.9880 mg / mL.

[0025] S4: Chromatographic column: Luna 5 μm NH2 100 A (4.6×250 mm, 5 μm); mobile phase: acetonitrile + water = 70 + 30 (volume ratio). Flow rate: 1.0 mL / min; column temperature: 40 ℃; injection volume: 10 μL; detector: differential refractive index detector (40 ℃).

[0026] Linearity and Range: Inject the standard series working solutions into the HPLC, measure the peak area of ​​lactose, and draw a standard curve with the concentration of the standard working solution (lactose concentration) as the horizontal axis and the lactose peak area as the vertical axis. Figure 1 .

[0027] As can be seen in the figure, the linear equation of lactose is y=1.2629x-0.0705, and the correlation coefficient is 0.9992. The results show that in the range of 0.2 mg / mL~4.0 mg / mL, a good linear relationship is presented.

[0028] Spiked recovery rate: Using methods S1, S2, S3, and S4, 2.0 mL of lactose standard stock solution (19.94 mg / mL) was added. The test results showed that the recoveries were 102.23% and 109.80%, respectively, with a recovery rate of 90%-110%.

[0029] The HPLC chromatogram of lactose standard solution in Example 1 is shown in Figure 2 , the HPLC chromatogram of lactose in protein component formula food for special medical purposes is shown in Figure 3 As can be seen from the figure, the lactose peak can be effectively separated and the peak shape is normal.

[0030] In Example 1, the lactose content of 40 batches of protein component formula foods for special medical purposes (powders) was continuously measured, and the measurement results were all within 2.0±0.2 g / 100g, indicating that the measurement results were stable. Example 2

[0031] The same detection method as in Example 1 was used, except that the chromatographic column used in this example was UltimateXB-NH2 (4.6×250 mm, 5 μm).

[0032] Linearity and Range: The standard series working solutions were injected into the high performance liquid chromatograph respectively, and the peak area of ​​lactose was measured. The standard curve was drawn with the concentration of the standard working solution (lactose concentration) as the abscissa and the lactose peak area as the ordinate. The standard curve of Example 2 is shown in Figure 4 .

[0033] As can be seen in the figure, the linear equation of lactose is y=1.4970x-0.0037, and the correlation coefficient is 1.0000. The results show that in the range of 0.2 mg / mL~4.0 mg / mL, there is a good linear relationship.

[0034] Spiked recovery rate: The same detection method as in Example 1 was adopted, using a chromatographic column Ultimate XB-NH2 (4.6×250 mm, 5 μm), and 2.0 mL of lactose standard stock solution (19.94 mg / mL) was added. The detection results showed that the recoveries were 95.43% and 94.41%, respectively, with a recovery rate of 90%-110%.

[0035] The HPLC chromatogram of the lactose standard solution of Example 2 is shown in Figure 5 , the HPLC chromatogram of lactose in protein component formula food for special medical purposes is shown in Figure 6 As can be seen from the figure, the lactose peak can be effectively separated and the peak shape is normal.

[0036] In Example 2, the lactose content of 10 batches of protein component formula foods for special medical purposes (powders) was measured continuously, and the measured results were all 1.9±0.1 g / 100g, indicating that the measured results were stable. Example 3

[0037] Repeatability: The method of Example 1 was used to process the protein component formula food for special medical purposes 8 times in parallel. The test results are shown in Table 1.

[0038]

[0039] It can be seen from the test results that the special medical purpose protein component formula food was processed in parallel 8 times, and the lactose content test results were in the range of 1.87~1.99 g / 100 g, with an RSD of 2.5%, which was less than 2.7%, and had good repeatability.

[0040] Comparative Example 1: The GB 5009.8 standard method was used to detect the lactose content in protein component formula foods (powders) for special medical purposes. During the detection process, it was found that the column pressure gradually increased to an alarm, and the separation effect became worse. Analysis found that the protein precipitation method using this standard method was incomplete, and protein still precipitated after the filtrate after passing the membrane was added to the mobile phase.

[0041] Comparative Example 2: The same detection method as in Example 1 was used, except that 2.0 g of the sample was weighed and dissolved in 50-60°C water, and the volume was fixed with acetonitrile after ultrasonication. The results showed that an inverted peak was produced in the HPLC graph, and a lactose peak with good separation effect and qualitative and quantitative properties could not be obtained.

[0042] The specific embodiments described above provide a series of detailed descriptions of the technical problems, technical solutions and beneficial effects solved by the present invention. However, the description is only for the specific embodiments of the present invention, and any equivalent embodiments or changes that do not deviate from the technical basis, principle and spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining the lactose content in a protein component formula food for special medical purposes, characterized in that: The specific steps of the determination method are: S1: After the sample is weighed, trichloroacetic acid solution and acetonitrile are added to precipitate the protein; S2: After ultrasonic extraction, the volume was fixed with trichloroacetic acid solution, centrifuged, and filtered through a membrane to obtain an injection solution; S3: Preparation of standard solution: weigh lactose standard to prepare lactose standard solution; S4: Determination by high performance liquid chromatography with a differential refractometer. Mobile phase: a mixture of acetonitrile and water in a volume ratio of 70:

30.

2. The measuring method according to claim 1, characterized in that The protein component formula food for special medical purposes is a powder containing concentrated whey protein and isolated soy protein.

3. The measuring method according to claim 1, characterized in that In the S1, the concentration of trichloroacetic acid added is 1 wt %.

4. The measuring method according to claim 1, characterized in that In S2, the ultrasonic extraction time is 10 min, the centrifugal speed is 8000-10000 rpm / min, and the centrifugal time is 10 min.

5. The measuring method according to claim 1, characterized in that In the above-mentioned S2, the filter membrane is a 0.22 μm organic filter membrane.

6. The measuring method according to claim 1, characterized in that The steps for preparing the lactose standard solution in S3 are as follows: (1) 20.0 mg / mL lactose standard stock solution: weigh 0.5 g of lactose standard, dissolve it in water and transfer it to a 25 mL volumetric flask, add 1.25 mL of acetonitrile, and dilute to the mark with water; (2) standard series working solution: pipette 0.100 mL, 0.50 mL, 1.00 mL, 1.50 mL and 2.00 mL of lactose standard stock solution into a 10.0 mL volumetric flask, dilute to the mark with water, and obtain a standard series working solution with a lactose mass concentration of 0.200 mg / mL, 1.00 mg / mL, 2.00 mg / mL, 3.00 mg / mL and 4.00 mg / mL.

7. The assay method according to claim 1, characterized in that The chromatographic column in S4 is an amino chromatographic column, 4.6 mm×250 mm, with a particle size of 5 μm, and aminosilane bonded silica gel is used as a filler.

8. The measuring method according to claim 1, characterized in that The flow rate of the mobile phase in S4 is 1.0 mL / min; the injection volume is 10 μL; the column temperature is 40° C., and the detection condition of the differential refractometer is 40° C.

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