A method for detecting the content of lactoferrin in hydrolyzed milk powder

Abstract

The present application belongs to the field of food quality and nutrition detection, and provides a method for detecting the content of lactoferrin in hydrolyzed milk powder, comprising: step S1, dissolving the sample to be tested, hydrolyzed milk powder, in an extraction solution, centrifuging to remove fat after complete dissolution to obtain supernatant; step S2, passing the supernatant through a heparin affinity column and collecting the eluate; step S3, quantifying the lactoferrin in the eluate by the external standard method of high performance liquid chromatography to obtain the weight of lactoferrin, and then obtaining the content of lactoferrin in the hydrolyzed milk powder according to the weight and the weight of the sample to be tested. The recovery rate of lactoferrin can reach more than 90%, which can effectively solve the problem that the latest national standard "GB 5009.299-2024 Food Safety National Standard Determination of Lactoferrin in Food" cannot be applied to hydrolyzed milk powder samples, and meet the requirements of food safety detection.

Description

Technical Field

[0001] The invention belongs to the field of food quality and nutrition detection, and particularly relates to a method for detecting the content of lactoferrin in hydrolyzed milk powder. Background Art

[0002] Lactoferrin (LF) is an iron-binding protein and a core immune function protein. It competes with pathogens for iron, defending against pathogenic microbial invasion and thus protecting infants and young children from immune damage. Therefore, as a functional nutrient, it is widely added to foods, particularly infant formula.

[0003] According to clinical trials in the 2022 "Chinese Expert Consensus on the Clinical Application of Lactoferrin," a lactoferrin content of 0.6g / L will effectively reduce the risk of respiratory and gastrointestinal infections in infants and young children. A lactoferrin content of 0.6g / L is equivalent to the lactoferrin content of infant formula containing 450mg / 100g lactoferrin, after being diluted into milk according to the feeding schedule. Therefore, lactoferrin plays an extremely important role, and infant formula containing lactoferrin requires accurate testing and quantification to ensure food safety. Based on this, my country promulgated the national standard "GB 5009.299-2024 National Food Safety Standard - Determination of Lactoferrin in Foods" in 2024, which can be used to determine the content of lactoferrin in samples such as sterilized milk, milk-containing beverages, adjusted milk powder, and infant formula.

[0004] However, some infants and young children are allergic to cow's milk protein and therefore need to consume hydrolyzed milk powder. Since the protein in hydrolyzed milk powder is hydrolyzed into smaller molecular weight polypeptides and amino acid fragments, its sample matrix is ​​essentially different from that of conventional milk powder. For hydrolyzed milk powder samples, when the national standard is used to detect lactoferrin, it will be found that the lactoferrin recovery rate cannot meet the national standard recovery rate requirements because the polypeptides and amino acid fragments contained in them have an impact on the detection of lactoferrin. For this reason, the standard-setting unit also issued a notice stating that this standard is not applicable to hydrolyzed milk powder, resulting in the lactoferrin detection of hydrolyzed milk powder becoming a blind spot in food safety. Therefore, the development of a detection method for lactoferrin in hydrolyzed milk powder samples is of great practical significance. Summary of the Invention

[0005] In view of the above technical problems, the present invention provides a method for detecting the content of lactoferrin in hydrolyzed milk powder, comprising the following steps:

[0006] Step S1, dissolving the hydrolyzed milk powder sample to be tested in the extract, and after sufficient dissolution, centrifuging to remove fat to obtain a supernatant;

[0007] Step S2, loading the supernatant onto a heparin affinity column; after loading, eluting with an extraction solution, discarding the effluent; eluting with an eluent, and collecting the eluate;

[0008] Step S3, quantifying the lactoferrin in the eluate by an external standard method of high performance liquid chromatography to obtain the weight of the lactoferrin, and obtaining the lactoferrin content in the hydrolyzed milk powder according to the weight of the lactoferrin and the weight of the sample to be tested.

[0009] Preferably, in step S1, the extract comprises a buffer salt, an amphiphilic surfactant and urea, and has a pH of 5.0 to 7.5, more preferably 6.0 to 6.5.

[0010] Preferably, the concentration of the buffer salt in the extract is 0.05 mol / L to 0.5 mol / L, more preferably 0.1 mol / L to 0.3 mol / L.

[0011] Preferably, in step S1, the weight percentage of the amphiphilic surfactant in the extract is 0.1% to 1.0%, more preferably 0.3% to 0.6%.

[0012] Preferably, in step S1, the concentration of urea in the extract is 1.0 mol / L to 5.0 mol / L, more preferably 2.0 mol / L to 3.0 mol / L.

[0013] Preferably, the buffer salt is ammonium acetate.

[0014] Preferably, the amphiphilic surfactant is any one or more of 3-(1-methylpiperidinium-1-yl)propane-1-sulfonate (NDSB-221), aminosulfobetaine-14 (ASB-14), or dodecyldimethylsulfopropyl betaine.

[0015] Preferably, the ratio of hydrolyzed milk powder to extract is (1.0-10.0 g):30 mL, more preferably (5.0-6.0 g):30 mL.

[0016] Preferably, the specific operation of step S2 is as follows: before use, the heparin affinity column is activated with 5 mL of the extract, the supernatant after centrifugation is loaded onto the heparin affinity column at a flow rate of 1 mL / min to 3 mL / min; after loading, the column is rinsed with 10 mL of the extract, the effluent is discarded, and the column is eluted with 5.0 mL of the eluent, and the eluent is collected. The purpose of eluting with the extract is to remove impurities other than lactoferrin, thereby reducing interference of impurities with subsequent high-performance liquid chromatography.

[0017] More preferably, the eluent is prepared from ammonium acetate and sodium chloride, wherein the concentration of ammonium acetate is 0.1 mol / L, the concentration of sodium chloride is 1.0 mol / L, and the pH value is 6.5-7.0.

[0018] Preferably, in step S3, mobile phase A is 0.1% trifluoroacetic acid, mobile phase B is acetonitrile, and the elution procedure of high performance liquid chromatography is:

[0019] .

[0020] Preferably, in step S3, the peak area of ​​lactoferrin in the eluate is obtained by an external standard method of high performance liquid chromatography, and then the abscissa value corresponding to the peak area, i.e., the lactoferrin concentration, is obtained according to a lactoferrin standard curve, and then the weight of lactoferrin in the eluate is calculated according to the volume of the eluate, and then the lactoferrin content in the sample to be tested is obtained according to the weight of the lactoferrin and the weight of the sample to be tested.

[0021] Preferably, the method further comprises step S0 of drawing a bovine lactoferrin standard curve. The specific experimental process is as follows:

[0022] According to GB 5009.299-2024 National Food Safety Standard - Determination of Lactoferrin in Foods, a series of standard working solutions of lactoferrin standards were prepared using PBS (phosphate) buffer at pH 7.0-7.4 to obtain concentrations of 1.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 50.0 μg / mL, 100.0 μg / mL, and 200.0 μg / mL, respectively. A standard curve was generated using the following HPLC detection conditions. The HPLC detection conditions and elution procedure were the same as those in step S3.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects:

[0024] The present invention detects lactoferrin in a hydrolyzed milk powder sample matrix. First, during sample pretreatment, an extracting solution consisting of a buffer salt, an amphiphilic surfactant, and urea is used for extraction, and the pH of the extracting solution used is 5.0 to 7.5. Then, an eluent consisting of ammonium acetate and sodium chloride is used for elution, and the pH of the eluent is 6.5 to 7.0. Through this operation, the recovery rate can reach over 90%, which can effectively solve the problem that the latest national standard "GB 5009.299-2024 National Food Safety Standard Determination of Lactoferrin in Foods" is not applicable to hydrolyzed milk powder samples, and meet food safety testing requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a standard curve diagram of lactoferrin in Example 1 of the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0027] Unless otherwise specified, the raw materials and materials used in the examples of the present invention were purchased through general commercial channels.

[0028] The sources of the raw materials, materials and instruments involved in the following examples are as follows:

[0029] Hydrolyzed milk powder, Nestlé, Aptamil, Mead Johnson, Feihe, Abbott and Wyeth;

[0030] Lactoferrin standard (cas number: 146897-68-9);

[0031] Heparin affinity column, 6 mL / tube, Shanghai Anpu Laboratory Technology Co., Ltd.

[0032] Agilent 1290 high performance liquid chromatography instrument equipped with UV detector;

[0033] Chromatographic column (Athena C4 4.6×150mm, 5μm, 300A);

[0034] HPLC grade water, Shanghai Anpu Laboratory Technology Co., Ltd.

[0035] <Example 1>

[0036] In this embodiment, the hydrolyzed milk powder sample was prepared in the laboratory: a certain amount of lactoferrin standard was weighed and added to Nestlé milk powder that did not contain lactoferrin. The lactoferrin concentration calculated based on the added amount was the theoretical added lactoferrin concentration.

[0037] In this embodiment, the composition of the extract is shown in Table 1 below, and the composition of the eluent is shown in Table 2.

[0038] Table 1. Extraction solution formula

[0039]

[0040] Table 2. Eluent composition

[0041]

[0042] Experimental process:

[0043] Step S0, drawing a bovine lactoferrin standard curve: The specific experimental process is:

[0044] According to the standard of GB 5009.299-2024 National Food Safety Standard Determination of Lactoferrin in Food, the lactoferrin standard was prepared into a standard series of working solutions with concentrations of 1.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 50.0 μg / mL, 100.0 μg / mL and 200.0 μg / mL using PBS (phosphate) buffer solution with a pH of 7.0-7.4. The standard curve was obtained according to the following liquid phase detection conditions. The results are as follows: Figure 1 Wherein, the horizontal axis is the concentration of the lactoferrin standard product, and the vertical axis is the peak area of ​​the lactoferrin standard product measured by high performance liquid chromatography.

[0045] Liquid phase detection conditions are as follows:

[0046] Flow rate: 1.0 mL / min;

[0047] Wavelength: 280nm;

[0048] Injection volume: 30 μL;

[0049] Column temperature: 30°C;

[0050] Mobile phase: A: 0.1% trifluoroacetic acid, B: acetonitrile;

[0051] The liquid phase elution procedure is shown in Table 3 below:

[0052] Table 3. Liquid phase elution program

[0053]

[0054] Step S1: Weigh 5 g of hydrolyzed milk powder sample per group, add 30 mL of the extract, and vortex for 10 minutes until completely dissolved. Then, centrifuge at 10,000 rpm at 4°C for 8-10 minutes. Aspirate 10 mL of the supernatant for use in a heparin affinity column.

[0055] In step S2, the heparin affinity column is activated with 5 mL of the extract before use. The sample solution after centrifugation is loaded onto the heparin affinity column at a flow rate of 1-3 mL / min. After loading, the column is rinsed with 10 mL of the extract, the effluent is discarded, and the column is eluted with 5.0 mL of the eluent. The eluent is collected and filtered through a 0.45 μm aqueous syringe filter for HPLC analysis.

[0056] Step S3, injecting the eluate into a high performance liquid chromatograph, and quantifying the lactoferrin in the eluate by an external standard method of high performance liquid chromatography to obtain the weight of the lactoferrin, and obtaining the lactoferrin content in the hydrolyzed milk powder according to the weight of the lactoferrin and the weight of the sample to be tested. The specific calculation method is:

[0057] The peak area of ​​lactoferrin in the eluate was obtained by the external standard method of high performance liquid chromatography, and then Figure 1 The lactoferrin standard curve is used to obtain the abscissa value corresponding to the peak area, i.e., the lactoferrin concentration. The weight of the lactoferrin in the eluate is then calculated based on the volume of the eluate. The lactoferrin content in the sample is then determined based on the weight of the lactoferrin and the weight of the sample. The HPLC detection conditions and elution procedure are the same as those in step S0.

[0058] The recovery rate can also be calculated based on the measured lactoferrin concentration using the formula: X = C / A * 100%, where X is the recovery rate, C is the lactoferrin concentration of the sample obtained from the standard curve after liquid chromatography testing, and A is the theoretical added lactoferrin concentration.

[0059] Based on the above experimental operation method and recovery rate calculation, the lactoferrin recovery results of hydrolyzed milk powder according to different extraction solution formulas are shown in Table 4 below.

[0060] Table 4. Recovery results

[0061]

[0062] From the results in Table 4, it can be seen that, using the above-mentioned extraction solution and detection method, the recovery rate of lactoferrin in hydrolyzed milk powder reaches over 90%, which meets the detection requirements of the national standard.

[0063] <Example 2>

[0064] In this example, the method of Example 1 was used to test commercially available hydrolyzed milk powder with a known lactoferrin content. The lactoferrin-containing hydrolyzed milk powder used was from Nestlé, whose product ingredients clearly stated a lactoferrin content of 20 mg / 100 g. The optimal extract, Extract 2, was used as the extract. The remaining experimental procedures and parameters were the same as in Example 1.

[0065] Weigh 5g of hydrolyzed milk powder, process according to steps S1 and S2, and collect the eluate. Then proceed to step S3 to obtain the peak area of ​​lactoferrin in the eluate. Combined with the standard curve, the concentration of lactoferrin in the eluate is quantitatively determined to be 96.8μg / mL with an RSD of 3.98%. Since the eluate volume is 10mL, the weight of the eluted lactoferrin is 96.8μg / mL × 10mL = 96.8μg. Based on the weight of the tested hydrolyzed milk powder of 5g, it can be determined that the lactoferrin content of this Nestlé hydrolyzed milk powder containing lactoferrin is 19.4mg / 100g. This result is consistent with the product label, demonstrating the accuracy of this detection method.

[0066] <Example 3>

[0067] Results of different brands of hydrolyzed milk

[0068] Commercially available hydrolyzed milk powders from different brands (Nestlé, Aptamil, Mead Johnson, Feihe, Abbott, and Wyeth) were subjected to lactoferrin recovery experiments using the detection method of Example 1, with the optimal extract 2 selected as the extract. The results are shown in Table 5. As can be seen, the lactoferrin recovery rates for the different brands of hydrolyzed milk powders using the detection method of Example 1 were all greater than 90%, meeting the national standard detection requirements.

[0069] Table 5. Lactoferrin recovery test results

[0070]

[0071] <Example 4>

[0072] Comparison results

[0073] The recovery rate of lactoferrin was tested using Nestlé hydrolyzed milk powder using the methods of GB 5009.299-2024 National Food Safety Standard Determination of Lactoferrin in Foods, T / TDSTIA 006-2019 Determination of Lactoferrin in Milk and Dairy Products by Liquid Chromatography, patent CN114414687A, and Example 1. The results are shown in Table 6 below.

[0074] Table 6. Comparison of results of different test methods

[0075]

[0076] As can be seen from Table 6, the detection methods using national standard method, group standard method and open patent CN114414687A are unable to effectively detect such matrices as hydrolyzed milk powder, and their spiked recovery is less than 50%, which is far from meeting the index requirement of more than 90% recovery of national standard. However, the detection method using Example 1 can achieve more than 95% recovery when detecting lactoferrin for this type of hydrolyzed milk powder, which has great advantages, embodies specificity and can realize efficient detection of lactoferrin in hydrolyzed milk powder.

[0077] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for detecting lactoferrin content in hydrolyzed milk powder, characterized in that, The following steps are involved: Step S1, dissolving the hydrolyzed milk powder sample to be tested in the extract, and after sufficient dissolution, centrifuging to remove fat to obtain a supernatant; Step S2, loading the supernatant onto a heparin affinity column; After the sample is loaded, the extraction solution is used for washing, and the effluent is discarded; the eluent is used for elution, and the eluate is collected; Step S3, quantitatively measuring the lactoferrin in the eluate by an external standard method of high performance liquid chromatography to obtain the weight of the lactoferrin, and obtaining the lactoferrin content in the hydrolyzed milk powder according to the weight of the lactoferrin and the weight of the sample to be tested; In step S1, the pH of the extract is 5.0-7.5, and the extract includes a buffer salt, an amphiphilic surfactant, and urea. The concentration of the buffer salt in the extract is 0.05 mol / L-0.5 mol / L, the weight percentage of the amphiphilic surfactant in the extract is 0.1%-1.0%, and the concentration of urea in the extract is 1.0 mol / L-5.0 mol / L.

2. The method for detecting lactoferrin content in the hydrolyzed milk powder as claimed in claim 1, wherein The buffer salt is ammonium acetate.

3. The method for detecting lactoferrin content in the hydrolyzed milk powder as claimed in claim 1, wherein The amphiphilic surfactant is any one or more of 3-(1-methylpiperidinium-1-yl)propane-1-sulfonate, aminosulfobetaine-14 or dodecyldimethylsulfopropylbetaine.

4. The method for detecting lactoferrin content in the hydrolyzed milk powder as claimed in claim 1, wherein The concentration of the buffer salt in the extract is 0.1 mol / L~0.3 mol / L, the weight percentage of the amphiphilic surfactant in the extract is 0.3%~0.6%, and the concentration of urea in the extract is 2.0 mol / L~3.0 mol / L; the pH of the extract is 6.0~6.

5.

5. The method for detecting lactoferrin content in the hydrolyzed milk powder as claimed in claim 1, wherein The ratio of the hydrolyzed milk powder to the extract is (1.0-10.0 g):30 mL.

6. The method for detecting lactoferrin content in hydrolyzed milk powder according to claim 5, wherein The ratio of the hydrolyzed milk powder to the extract is (5.0-6.0 g):30 mL.

7. The method for detecting lactoferrin content in hydrolyzed milk powder according to claim 1, wherein The specific operation of step S2 is as follows: before use, the heparin affinity column is activated with 5 mL of the extract, and the supernatant after centrifugation is loaded onto the heparin affinity column at a flow rate of 1 mL / min to 3 mL / min; after loading, the column is rinsed with 10 mL of the extract and the effluent is discarded; the column is then eluted with 5.0 mL of the eluent and the eluate is collected.

8. The method for detecting lactoferrin content in hydrolyzed milk powder according to claim 7, wherein The eluent is prepared from ammonium acetate and sodium chloride, wherein the concentration of ammonium acetate is 0.1 mol / L, the concentration of sodium chloride is 1.0 mol / L, and the pH value is 6.5-7.

0.

9. The method for detecting lactoferrin content in hydrolyzed milk powder according to claim 1, wherein In step S3, mobile phase A is 0.1% trifluoroacetic acid, mobile phase B is acetonitrile, and the elution procedure of the high performance liquid chromatography is: 。 10. The method for detecting the content of lactoferrin in hydrolyzed milk powder according to claim 1, wherein In step S3, the peak area of ​​lactoferrin in the eluate is obtained by an external standard method of high performance liquid chromatography, and then the abscissa value corresponding to the peak area, i.e., the lactoferrin concentration, is obtained according to a lactoferrin standard curve. The weight of lactoferrin in the eluate is then calculated according to the volume of the eluate, and the lactoferrin content in the sample is obtained according to the weight of the lactoferrin and the weight of the sample.

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