A method for detecting allergens in hydrolyzed protein milk powder by combining electrophoresis and mass spectrometry

Abstract

The present invention relates to a method for detecting allergens in hydrolyzed milk powder by electrophoresis combined with liquid phase mass spectrometry analysis. The electrophoresis results presented by the detection method can firstly visually observe the distribution range of protein components and molecular weight in the milk powder sample, and preliminarily judge the residual allergens; then the milk powder sample is pre-treated, and the peptide segment after hydrolysis with a confidence interval of 80% or more is selected by LC-MS / MS full scan analysis, and it is compared with the antigenic epitopes of each allergen in cow's milk, and then the allergenic residual situation of the hydrolyzed milk powder is inferred. The hydrolyzed milk powder allergen detection method of the present invention has the advantages of high accuracy, high sensitivity, good stability, high throughput, etc., and can provide a theoretical basis and technical support for the detection of allergens in low-allergenic formula milk powder.

Description

Technical Field

[0001] The present invention relates to the field of food detection, and in particular to a method for detecting allergens in hydrolyzed protein milk powder. Background Art

[0002] Milk protein is a general term for various proteins in milk, mainly composed of casein and whey protein. Breast milk does not contain αS-casein and β-lactoglobulin, while cow's milk contains a high content of αS-casein and β-lactoglobulin. These two proteins are currently recognized allergens. Under normal circumstances, these two proteins can be digested and absorbed by the human body, but if the human body (especially infants) has insufficient digestive capacity, these two proteins enter the human body in an undigested form and cause allergic reactions. Common allergic symptoms include eczema, allergic rhinitis, asthma, etc.

[0003] Scientists have found through extensive research that the allergenicity of milk can be greatly reduced by using a unique hydrolysis technology to convert large molecular proteins of milk into short fragments. The hydrolysis methods and hydrolysis degrees on the market vary, and there are still cases where hydrolyzed formula milk powder contains antigen epitopes that cause allergies. Therefore, it is urgent to establish a detection method to prevent people with severe allergies from coming into contact with milk allergens.

[0004] Liquid chromatography-mass spectrometry (LC-MS) is an analytical method that has developed rapidly in recent years. This method uses liquid chromatography as the separation system and mass spectrometry as the detection system. After the sample is separated and ionized, the ion fragments are separated by mass number through the mass analyzer of the mass spectrometer, and the mass spectrum is obtained through the detector. This method has the characteristics of high sensitivity, specificity, reproducibility, and recovery rate. At present, there are no related research reports on the detection of hydrolyzed protein milk powder allergens by electrophoresis combined with liquid chromatography-mass spectrometry. The combination of the two technologies can effectively identify and identify allergens in hydrolyzed protein powder. Summary of the invention

[0005] The present invention aims to provide a method for detecting allergens in hydrolyzed milk powder. In view of the above-mentioned invention object, the first aspect of the present invention provides a method for detecting allergens in hydrolyzed protein milk powder, which comprises the following steps:

[0006] (1) using a separation gel and a concentrated gel to perform electrophoresis analysis on the milk powder sample, staining with Coomassie Brilliant Blue after the electrophoresis, decolorizing with a decolorizing solution, and then imaging the gel in a gel imager;

[0007] (2) dissolving the hydrolyzed milk powder sample in water and mixing it evenly, dissolving it by ultrasonication, taking out the lower layer liquid after centrifugation, and filtering it;

[0008] (3) ultrafiltration of the filtrate sample, rotary evaporation of the ultrafiltration liquid, reconstitution with a reconstitution reagent, and analysis of the reconstituted liquid in a liquid chromatography-mass spectrometer;

[0009] (4) The data obtained by the liquid chromatography-mass spectrometry instrument were imported into the Proteinpilot software, and the milk protein in UniprotKB was used as the library to search and identify the peptides, and the peptides with high response and confidence > 80% were screened out;

[0010] (5) Summarize the amino acid sequences of the antigenic epitopes of the major milk allergen proteins reported, compare the peptides screened in step (4) with the summarized allergen antigenic epitopes one by one, and calculate the number of amino acid overlaps and the repetition rate between the two.

[0011] Preferably, in the step (1), the milk powder sample is subjected to electrophoresis analysis using 15% separation gel and 5% concentration gel, the sample loading amount is 15 μL, and after electrophoresis separation is performed at 80 V for 30 min, the voltage is adjusted to 120 V, and separation is continued for 100 min; after the electrophoresis is completed, it is stained with Coomassie Brilliant Blue for 2 h, and then decolorized with a decolorizing solution, the decolorizing solution is replaced every 1 h, and decolorization is performed 3 times in total, and finally the gel is imaged in a gel imager.

[0012] Preferably, in step (2), 10 mg of the hydrolyzed milk powder sample is accurately weighed, dissolved in 1 mL of mass spectrometry water, vortexed for 2 minutes until no obvious large particles are present, and then sonicated for 10 minutes.

[0013] Preferably, the centrifugation condition of the solution after ultrasound in step (2) is at 4° C. and 12,000 g for 10 min.

[0014] Preferably, the filtering condition in step (2) is to filter through a 0.22 μm filter membrane to remove large molecular weight impurities, undissolved milk powder and unremoved fat particles.

[0015] Preferably, in step (3), the filtered sample is transferred to a 10 kDa ultrafiltration centrifuge tube and centrifuged at 12000 g for 10 min to remove high molecular weight proteins.

[0016] Preferably, in step (3), 200 μL of the ultrafiltered liquid is taken and dried in a rotary evaporator at 30° C. for 2 h.

[0017] Preferably, in step (3), 100 μL of an aqueous solution of 2% acetonitrile and 0.1% formic acid is used for re-dissolution, and after vortex mixing, the liquid is transferred to a liquid phase vial and placed in a sample tray for liquid chromatography-mass spectrometry analysis.

[0018] Preferably, the liquid phase conditions of the liquid chromatography-mass spectrometer in step (3) are as follows:

[0019] Japan Shimadzu NexeraX2 liquid chromatograph, PeptideBEHC18 Column( 3.5μm, 4.6mm×150mm), mobile phase A is 2% acetonitrile and 0.1% formic acid in water, mobile phase B is 2% water and 0.1% formic acid in acetonitrile; column temperature is 40°C, flow rate is 0.25mL / min; injection volume is 10μL; mobile phase gradient elution program is shown in the table below:

[0020]

[0021]

[0022] Preferably, the mass spectrometry conditions of the liquid chromatography-mass spectrometer in step (3) are as follows:

[0023] A TripleTOF6600 high-resolution mass spectrometer was used, and information dependence acquisition (IDA) scanning was selected in positive ionization mode; ion source gas 1 (GS1): 55 psi; ion source gas 2 (GS2): 55 psi; curtain gas: 25 psi; declustering voltage (DP): 80 V; precursor ion scan m / z: 350-1500; precursor ion scan accumulation time: 0.25 s; product ion scan m / z: 100-1500; product ion scan IDA number: 20; product ion scan accumulation time: 0.08 s; rolling collision energy (CE): enabled.

[0024] Preferably, the parameters for searching and identifying peptides in step (4) are set as follows: SampleType: Identification; CysAlkylation: Iodoacetamide; Digestion: None; SearchEffort: RapidID; IDFocus: Biologicalmodifications.

[0025] Preferably, when searching and identifying peptide segments in step (4), the selected allergen protein type is selected from one or more of α-lactalbumin, β-lactoglobulin, αS1-casein, αS2-casein, β-casein and κ-casein.

[0026] More preferably, when searching and identifying peptide segments in step (4), the selected allergen protein types are α-lactalbumin, β-lactoglobulin, αS1-casein, αS2-casein, β-casein and κ-casein.

[0027] Further preferably, when searching and identifying peptide segments in step (4), the allergen protein types selected are β-lactoglobulin, β-casein and κ-casein.

[0028] Preferably, the antigen epitope amino acid sequence in step (5) is selected from one or more of the following sequences:

[0029] α-lactalbumin antigen epitope 1: EQLTKCEVFRELKDLK (SEQ ID NO. 1);

[0030] α-lactalbumin antigen epitope 2: KDLKGYGGVSLPEW (SEQ ID NO. 2);

[0031] α-lactalbumin antigen epitope 3: STEYGLFQINNK (SEQ ID NO. 3);

[0032] α-lactalbumin antigen epitope 4: KKILDKVGIN (SEQ ID NO. 4);

[0033] α-lactalbumin antigen epitope 5: EVFRELKDLKGY (SEQ ID NO. 5);

[0034] α-lactalbumin antigen epitope 6: GLFQINNKIWC (SEQ ID NO. 6);

[0035] α-lactalbumin antigen epitope 7: IMCVKKILDKVGINYWLAHK (SEQ ID NO. 7);

[0036] β-lactoglobulin antigen epitope 1: LIVTQTMK (SEQ ID NO. 8);

[0037] β-lactoglobulin antigen epitope 2: MAASDISLLDAQSAPL (SEQ ID NO. 9);

[0038] β-lactoglobulin antigen epitope 3: PLRVYVEELKPIEGDLE (SEQ ID NO. 10);

[0039] β-lactoglobulin antigen epitope 4: LVLDTDYKK (SEQ ID NO. 11);

[0040] β-lactoglobulin antigen epitope 5: LLPCMENSAEPEQSLVQCLVRTP (SEQ ID NO. 12);

[0041] β-lactoglobulin antigen epitope 6: MHIRLSPNPTQLEE (SEQ ID NO. 13);

[0042] β-lactoglobulin antigen epitope 7: EGDLEILLQKWENG (SEQ ID NO. 14);

[0043] β-lactoglobulin antigen epitope 8: AQKKIIAEKTKIPAVFKIDALN (SEQ ID NO. 15);

[0044] β-lactoglobulin antigen epitope 9: RTPEVDDEALEKFDKALKALP (SEQ ID NO. 16);

[0045] β-lactoglobulin antigen epitope 10: ALKALPMHIRLSFNPTQL (SEQ ID NO. 17);

[0046] αS1-casein antigen epitope 1: NENLLRFFVAPFPEVFGKEK (SEQ ID NO. 18);

[0047] αS1-casein antigen epitope 2: EEIVPNSVEQ (SEQ ID NO. 19);

[0048] αS1-casein antigen epitope 3: LEIVPNSAEERL (SEQ ID NO. 20);

[0049] αS1-casein antigen epitope 4: TDAPSFSDIPNPIGSENSEKT (SEQ ID NO. 21);

[0050] αS2-casein antigen epitope 1: NEINQFYQKFPQYLQYLY (SEQ ID NO. 22);

[0051] αS2-casein antigen epitope 2: STEVFTKKTKLTEEE (SEQ ID NO. 23);

[0052] αS2-casein antigen epitope 3: EKNRLNFLKKISQRYQ (SEQ ID NO. 24);

[0053] αS2-casein antigen epitope 4: KKISQRYQKFALPQYLKTVVQHQK (SEQ ID NO. 25);

[0054] β-casein antigen epitope 1: RELEELNVPGEIVESL (SEQ ID NO. 26);

[0055] β-casein antigen epitope 2: PVVVPPFLQPEV (SEQ ID NO. 27);

[0056] β-casein antigen epitope 3: LPLPLLQSWMH (SEQ ID NO. 28);

[0057] β-casein antigen epitope 4: QPLPPTVMFPPQSVLS (SEQ ID NO. 29);

[0058] κ-casein antigen epitope 1: IRCEKDERFFSDKIAKYI (SEQ ID NO. 30);

[0059] κ-casein antigen epitope 2: KIAKYIPIQYVLSRYPSYGLNYYQ (SEQ ID NO. 31);

[0060] κ-casein antigen epitope 3: PVALINNQFLPVPYYAKPAAVR (SEQ ID NO. 32);

[0061] κ-casein antigen epitope 4: FLPYPYYAKPAAVRSPAQILQWQVLS (SEQ ID NO. 33); and

[0062] κ-Casein antigen epitope 5: SFMAIPPKKNQD (SEQ ID NO. 34).

[0063] More preferably, the antigen epitope amino acid sequence in step (5) is a combination of sequences SEQ ID NO.1 to SEQ ID NO.34.

[0064] Further preferably, the antigen epitope amino acid sequence in step (5) is a combination of sequences SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28.

[0065] Further preferably, the antigen epitope amino acid sequence in step (5) is a combination of sequences SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.15 and SEQ ID NO.34.

[0066] Further preferably, the antigen epitope amino acid sequence in step (5) is a combination of sequences SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

[0067] The present invention has the following beneficial effects:

[0068] The present invention analyzes the residual allergens in hydrolyzed milk powder based on electrophoresis combined with liquid phase mass spectrometry. The electrophoresis results presented by the detection method of the present invention can firstly visually observe the distribution range of protein components and molecular weight in the milk powder sample, and preliminarily judge the residual allergens. Then the milk powder sample is pre-treated, and the peptide segment after hydrolysis with a confidence interval of 80% or more is selected by LC-MS / MS full scan analysis, and the peptide segment is compared with the main IgE and IgG sensitizing epitopes of the main allergens (casein, α-lactalbumin, β-lactoglobulin, etc.) in cow's milk, and then the allergenic residual situation of the hydrolyzed milk powder is inferred. The hydrolyzed milk powder allergen detection method of the present invention has the advantages of high accuracy, high sensitivity, good stability, high throughput, etc., and can provide a theoretical basis and technical support for the detection of allergens in low-allergenic formula milk powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 is the SDS-PAGE image of the unhydrolyzed milk powder (1), the partially hydrolyzed milk powder (2) and the completely hydrolyzed milk powder (3) in Example 1;

[0070] Figure 2 is the total ion current chromatogram of partially hydrolyzed milk powder (A) and completely hydrolyzed milk powder (B) in Example 1;

[0071] Figure 3 is the SDS-PAGE image of one common milk powder sample (1) and five partially hydrolyzed milk powder samples (2-6) in Example 2;

[0072] Figure 4 It is the total ion current chromatogram (A) and peptide ion current chromatogram (B) of the partially hydrolyzed milk powder sample 6 in Example 2. DETAILED DESCRIPTION

[0073] Test Example 1: Detection methods for allergens in various protein milk powders

[0074] 1. Test methods

[0075] The milk powder used in this test example is lactose-free formula milk powder from company A, partially hydrolyzed whey protein powder from company B, and deeply hydrolyzed whey protein powder from company C. Weigh the three kinds of milk powder according to the protein content in the milk powder ingredient list to make the protein concentration reach 1mg / ml. Electrophoresis analysis of the milk powder sample was performed using 15% separation gel and 5% concentrated gel, with a sample volume of 15μL. After electrophoresis separation at 80V for 30min, the voltage was adjusted to 120V, and separation was continued for 100min; after electrophoresis, it was stained with Coomassie brilliant blue for 2h, and then decolorized with decolorizing solution, and the decolorizing solution was replaced every 1h, for a total of 3 decolorizations, and finally the gel was imaged in a gel imager.

[0076] Accurately weigh 10 mg of hydrolyzed milk powder sample, dissolve it in 1 mL of mass spectrometry water, vortex mix for 2 minutes until there are no obvious large particles, and then ultrasonicate for 10 minutes. Centrifuge the solution at 4 ° C and 12000g for 10 minutes, avoid the white fat layer on the surface, and take out the lower layer of liquid. Use a syringe to draw the lower layer of liquid, pass it through a 0.22μm filter membrane, and remove large molecular weight impurities, undissolved milk powder, and unremoved fat particles. Transfer the filtered sample to a 10kDa ultrafiltration centrifuge tube, centrifuge it at 12000g for 10 minutes, remove large molecular weight proteins, take 200μL of the ultrafiltered liquid, spin dry it in a rotary evaporator at 30 ° C for 2 hours, and re-dissolve it with 100μL of 2% acetonitrile and 0.1% formic acid in water. After vortex mixing, transfer the liquid to a liquid phase vial and put it into a sample tray for liquid phase mass spectrometry analysis. The liquid phase conditions are as follows:

[0077] A Shimadzu NexeraX2 liquid chromatograph was used. PeptideBEHC18 Column( 3.5μm, 4.6mm×150mm), mobile phase A is 2% acetonitrile and 0.1% formic acid in water, mobile phase B is 2% water and 0.1% formic acid in acetonitrile; column temperature is 40°C, flow rate is 0.25mL / min; injection volume is 10μL; mobile phase gradient elution program is shown in the table below:

[0078] Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 0.5 92 8 0.6 88 12 25 70 30 30 65 35 30.5 20 80 38 20 80 38.5 95 5 50 95 5

[0079] The mass spectrometry conditions are as follows: using TripleTOF6600 high-resolution mass spectrometer, selecting information dependence acquisition (IDA) scanning, positive ionization mode; ion source gas 1 (GS1): 55psi; ion source gas 2 (GS2): 55psi; curtain gas: 25psi; declustering voltage (DP): 80V; precursor ion scan m / z: 350-1500; precursor ion scan accumulation time: 0.25s; product ion scan m / z: 100-1500; product ion scan IDA number: 20; product ion scan accumulation time: 0.08s; rolling collision energy (CE): enabled.

[0080] The data obtained by liquid chromatography-mass spectrometry were imported into Proteinpilot software, and the milk protein in UniprotKB was used as the library to search and identify the peptides; the parameters were set as follows: SampleType: Identification; CysAlkylation: Iodoacetamide; Digestion: None; SearchEffort: RapidID; IDFocus: Biologicalmodifications; the selected allergen proteins were as follows: α-lactalbumin, β-lactoglobulin, αS1-casein, αS2-casein, β-casein and κ-casein, and peptides with high response and credibility >80% were screened out.

[0081] The amino acid sequences of the antigenic epitopes of the major milk allergen proteins reported are summarized as follows:

[0082] α-Lactalbumin antigen epitope 1: EQLTKCEVFRELKDLK (SEQ ID NO.1)

[0083] α-Lactalbumin antigen epitope 2: KDLKGYGGVSLPEW (SEQ ID NO.2)

[0084] α-Lactalbumin antigen epitope 3: STEYGLFQINNK (SEQ ID NO.3)

[0085] α-lactalbumin antigen epitope 4: KKILDKVGIN (SEQ ID NO.4)

[0086] α-Lactalbumin antigen epitope 5: EVFRELKDLKGY (SEQ ID NO.5)

[0087] α-lactalbumin antigen epitope 6: GLFQINNKIWC (SEQ ID NO.6)

[0088] α-Lactalbumin antigen epitope 7: IMCVKKILDKVGINYWLAHK (SEQ ID NO.7)

[0089] β-lactoglobulin antigen epitope 1: LIVTQTMK (SEQ ID NO.8)

[0090] β-lactoglobulin antigen epitope 2: MAASDISLLDAQSAPL (SEQ ID NO.9)

[0091] β-lactoglobulin antigen epitope 3: PLRVYVEELKPIEGDLE (SEQ ID NO.10)

[0092] β-lactoglobulin antigen epitope 4: LVLDTDYKK (SEQ ID NO.11)

[0093] β-lactoglobulin antigen epitope 5: LLPCMENSAEPEQSLVQCLVRTP (SEQ ID NO.12)

[0094] β-lactoglobulin antigen epitope 6: MHIRLSPNPTQLEE (SEQ ID NO.13)

[0095] β-lactoglobulin antigen epitope 7: EGDLEILLQKWENG (SEQ ID NO.14)

[0096] β-lactoglobulin antigen epitope 8: AQKKIIAEKTKIPAVFKIDALN (SEQ ID NO.15)

[0097] β-lactoglobulin antigen epitope 9: RTPEVDDEALEKFDKALKALP (SEQ ID NO.16)

[0098] β-lactoglobulin antigen epitope 10: ALKALPMHIRLSFNPTQL (SEQ ID NO.17)

[0099] αS1-casein antigen epitope 1: NENLLRFFVAPFPEVFGKEK (SEQ ID NO.18)

[0100] αS1-casein antigen epitope 2: EEIVPNSVEQ (SEQ ID NO.19)

[0101] αS1-casein antigen epitope 3: LEIVPNSAEERL (SEQ ID NO.20)

[0102] αS1-casein antigen epitope 4: TDAPSFSDIPNPIGSENSEKT (SEQ ID NO.21)

[0103] αS2-casein antigen epitope 1: NEINQFYQKFPQYLQYLY (SEQ ID NO.22)

[0104] αS2-casein antigen epitope 2: STEVFTKKTKLTEEE (SEQ ID NO.23)

[0105] αS2-casein antigen epitope 3: EKNRLNFLKKISQRYQ (SEQ ID NO.24)

[0106] αS2-casein antigen epitope 4: KKISQRYQKFALPQYLKTVVQHQK (SEQ ID NO.25)

[0107] β-Casein antigen epitope 1: RELEELNVPGEIVESL (SEQ ID NO.26)

[0108] β-Casein antigen epitope 2: PVVVPPFLQPEV (SEQ ID NO.27)

[0109] β-Casein antigen epitope 3: LPLPLLQSWMH (SEQ ID NO.28)

[0110] β-Casein antigen epitope 4: QPLPPTVMFPPQSVLS (SEQ ID NO.29)

[0111] κ-Casein antigen epitope 1: IRCEKDERFFSDKIAKYI (SEQ ID NO.30)

[0112] κ-Casein antigen epitope 2: KIAKYIPIQYVLSRYPSYGLNYYQ (SEQ ID NO.31)

[0113] κ-casein antigen epitope 3: PVALINNQFLPVPYYAKPAAVR (SEQ ID NO.32)

[0114] κ-Casein antigen epitope 4: FLPYPYYAKPAAVRSPAQILQWQVLS (SEQ ID NO.33)

[0115] κ-casein antigen epitope 5: SFMAIPPKKNQD (SEQ ID NO. 34);

[0116] The screened peptides were compared with the allergen antigen epitopes summarized above one by one, and the number of amino acid overlaps and the overlap percentage between the two were calculated.

[0117] 2. Test results

[0118] The electrophoresis diagrams of unhydrolyzed milk powder (1), partially hydrolyzed milk powder (2), and fully hydrolyzed milk powder (3) are as follows: Figure 1As shown. Compared with unhydrolyzed milk powder, the partially hydrolyzed milk powder sample did not show obvious protein bands, and the molecular weight was less than 15kDa, showing a tailing shape. This result shows that the milk powder was decomposed into small molecules with a molecular weight below 15kDa. However, there was no obvious trace of completely hydrolyzed milk powder in the swimming lane, indicating that it was hydrolyzed into small peptides with a smaller molecular weight. In order to explore the residual allergenic protein epitopes in milk powder after hydrolysis, partially hydrolyzed milk powder and completely hydrolyzed milk powder were further analyzed by mass spectrometry.

[0119] In this example, partially hydrolyzed milk powder and fully hydrolyzed milk powder were subjected to mass spectrometry detection. According to the screening principle of "peptide confidence (conf)>80%, and peptide fragment ion intensity>1000", no peptides meeting the conditions were detected in the fully hydrolyzed milk powder (see Figure 2 B), a total of 7 peptides that met the criteria were obtained from partially hydrolyzed milk powder (see Figure 2 A), including 4 β-lactoglobulin and 3 β-casein.

[0120] The overlap between the qualified peptide segments and antigen epitopes in partially hydrolyzed milk powder is shown in the following table:

[0121]

[0122]

[0123] Example 2: Method for detecting allergens in partially hydrolyzed protein milk powder

[0124] 1. Test methods

[0125] The milk powders used in this test example are 1 ordinary milk powder and 5 milk powders with different hydrolysis degrees from Enterprise D, and the rest of the test methods are the same as in Example 1.

[0126] 2. The test results were compared by comparing the electrophoresis diagrams of ordinary milk powder (sample 1) and five kinds of partially hydrolyzed whey protein milk powders (samples 2-6) (see Figure 3 ), among the five partially hydrolyzed milk powder samples, the average molecular weight of samples 2-4 is significantly smaller than that of samples 5-6, which is related to their degree of hydrolysis (samples 2-4 are 10-15, and sample 5-6 is 9.6). Most of the casein in the partially hydrolyzed whey protein powder is removed, with only a small amount of residue. Proteins above 50KDa are mainly bovine serum albumin, and proteins below 50KDa are concentrated between 10-20KDa. Clear β-lactoglobulin bands can still be seen. Since some proteins are hydrolyzed into peptides, the protein bands below are aggregated.

[0127] Sample 6 was tested by LC-MS, and the screening principle was the same as in Example 1. A total of 12 peptides meeting the criteria were obtained from the partially hydrolyzed whey protein powder, including 11 β-lactoglobulin and 1 κ-casein (see Figure 4 A), Figure 4 B is the ion flow chart of the peptide (SFMAIPPKKNQD) extracted in a certain period of time. The overlap between the peptides that meet the conditions and the antigen epitopes in the partially hydrolyzed milk powder is shown in the following table:

[0128]

[0129]

[0130] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. A method for detecting allergens in hydrolyzed protein milk powder, It is characterized in that The detection method comprises the following steps: (1) Using separation gel and concentrated gel to perform electrophoresis analysis on partially hydrolyzed milk powder and completely hydrolyzed milk powder samples respectively, staining with Coomassie Brilliant Blue after electrophoresis, decolorizing with decolorizing solution, and then imaging the gel using a gel imager; (2) Performing mass spectrometry on the partially hydrolyzed milk powder and the completely hydrolyzed milk powder samples, respectively, dissolving the partially hydrolyzed milk powder and the completely hydrolyzed milk powder in water, mixing them evenly, dissolving them by ultrasonication, taking out the lower layer of liquid after centrifugation, and filtering them; (3) ultrafiltration of the filtrate sample, rotary evaporation of the ultrafiltration liquid, reconstitution with a reconstitution reagent, and analysis of the reconstituted liquid in a liquid chromatography-mass spectrometer; (4) The data obtained by the liquid chromatography-mass spectrometry instrument were imported into the Proteinpilot software, and the milk protein in UniprotKB was used as the library to search and identify the peptides, and the peptides with high response and confidence > 80% were screened out; (5) Summarize the amino acid sequences of the antigenic epitopes of the major milk allergen proteins reported, compare the peptides screened in step (4) with the summarized allergen antigenic epitopes one by one, and calculate the number of amino acid overlaps and the repetition rate between the two; The antigen epitope amino acid sequence in step (5) is a combination of sequences SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 13, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO.

28.

2. The detection method according to claim 1, It is characterized in that In the step (1), the milk powder sample is subjected to electrophoresis analysis using 15% separation gel and 5% concentration gel, the sample loading volume is 15 μL, and the electrophoresis separation is performed at 80 V for 30 min, then the voltage is adjusted to 120 V, and the separation is continued for 100 min; after the electrophoresis is completed, the sample is stained with Coomassie Brilliant Blue for 2 h, and then decolorized with a decolorizing solution, the decolorizing solution is replaced every 1 h, and decolorization is performed 3 times in total, and finally the gel is imaged in a gel imaging instrument.

3. The detection method according to claim 1, It is characterized in that The filtering condition in step (2) is to filter through a 0.22 µm filter membrane to remove large molecular weight impurities, undissolved milk powder and unremoved fat particles.

4. The detection method according to claim 1, It is characterized in that In step (3), 200 µL of the ultrafiltered liquid was taken and dried in a rotary evaporator at 30°C for 2 h.

5. The detection method according to claim 1, It is characterized in that In step (3), the sample was reconstituted with 100 µL of an aqueous solution of 2% acetonitrile and 0.1% formic acid, vortexed and mixed, and then the liquid was transferred to a liquid phase vial and placed in a sample tray for liquid phase mass spectrometry analysis.

6. The detection method according to claim 1, It is characterized in that The liquid phase conditions of the liquid chromatography-mass spectrometry instrument in step (3) are as follows: Shimadzu Nexera X2 liquid chromatograph, XBridge® Peptide BEH C18 column, column specifications: 300Å, 3.5µm, 4.6mm×150mm, mobile phase A: 2% acetonitrile and 0.1% formic acid in water, mobile phase B: 2% water and 0.1% formic acid in acetonitrile; column temperature: 40°C, flow rate: 0.25mL / min; injection volume: 10µL; mobile phase gradient elution program: see the table below: 。 7. The detection method according to claim 1, It is characterized in that The mass spectrometry conditions of the liquid chromatography-mass spectrometer in step (3) are as follows: A TripleTOF 6600 high-resolution mass spectrometer was used, and information correlation acquisition scanning was selected, with positive ionization mode; ion source gas 1: 55 psi; ion source gas 2: 55 psi; curtain gas: 25 psi; declustering voltage: 80 V; precursor ion scan m / z: 350-1500; precursor ion scan accumulation time: 0.25 s; product ion scan m / z: 100-1500; product ion scan IDA number: 20; product ion scan accumulation time: 0.08 s; rolling collision energy: enabled.

8. The detection method according to claim 1, It is characterized in that The parameters for searching and identifying peptides in step (4) are set as follows: Sample Type: Identification; Cys Alkylation: Iodoacetamide; Digestion: None; Search Effort: Rapid ID; ID Focus: Biological modifications.

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