Method for quantifying beta-casein in food based on isotopic internal standard protein

Abstract

The application discloses a kind of based on isotopic internal standard protein β-casein quantitative analysis method in food, belong to food detection technical field.The present application is based on liquid chromatography-tandem mass spectrometry technology, in combination with recombinant protein isotopic marking technology establishes a kind of precision quantitative analysis technology for milk allergen β-casein, the technology has the characteristics of accurate, sensitive, reliable, can accurately detect the content of β-casein in a variety of complex food matrix, provides scientific basis for milk allergen risk assessment and guarantee allergic population health, and can carry out label consistency evaluation to β-casein in commercially available processed food.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a quantitative analysis method for β-casein in food based on isotope internal standard protein. Background Technology

[0002] Food allergies are a prominent issue in food safety and public health, affecting approximately 1-2% of adults and 5-8% of children worldwide. Milk allergy is the most common type of food allergy in infants and children, severely impacting the quality of life and health of those with allergies. Currently, many countries place great emphasis on research into reducing the allergenicity of dairy products, striving to develop precise analytical and detection methods for milk allergens and improve food labeling systems. Therefore, developing a highly sensitive and specific analytical detection method is crucial for ensuring the health and safety of people with milk allergies and verifying the authenticity and validity of pre-packaged allergen labels.

[0003] Common milk allergens are mainly divided into two categories: casein and whey protein. Other potential allergens include lactoferrin. Casein mainly includes β-casein, α-casein, and κ-casein. Whey protein mainly includes α-lactalbumin and β-lactoglobulin, among others.

[0004] Currently, the commonly used food allergen detection technologies are mainly immunoassay and gene assay. Gene assay is a molecular biology method based on DNA amplification technology. It has high specificity and is not easily affected by cross-reaction. However, for samples such as milk and its processed foods where DNA exists in trace amounts, the quantitative accuracy still needs to be further improved. Immunoassay utilizes the specific binding of antigen and antibody and uses the color change produced by the action of enzymes on the substrate to perform qualitative and quantitative analysis of the allergens to be tested in the sample. However, this method is easily affected by cross-reaction and complex matrices, and is prone to false positive or false negative results.

[0005] With the development of science and technology, liquid chromatography-tandem mass spectrometry (LC-MS / MS) has been widely used in the detection of food allergens. This technique combines liquid chromatography with mass spectrometry, first separating peptides in the sample using liquid chromatography, and then ionizing and detecting them using mass spectrometry. Tandem mass spectrometry can further fragment the peptides, providing more detailed peptide sequence information, significantly improving sensitivity and specificity. This method can perform multi-target detection with high sensitivity and specificity. However, since most existing detection methods use artificially synthesized peptides as internal standards, they cannot correct for errors in the extraction and enzymatic digestion of allergens in food, resulting in low accuracy in the quantification of allergen proteins.

[0006] To improve the current situation, this study introduced stable isotope labeling by amino acids in cell culture (SILAC). By constructing isotope proteins as internal standards in E. coli with defects in key amino acid synthesis genes, errors generated in sample extraction, enzymatic digestion, and purification were effectively corrected. This solved the problem of inaccurate quantification of food allergens in complex food matrices and established a highly sensitive and specific trace allergen precise quantification detection technology. Summary of the Invention

[0007] This invention aims to establish a precise quantitative analysis technique for β-casein, a milk allergen, using liquid chromatography-tandem mass spectrometry combined with recombinant protein isotope labeling. This technique is accurate, sensitive, and reliable, and can accurately detect the content of β-casein in various complex food matrices. It provides a scientific basis for milk allergen risk assessment and protecting the health of allergy sufferers, and can also evaluate the label consistency of β-casein in commercially processed foods.

[0008] This invention includes the following technical solutions:

[0009] In a first aspect, the present invention provides a method for quantitative analysis of β-casein in food, the method comprising adding isotopically labeled recombinant β-casein protein as an internal standard to the sample to be tested, performing LC-MS / MS analysis after trypsin digestion, selecting characteristic peptides of β-casein, and calculating the β-casein content according to the internal standard method.

[0010] The food products described in this invention include any food products that may contain the milk allergen β-casein, including but not limited to milk powder ingredients, formula milk powder, jam, bread, biscuits, cakes, steamed buns, snacks, oatmeal, rice porridge, or baking premixes.

[0011] The amino acid sequence of the isotopically labeled β-casein recombinant protein of the present invention is shown in SEQ ID NO.1, wherein lysine (K) and arginine (R) in the sequence are amino acids labeled with carbon and nitrogen isotopes.

[0012] The amino acid sequence (SEQ ID NO.1) of the isotopically labeled β-casein recombinant protein is as follows:

[0013] MGSSHHHHHHSSGLVPR*GSHMASMK*VLILACLVALALAR*ELEELNVPGEIVESLSSSEESITR*INK*K*IEK*FQSEEQQQTEDELQDK*IHPFAQTQSLVYPFPGPIHNSLPQNIPPLTQTPVVVPP FLQPEVMGVSK*VK*EAMAPK*HK*EMPFPK*YPVEPFTESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSK*VLPVPQK*AVPYPQR*DMPIQAFLLYQEPVLGPVR*GPFPIIV

[0014] In one specific embodiment of the present invention, the isotope-labeled β-casein recombinant protein is prepared by the following method:

[0015] 1) Using E. coli BL21(DE3) as the starting strain, construct the lysine and arginine auxotrophic strain E. coli BL21(DE3)ΔLys / ΔArg;

[0016] 2) The β-casein gene shown in SEQ ID NO.2 was introduced into a vector plasmid to construct a recombinant plasmid, and the recombinant plasmid was transformed into E. coli BL21(DE3)ΔLys / ΔArg strain to construct a recombinant engineered bacterium;

[0017] 3) Using a lysine and arginine-deficient culture medium, and adding isotopically labeled lysine and arginine as an isotopically labeled culture medium, the recombinant engineered bacteria from step 2) were passaged multiple times in the isotopically labeled culture medium to induce expression, and the isotopically labeled β-casein recombinant protein was obtained by isolation and purification.

[0018] In some optional embodiments of the present invention, the vector plasmid mentioned in step 2) is selected from the pET, pEX, GST, PEZZ18, or PKK223 series vector plasmids. Preferably, the vector plasmid is selected from pET.

[0019] In some optional embodiments of the present invention, the isotopically labeled lysine and arginine in step 3) includes C, N, H, or O elements labeled on lysine and arginine, and the isotopes include... 13 C, 15 N, 2 H, 17 O or 18 O can be used to label one or more atoms simultaneously, or to label one or more elements simultaneously.

[0020] In a preferred embodiment of the present invention, the isotope-labeled lysine and arginine are selected from... 13 C, 15 N-labeled lysine and arginine.

[0021] In this invention, the lysine and arginine-deficient culture medium mentioned in step 3) is a culture medium containing salts, sugars, nitrogen sources and all other essential amino acids required for the growth of Escherichia coli, except for lysine and arginine.

[0022] More preferably, the concentration of isotopically labeled lysine and arginine added in step 3) is selected from 20~100 mg / L.

[0023] According to the quantitative analysis method for β-casein in food according to the present invention, the method further includes sample extraction using a sample extraction solution before trypsin digestion. The sample extraction solution is an aqueous solution containing 10 mol / L urea and 200 mmol / L Tris-HCl, with a pH of 8.

[0024] According to the quantitative analysis method for β-casein in food according to the present invention, after trypsin digestion, sample purification is further included. The purification uses an Oasis HLB premium (3cc) purification column. After activation, the sample is added, and the sample is washed with 0.1% formic acid water or 0.1% ammonia water. The elution is performed using 40% ACN (containing 0.1% FA), 60% ACN (containing 0.1% FA), 80% ACN (containing 0.1% FA), or 90% ACN (containing 0.1% FA).

[0025] In a preferred embodiment of the present invention, 0.1% formic acid water is used for purification and washing, and 60% ACN (containing 0.1% FA) is used for purification and elution.

[0026] According to the quantitative analysis method for β-casein in food according to the present invention, the trypsin hydrolysis time is selected from 6-8 hours, preferably 6 hours.

[0027] In some embodiments of the present invention, the β-casein characteristic peptide is selected from AVPYPQR or VLPVPQK.

[0028] In the most preferred embodiment of the present invention, the β-casein characteristic peptide is selected from VLPVPQK.

[0029] According to the quantitative analysis method for β-casein in food according to the present invention, the LC-MS / MS conditions are as follows:

[0030] 1) Chromatographic conditions

[0031] Chromatographic column: ACQUITY UPLC® Peptide CSHTM C18 (2.1 mm × 100 mm, 1.7 μm); injection volume: 5 μL; flow rate: 0.3 mL / min; column temperature: 40℃. Mobile phase: 0.1% formic acid in water (A), 0.1% formic acid in acetonitrile (B); gradient elution program: 0–0.5 min, 5% A; 0.5–6.50 min, 5%–30% A; 6.50–8.00 min, 30%–95% A; 8.00–10.50 min, 95% A; 10.50–13.00 min, 95%–5% A.

[0032] 2) Mass spectrometry conditions

[0033] Electrospray ionization source (ESI+); atomizing gas is nitrogen, flow rate is 3.0 L / min; heating gas is air, flow rate is 10 L / min; drying gas is nitrogen, flow rate is 10 L / min; collision gas is argon; ion source interface temperature is 300 ℃; desolvation tube temperature is 526 ℃; heating module temperature is 400 ℃; scanning mode is multiple reaction monitoring (MRM).

[0034] Secondly, the present invention provides the application of the following combination in the quantitative analysis and detection of β-casein content in food:

[0035] 1) Isotope-labeled β-casein recombinant protein;

[0036] 2) Isotope-labeled β-casein characteristic peptides.

[0037] The isotope-labeled β-casein recombinant protein is used as an internal standard, and its amino acid sequence is shown in SEQ ID NO.1, wherein lysine (K) and arginine (R) in the sequence are amino acids labeled with carbon and nitrogen isotopes.

[0038] The isotopically labeled β-casein characteristic peptides are selected from VLPVPQK* or AVPYPQR*, wherein K* and R* are amino acids labeled with carbon and nitrogen isotopes.

[0039] Preferably, the isotopically labeled β-casein characteristic peptide is selected from VLPVPQK*.

[0040] This invention utilizes the specificity of trypsin to act only on arginine (R) and lysine (K) to enzymatically cleave milk proteins into peptide molecules, from which characteristic peptides are selected as quantitative targets. At the same time, this invention uses isotopically labeled β-casein recombinant protein as an internal standard to participate in enzymatic hydrolysis. After LC-MS / MS detection, the content of milk allergen β-casein in food can be accurately calculated according to the internal standard method. Attached Figure Description

[0041] Figure 1 These are daughter ions collected by the peptide VLPVPQK on a TOF time-of-flight device.

[0042] Figure 2 These are daughter ions collected by the peptide AVPYPQR on a TOF time-of-flight device.

[0043] Figure 3 These are daughter ions collected by EMPFPK peptides on TOF.

[0044] Figure 4 The results of 1% agarose gel electrophoresis were obtained for the amplification of the upstream and downstream homologous arms of lys.

[0045] Figure 5 Electrophoresis results of amplification of lys knockout homologous arms.

[0046] Figure 6 The results of wild-type and knockout bacterial amplification were used to identify lys-up-F / down-R and lys-ter-F / R strains.

[0047] Figure 7 The upstream and downstream homologous arms of arg were amplified and detected by 1% agarose gel electrophoresis.

[0048] Figure 8 The arg knockout was amplified and detected by electrophoresis using the repair homologous arm.

[0049] Figure 9 To identify wild-type and knockout bacteria using arg-up-F / down-R and arg-ter-F / R methods.

[0050] Figure 10 This is a schematic diagram of the β-casein protein sequence and the corresponding pET 28b-β-casein protein particle structure.

[0051] Figure 11 The results of Coomassie Brilliant Blue staining for β-casein.

[0052] Figure 12 Graphs showing the optimization of enzymatic hydrolysis and purification conditions; (A) Enzymatic hydrolysis curves of characteristic peptides of VLPVPQK and AVPYPQR; (B) Graphs showing the intensity of β-casein purification and washing response under different conditions; (C) Graphs showing the intensity of β-casein purification and elution response under different conditions.

[0053] Figure 13 The standard curves are (A) the full field of the protein and (B) the peptide, which serve as internal standards. Detailed Implementation

[0054] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] Sample source

[0056] Twelve types of processed food products sold in Beijing were included, comprising products with milk and related ingredients listed in their ingredient lists, as well as products with no milk-related ingredients listed in their ingredient lists and products that do not share production lines. Sample information is shown in Table 1.

[0057] Table 1 Sample Information

[0058]

[0059] Instruments and reagents

[0060] The system included a SCIEX TripleQaud™ 7500 liquid chromatography-tandem quadrupole linear ion trap mass spectrometer, a UPLC-TOF SYNAPT G2 time-of-flight mass spectrometer (TOF-MS, Waters Corporation, USA), an ACQUITY UPLC® Peptide CSH™ C18 column (2.1 mm × 100 mm, 1.7 μm), an X-30R benchtop refrigerated centrifuge (Beckman Corporation, USA), an electronic balance (Shanghai Sunny Hengping Technology Co., Ltd.), a vortex mixer (Scientific Industry Corporation, USA), a Milli-Q ultrapure water system (Millipore Corporation, USA), an Oasis HLB premium (3cc) solid-phase extraction column (Waters Corporation, USA), an ultrasonic cell disruptor SB25-12DTD (Ningbo Xinzhi Biotechnology Co., Ltd.), and a nitrogen evaporator.

[0061] Acetonitrile (LC / MS grade, Merck, Germany), ammonium bicarbonate (99% purity, Honeywell, USA), IPTG (Sigma-Aldrich, USA), dithiothreitol (DTT, Sigma-Aldrich, USA) solution (1 mol / L), iodoacetamide (IAA, Sigma-Aldrich, USA) solution, trypsin (Sigma-Aldrich, USA), and β-lactoglobulin standard (Sigma-Aldrich, USA).

[0062] TOF analysis was used to obtain mass spectrometry parameters from the fingerprinting of β-casein and its isotopic peptides.

[0063] The daughter and parent ions of β-casein were collected on a Time-of-Flight (TOF) sensor and compared with those predicted using Skyline software to simulate trypsin digestion. Peptides with high response intensity and no interfering peaks were selected as candidate characteristic peptides. Results are as follows: Figure 1-3 As shown.

[0064] Furthermore, based on the Uniprot protein database and Skyline simulated enzyme digestion software analysis, combined with TOF acquisition data, the final selected candidate characteristic peptides and mass spectrometry parameters are shown in Table 2.

[0065] Table 2 Candidate Feature Peptide Parameters

[0066]

[0067] Note: * indicates isotopic peptide segments.

[0068] Preparation of isotopically labeled β-casein recombinant protein

[0069] (I) Construction of the lysine and arginine auxotrophic strain E. coli BL21(DE3)ΔLys / ΔArg

[0070] Knockout of the lys gene in BL21(DE3) strain yielded a single knockout strain; knockout of the arg gene in this strain yielded a double deletion mutant strain of arg and lys.

[0071] The primer sequences involved in the experiment are as follows:

[0072] Table 3 Primer Sequences

[0073]

[0074] 1) Construction and screening of lys gene knockout strains

[0075] 1.1) Gene knockout using homologous arm amplification

[0076] Using extracted DNA from BL21(DE3) strain as a template, the upper and lower homologous arms for lys knockout were amplified using lys-19-F / lys-up-R and lys-down-F / lys-19-R. The amplification system conditions are as follows:

[0077]

[0078] Amplification conditions: 94℃ for 5 min, 30 cycles (94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 20 sec), 10℃ holdon.

[0079] The amplified lys UP and lys down fragments ( Figure 4Seamless cloning was performed, and Ecoli DH5alpha competent cells were transformed, plated on Amp-IPTG-xgal plates, and cultured overnight at 37°C. White clones were picked and sequenced, and clones with correct sequencing were named lys-UD-pUC19.

[0080] 1.2) Preparation of repaired homologous arms

[0081] Using primers lys-up-F / lys-down-R and the correctly sequenced lys-UD-pUC19 plasmid as a template, amplify the lys repair homologous arm. The amplification system and procedure are the same as above. The amplification product ( Figure 5 The PCR product was purified for later use.

[0082] 1.3) Preparation of electrocompetent cells of BL21(DE3) strain

[0083] BL21(DE3) bacteria were activated and cultured at 37℃. A single clone was inoculated into 5 ml of LB liquid medium. The next day, 1% was transferred to 50 ml of LB liquid medium. When the OD reached about 0.8, the cells were collected by centrifugation. The cells were washed three times with 10% glycerol and finally resuspended in 2 ml of 10% glycerol to obtain the prepared competent cells.

[0084] 1.4) Electroconversion of plasmids containing Cas9

[0085] Add 10 μl of plasmid to the prepared competent cells and place on ice for 5 min. Electroporate at 2500 V, add 1 ml of LB medium, and incubate at 30 °C for 1 h. Then spread on Kan resistance plates and prepare Cas9 BL21(DE3) competent cells according to the method in 1.3).

[0086] 1.5) Transformation of sgRNA and repair of homologous arms

[0087] The lys repair homologous arms and the prepared lys sgRNA plasmid were electroporated into the prepared Cas9 BL21(DE3) competent cells, and the cells were screened on Kan / Spc plates to select lys deletion lines.

[0088] 1.6) Knockout identification

[0089] lys-up-F / down-R: Homologous arm identification primers, identifying positive bacteria (766bp) and original bacteria (2029bp).

[0090] lys-ter-F / R: Primers for identifying the target gene. Positive bacteria cannot be amplified, while the original bacteria are identified at 1060bp.

[0091] The results are as follows Figure 6As shown in the figure, 1 and 3 refer to the amplification products of the BL21(DE3)△lys strain, and 2 and 4 refer to the amplification products of the original BL21(DE3) strain. 1 and 2 were amplified using lys-up-F / down-R, and 3 and 4 were amplified using lys-ter-F / R. Product 1 in the figure was sent for sequencing. The strain was named BL21(DE3)△lys, and BL21(DE3)△lys containing the Cas9 plasmid was also retained for future use.

[0092] 2) Construction and screening of arg gene knockout strains

[0093] 2.1) Gene knockout homologous arm amplification

[0094] Using the extracted DNA from BL21(DE3) strain as a template, the upper and lower homologous arms for arg knockout were amplified using arg-19-F / arg-up-R and arg-down-F / arg-19-R. The amplification system and conditions were the same as in 1.1.

[0095] The amplified arg UP and arg down fragments ( Figure 7 Seamless cloning was performed, and Ecoli DH5alpha competent cells were transformed, plated on Amp-IPTG-xgal plates, cultured overnight at 37°C, and white clones were picked for sequencing. The clones with correct sequencing were named arg-UD-pUC19.

[0096] 2.2) Preparation of repaired homologous arms

[0097] Using primers arg-up-F / arg-down-R as a template, amplify the arg-repaired homologous arms using the correctly sequenced arg-UD-pUC19 plasmid. The amplification system and procedure are the same as above. The amplification products ( Figure 8 The PCR product was purified for later use.

[0098] 2.3) Prepare electrocompetent cells of BL21(DE3)△lys pCas9 strain according to method 1.3).

[0099] 2.4) Transformation of sgRNA and repair of homologous arms

[0100] Arg repair homologous arms and prepared arg sgRNA plasmids were electroporated into prepared BL21(DE3)△lyspCas9 competent cells, and arg deletion lines were screened by plating on Kan / Spc plates.

[0101] 2.5) Knockout identification

[0102] arg-up-F / down-R: Homologous arm identification primers, identifying positive bacteria (885bp) and original bacteria (2217bp).

[0103] arg-ter-F / R: Primers for identifying the target gene. Positive bacteria cannot be amplified, while the original bacteria are identified at 964bp.

[0104] The results are as follows Figure 9 As shown in the figure, 1 and 3 refer to the amplification products of the BL21(DE3)△lys / △arg strain, and 2 and 4 refer to the amplification products of the original BL21(DE3)△lys strain. 1 and 2 were amplified using arg-up-F / down-R, and 3 and 4 were amplified using arg-ter-F / R. Product 1 in the figure was sent for sequencing, and the strain was named BL21(DE3)△lys / △arg.

[0105] Streaking BL21(DE3)Δlys / Δarg strain (arginine and lysine auxotrophs) onto LB agar plates and incubating overnight at 37°C. Single colonies were picked and inoculated into 3 mL of antibiotic-free LB liquid medium, and then expanded in 100 mL of LB broth at a 1:100 dilution until the logarithmic growth phase (OD600=0.5). The bacterial culture was aliquoted into 50 mL centrifuge tubes pre-chilled at 4°C, placed on ice for 10 minutes, centrifuged at 4000 r / min for 10 minutes, and the supernatant was discarded. The bacterial cells were resuspended in 25 mL of pre-chilled 75 mmol / L calcium chloride solution, placed on ice for 20 minutes, centrifuged at 4000 r / min for 10 minutes, and the bacterial cells were collected. The cells were resuspended in calcium chloride solution containing 20% ​​glycerol, aliquoted, and stored at -80°C.

[0106] (II) Construction of isotopically labeled β-casein recombinant expression vector

[0107] (1) Take 200 μL of competent cells BL21(DE3)Δlys / Δarg and thaw them on ice. Mix them with 10 μL of pET 28b-β-casein containing 60 ng / μL (β-casein gene is shown in SEQ ID NO.2, and plasmid schematic diagram is shown in the figure). Figure 10 Mix well and let stand on ice for 30 minutes;

[0108] (2) Place the centrifuge tube in a preheated 42 ℃ water bath for 90 seconds, then quickly transfer it to an ice bath to cool for 3 minutes. Add 800 μL of antibiotic-free LB liquid medium and incubate it in a shaker at 37 ℃ for 45 minutes.

[0109] (3) Centrifuge the bacterial solution at 6000 r / min for 10 minutes, discard the supernatant and resuspend the bacterial cells. Spread the bacterial solution evenly on LB solid medium containing 30 μg / mL kanamycin. Incubate in an incubator at 37 ℃ for 24 hours and white colonies can be observed.

[0110] (4) Select ten white single colonies from the culture medium and expand them in 1 mL LB liquid medium for 8 hours. Sequencing was used to verify the integrity and accuracy of the target gene sequence. Strains with correct sequences were stored in 20% glycerol broth at -80 ℃.

[0111] (III) Induced expression of recombinant proteins

[0112] (1) Transform the recombinant plasmid into competent cells, pick a single colony from BHI agar and inoculate it into M9 solid medium. After incubating at 37 °C upside down for 24 hours, passage it for two more generations on fresh M9 solid medium.

[0113] (2) Select larger single colonies and inoculate them into 3 ml of M9 liquid medium. Incubate them in a shaker at 37 ℃ with shaking at 200 r / min until OD600=0.5. Take out 1 mL of bacterial solution as an uninduced control. Add IPTG to the remaining bacterial solution to a final concentration of 1 mmol / L and induce expression for 18 hours in a shaker at 19 ℃ with shaking at 150 r / min.

[0114] (3) Place the centrifuge tube on ice for 5 minutes, centrifuge at 9000 r / min at 4℃ for 10 minutes to collect the bacterial cells, resuspend the bacterial cell pellet with pre-cooled PBS, centrifuge again, remove the supernatant, store the bacterial cells in a -80 ℃ freezer, and separate and purify the target protein.

[0115] The M9 culture medium used in this method was supplemented with isotopically labeled amino acids and essential nutrients. 13 C6 15 N4 arginine, 13 C6 15 N4 lysine, glucose, kanamycin, MgSO4, VB1, and CaCl2.

[0116] (iv) SDS-PAGE verification of recombinant protein expression

[0117] (1) Prepare 10% separating gel and 5% stacking gel using the SDS-PAGE gel preparation kit produced by Shanghai Beyotime Biotechnology Co., Ltd.

[0118] (2) Take 160 μL of the collected supernatant and precipitate directly, and add 40 μL of 5× loading buffer; after centrifuging the protein samples before and after induction at 6000 r / min for 10 minutes, discard the supernatant, add 160 μL of PBS to resuspend, and then add 40 μL of 5× loading buffer; take 160 μL of the purified protein sample and add 40 μL of 5× loading buffer. Place the above sample in a 100 ℃ metal bath for 15 minutes, cool to room temperature, centrifuge at 8000 r / min for 5 minutes, and take 20 μL of supernatant for loading;

[0119] (3) Set the initial voltage of the electrophoresis apparatus to 80 V. After 30 minutes, when the bromophenol blue enters the separating gel, increase the voltage to 120 V until the indicator reaches about 2 cm from the bottom of the gel and stop electrophoresis.

[0120] (4) After electrophoresis, take out the gel and place it flat in a petri dish. Add Coomassie brilliant blue staining solution and stain overnight on a shaker at room temperature. Then add destaining solution and place on a shaker to destain. Change the destaining solution every 2 hours until the protein bands on the gel are clear and the background is light. Take a picture to record.

[0121] The results are as follows Figure 11 As shown, no obvious band appeared before induction, but a clear band appeared around 27 kDa after induction, consistent with the theoretical molecular weight of β-casein, indicating successful expression of the target protein. After purification, a clear single band appeared at 27 kDa, indicating successful purification. The marker lanes covered the target protein's molecular weight range, verifying the reliability of the results.

[0122] Methods for the analysis and detection of β-casein in food based on isotope internal standard proteins

[0123] (a) Sample pretreatment

[0124] 1. Sample extraction

[0125] (1) Weigh 2 g of the sample to be tested and place it in a centrifuge tube. Add 10 mL of sample extraction solution (pH=8) containing 10 mol / L urea and 200 mmol / L Tris-HCl and extract in a shaker at 37 ℃ for 30 minutes.

[0126] (2) Remove from the shaker, sonicate for 30 minutes, centrifuge at 8000 r / min for 10 minutes, and transfer 500 μL of supernatant to a new 2 mL centrifuge tube.

[0127] 2. Enzymatic hydrolysis

[0128] (1) Take 500 μL of sample solution, add DTT (dithiothreitol) solution with a concentration of 1 mol / L to a final concentration of 10 mmol / L, and react at 37 ℃ for 1 hour;

[0129] (2) After cooling to room temperature, add 1 mol / L IAA (iodoacetamide) solution to a final concentration of 20 mmol / L, and let stand at room temperature in the dark for 30 minutes.

[0130] (3) Add 25 μL of trypsin with a concentration of 0.1 μg / mL, place at 37 ℃ and shake for 6 hours to digest, then place in a 4 ℃ refrigerator to terminate the reaction.

[0131] 3. Purification

[0132] For purification, an Oasis HLB premium (3cc) column was used. The column was activated sequentially with 1 mL of 80% ACN and 1 mL of ultrapure water. 200 μL of sample was added. During the washing stage, 0.1% formic acid solution was used for rinsing. During the elution stage, 60% ACN (containing 0.1% FA) was used for elution. The eluent was dried under nitrogen, and 100 μL of 0.1% formic acid solution was added and vortexed to reconstitute. The sample was centrifuged at 6000 r / min for 10 minutes, and the supernatant was collected for analysis.

[0133] Enzymatic hydrolysis time optimization

[0134] Two peptides, VLPVPQK and AVPYPQR, were selected. This experiment investigated the enzymatic hydrolysis levels of the selected target peptides at hydrolysis times of 2, 4, 6, 8, 12, 24, and 30 hours. The results are shown in [Figure number missing]. Figure 12 A. Of the two candidate characteristic peptides of β-casein, VLPVPQK showed the best enzymatic hydrolysis effect at 6 hours.

[0135] Optimization of purification treatment (purification washing, purification elution) conditions

[0136] This experiment also investigated the effects of washing with ultrapure water, 0.1% formic acid solution, and 0.1% ammonia solution during the washing stage of the purification process. Except for the different reagents used in the purification washing stage, the subsequent operations were identical. The response intensity was compared and measured on the instrument. The results are shown in […]. Figure 12 B. β-casein showed a higher response intensity after washing with 0.1% formic acid water.

[0137] During the purification and elution stage, the effects of using 40% ACN (containing 0.1% FA), 60% ACN (containing 0.1% FA), 80% ACN (containing 0.1% FA), and 90% ACN (containing 0.1% FA) as eluents were investigated, and the response intensity was compared. Figure 12 As shown in C, β-casein is more effective when eluted with 60% ACN (containing 0.1% FA).

[0138] (II) LC-MS / MS detection conditions

[0139] Chromatographic conditions

[0140] Chromatographic column: ACQUITY UPLC® Peptide CSHTM C18 (2.1 mm × 100 mm, 1.7 μm); injection volume: 5 μL; flow rate: 0.3 mL / min; column temperature: 40℃. Mobile phase: 0.1% formic acid in water (A), 0.1% formic acid in acetonitrile (B); gradient elution program: 0–0.5 min, 5% A; 0.5–6.50 min, 5%–30% A; 6.50–8.00 min, 30%–95% A; 8.00–10.50 min, 95% A; 10.50–13.00 min, 95%–5% A.

[0141] Mass spectrometry conditions

[0142] Electrospray ionization source (ESI+); atomizing gas is nitrogen, flow rate is 3.0 L / min; heating gas is air, flow rate is 10 L / min; drying gas is nitrogen, flow rate is 10 L / min; collision gas is argon; ion source interface temperature is 300 ℃; desolvation tube temperature is 526 ℃; heating module temperature is 400 ℃; scanning mode is multiple reaction monitoring (MRM).

[0143] Preparation of standard curve using internal standard method

[0144] Standard solutions with mass-to-volume concentrations of 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL were prepared using β-casein standards. The isotopically labeled recombinant β-casein protein (full-length protein) prepared according to this invention was added as an internal standard. Pretreatment was performed according to the aforementioned method, followed by instrumental analysis.

[0145] A linear regression equation was obtained by plotting the protein mass concentration on the x-axis and the peak area of ​​the target peptide on the y-axis. The standard curves and correlation coefficients of the isotopically labeled β-casein recombinant protein prepared in this invention as internal standards, with the characteristic peptides AVPYPQR and VLPVPQK, are shown in the table below.

[0146] Table 4. Linear relationships of characteristic peptides of β-casein

[0147]

[0148] As shown in the table above, using β-casein recombinant protein (full-length protein) as an internal standard and AVPYPQR and VLPVPQK as characteristic peptides to construct standard curves, the linear relationship between the two standard curves is shown in Table 4. The standard curve using VLPVPQK as the characteristic peptide is shown in Table 4. Figure 13As shown in Table A, the data in Table 4 shows that the correlation coefficients of the standard curves using AVPYPQR and VLPVPQK as characteristic peptides are quite similar, with no significant difference.

[0149] In addition, this invention uses isotopically labeled VLPVPQK* (peptide) as an internal standard, and simultaneously uses VLPVPQK as the characteristic peptide to prepare a standard curve, the results of which are as follows. Figure 13 As shown in B. Compare Figure 13 A and Figure 13 B reveals that, compared to using peptides (VLPVPQK*) as internal standards, using the full-length isotopically labeled protein as an internal standard demonstrates a significant advantage in standard curve fitting. Current detection methods typically use β-casein peptides as internal standards. This invention innovatively uses the full-length isotopically labeled β-casein protein as an internal standard, and the results show that using the full-length protein as the internal standard yields better detection results.

[0150] Limit of detection and limit of quantitation

[0151] Blueberry jam, chickpea biscuits, and rice cereal (containing no milk protein or related ingredients) were used as blank matrices. Low-concentration protein standard solutions were added, and after pretreatment, the signal-to-noise ratio (S / N) of each peptide at different concentrations was obtained. The concentration at which S / N ≥ 3 was the limit of detection (LOD) of the peptide detection method, and the concentration at which S / N ≥ 10 was the limit of quantitation (LOQ) of the method.

[0152] Precision and accuracy

[0153] The precision and accuracy of the method were verified through spiked recovery experiments. 500 μL of blank matrix solution was used, and low concentrations of the corresponding β-casein standard solution and 10 μg / mL of isotope internal standard were added, with three replicates for each concentration. After pretreatment, the samples were analyzed. The recoveries and relative standard deviations (RSD) for each concentration were calculated.

[0154] Table 5. Methodological evaluation in different matrices

[0155]

[0156] The results above show that when the quantitative peptides of β-casein are AVPYPQR and VLPVPQK, the limits of quantification and detection are the lowest in the three food matrices.

[0157] Sample testing

[0158] This experiment collected 12 kinds of commercially available processed foods in Beijing as samples, including various biscuits, cheese products and cheese slices, goat milk powder, and soy milk powder. The various biscuits were ground into powder using a grinder and then stored in 50 mL centrifuge tubes; cheese blocks, cheese slices, and other samples were removed from their packaging and stored in 50 mL centrifuge tubes; goat milk powder and soy milk powder were directly poured into 50 mL centrifuge tubes for storage.

[0159] Weigh 2 g of the above samples, add the isotopically labeled β-casein recombinant protein prepared in this invention as an internal standard, perform pretreatment according to the method provided in this invention, and then perform instrument detection. Select VLPVPQK as the characteristic quantitative peptide to obtain the β-casein presence level in the sample.

[0160] Table 6. Quantitative Detection Results of β-Casein in Actual Samples

[0161]

[0162] The test results above show that the commercially available sample 12 rice cakes has a problem where the milk allergen is inconsistent with the product label.

[0163] The above specific embodiments are merely illustrative of the invention and do not represent a limitation thereof. Those skilled in the art will recognize that other variations of the specific structure of this invention are possible.

Claims

1. A method for quantitative analysis of β-casein in food, the method comprising adding isotopically labeled recombinant β-casein protein as an internal standard to the sample to be tested, followed by trypsin digestion and LC-MS / MS analysis, selecting characteristic peptides of β-casein, and calculating the β-casein content according to the internal standard method; the amino acid sequence of the isotopically labeled recombinant β-casein protein is shown in SEQ ID NO.1, wherein, Lysine and arginine in the sequence are carbon and nitrogen isotope labeled amino acids.

2. The method according to claim 1, characterized in that, The food products mentioned include any food that may contain the milk allergen β-casein, including milk powder ingredients, formula milk powder, jam, bread, biscuits, cakes, steamed buns, snacks, oatmeal, rice porridge, or baking premixes.

3. The method according to claim 1, characterized in that, The β-casein characteristic peptides are selected from AVPYPQR or VLPVPQK.

4. The method according to claim 1, characterized in that, Prior to trypsin digestion, sample extraction is performed using a sample extraction solution, which is an aqueous solution containing 10 mol / L urea and 200 mmol / L Tris-HCl, with a pH of 8.

5. The method according to claim 1, characterized in that, Following trypsin digestion, sample purification is also included. The purification uses an Oasis HLB premium purification column. After activation, the sample is added and washed with 0.1% formic acid solution or 0.1% ammonia solution. Elution is performed using 40% ACN (containing 0.1% FA), 60% ACN (containing 0.1% FA), 80% ACN (containing 0.1% FA), or 90% ACN (containing 0.1% FA).

6. The method according to claim 1, characterized in that, The digestion time for trypsin is selected from 6-8 hours.

7. The method according to claim 1, characterized in that, The isotopically labeled β-casein recombinant protein was prepared by the following method: 1) Using E. coli BL21(DE3) as the starting strain, construct the lysine and arginine auxotrophic strain E. coli BL21(DE3)ΔLys / ΔArg; 2) The β-casein gene shown in SEQ ID NO.2 was introduced into a vector plasmid to construct a recombinant plasmid, and the recombinant plasmid was transformed into E. coli BL21(DE3)ΔLys / ΔArg strain to construct a recombinant engineered bacterium; 3) Using a lysine and arginine-deficient culture medium, and adding isotopically labeled lysine and arginine as an isotopically labeled culture medium, the recombinant engineered bacteria from step 2) were passaged multiple times in the isotopically labeled culture medium to induce expression, and the isotopically labeled β-casein recombinant protein was obtained by isolation and purification.

8. The method according to claim 7, characterized in that, The vector plasmid mentioned in step 2) is selected from the pET, pEX, GST, PEZZ18 or PKK223 series vector plasmids.

9. The method according to claim 7, characterized in that, The isotope-labeled lysine and arginine mentioned in step 3) include the C, N, H, or O elements labeled on lysine and arginine, and their isotopes include 13 C, 15 N, 2 H, 17 O or 18 O.

10. The following combination is used in the quantitative analysis and detection of β-casein content in food: 1) Isotope-labeled β-casein recombinant protein; 2) Isotope-labeled characteristic peptides of β-casein; The isotope-labeled β-casein recombinant protein is used as an internal standard, and its amino acid sequence is shown in SEQ ID NO.1, wherein, The lysine and arginine in the sequence are carbon and nitrogen isotope-labeled amino acids; the isotope-labeled β-casein characteristic peptides are selected from VLPVPQK* or AVPYPQR*, wherein K* and R* are carbon and nitrogen isotope-labeled amino acids.

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