CN114414688A

CN114414688A - Method for detecting contents of A1 beta-casein and A2 beta-casein in milk and dairy products

Info

Publication number: CN114414688A
Application number: CN202210037245.6A
Authority: CN
Inventors: 李明; 吴佩泽
Original assignee: National Institute of Metrology
Current assignee: National Institute of Metrology
Priority date: 2022-01-13
Filing date: 2022-01-13
Publication date: 2022-04-29

Abstract

The invention discloses a method for detecting the contents of A1 beta-casein and A2 beta-casein in milk and dairy products. The method comprises the following steps: performing enzyme digestion treatment on milk or dairy products to be detected by using thermolysin so as to obtain enzyme digested protein liquid to be detected; and detecting the enzyme-digested protein solution to be detected by using a liquid chromatography-tandem mass spectrometry system, and based on the detection information of the characteristic peptide fragment, so as to obtain the contents of A1 beta-casein and A2 beta-casein in the milk or dairy product. The method adopts thermolysin to carry out enzyme digestion on the protein of milk or dairy products, the enzyme digestion speed is high, the time is short, the impurities are few, the length and the composition difference of the characteristic peptide segments of the obtained A1 beta-casein and A2 beta-casein are obvious, the quality of the peptide segments is small, the two are easy to separate and detect, and the detection sensitivity and the detection accuracy are high.

Description

Method for detecting contents of A1 beta-casein and A2 beta-casein in milk and dairy products

Technical Field

The invention relates to the fields of analytical chemistry and food, in particular to a method for detecting the contents of A1 beta-casein and A2 beta-casein in milk and dairy products.

Background

There are two major components in milk proteins: casein and whey protein, wherein casein accounts for about 80% of the total milk protein. Casein is further classified into 4 classes according to the homology of the casein primary structure (amino acid sequence): α s1-, α s2-, β -and κ -caseins. Beta-casein accounts for about 40 percent of all casein, is a substance with complete phosphorylation, and has the obvious characteristic of providing help for mineral absorption, growth and development of newborns, so that a plurality of dairy manufacturers begin to pertinently improve the content of the beta-casein in products, so that the products can provide richer nutrition and better marketing direction. However, due to the difference of biological enzymes and gene mutation in cows in nature, the amino acid types at individual positions in the sequence are different, so that the beta-casein exists in many different variant forms, and more than ten variants are found at present, such as: a1, A2, A3, H1, H2, I, etc. Of these variants, a1 and a2 β -casein are abundantly present in common milk and dairy products as the two most closely related and most widely present variant forms, and the effects of these substances on the human body have received extensive attention due to the large-scale use of milk.

Originally, a1 β -casein was not found in nature, and genetic mutations in a2 β -casein were the root cause for its occurrence. Because the milk is not screened in a targeted manner in the natural iteration and artificial breeding processes, the situation that A1 beta-casein is taken as the leading situation in the existing milk is finally caused. Research shows that the A1 beta-casein is subjected to enzymolysis by human bodies to generate beta-casomorphin-7 (BCM-7), and the substance is considered to be the causative factor of a plurality of diseases all the time, but the A2 beta-casein does not generate the substance under the same condition. Thus, the detection of the contents of A1 and A2 beta-casein in milk and dairy products is to be investigated.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, one purpose of the invention is to provide a method for detecting the contents of A1 beta-casein and A2 beta-casein in milk and dairy products, the method utilizes thermolysin to carry out enzyme digestion on proteins in the milk or dairy products at specific positions to form characteristic peptide fragments, and the characteristic peptide fragments are detected by a liquid chromatography-tandem mass spectrometry system to obtain the contents of A1 beta-casein and A2 beta-casein, and the detection method is simple and has high accuracy.

According to one aspect of the invention, the invention provides a method for detecting the content of A1 beta-casein and A2 beta-casein in milk and dairy products. According to an embodiment of the invention, the method comprises: performing enzyme digestion treatment on milk or dairy products to be detected by using thermolysin so as to obtain enzyme digested protein liquid to be detected; and detecting the enzyme-digested protein solution to be detected by using a liquid chromatography-tandem mass spectrometry system, and based on the detection information of the characteristic peptide fragment, so as to obtain the contents of A1 beta-casein and A2 beta-casein in the milk or dairy product.

According to the method for detecting the contents of A1 beta-casein and A2 beta-casein in milk and dairy products, thermolysin is adopted to carry out enzyme digestion on the protein of the milk or dairy products, the enzyme digestion speed is high, the time is short, the types of used chemical reagents are few, impurities are few, the length and the composition difference of the characteristic peptide sections of the obtained A1 beta-casein and A2 beta-casein are obvious, the quality of the peptide sections is small, the A1 beta-casein and the A2 beta-casein are easy to carry out chromatographic separation and mass spectrum detection, and the detection sensitivity and the detection accuracy are high.

In addition, the method for detecting the contents of A1 beta-casein and A2 beta-casein in milk and dairy products according to the embodiment of the invention can also have the following additional technical characteristics:

according to the embodiment of the invention, the mass ratio of the thermolysin to the protein in the dairy product to be detected is 1: 10-25, preferably 1: 20.

According to the embodiment of the invention, the time of the enzyme digestion treatment is 3-6 hours.

According to an embodiment of the invention, the enzymatic cleavage is carried out at 50-70 ℃, preferably 60 ℃.

According to an embodiment of the invention, the amino acid sequence of said characteristic peptide stretch of said a1 β -casein comprises SEQ ID NO: 1.

According to an embodiment of the invention, the amino acid sequence of said characteristic peptide stretch of said a2 β -casein comprises SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

According to an embodiment of the present invention, the detection information of the characteristic peptide fragment is a mass-to-charge ratio of the characteristic peptide fragment.

According to an embodiment of the invention, the mass to charge ratio of the characteristic peptide stretch first isotope of the a1 β -casein is 1229.66 ± 0.2, 615.34 ± 0.2, 410.56 ± 0.2.

According to an embodiment of the invention, the mass to charge ratio of the characteristic peptide stretch first isotope of the a2 β -casein is 1947.04 ± 0.2, 974.03 ± 0.2, 649.68 ± 0.2.

According to the embodiment of the invention, the liquid chromatography detection conditions of the liquid chromatography tandem mass spectrometry system are as follows: a chromatographic column: c18 column, flow rate: 100 μ L/min-500 μ L/min, preferably 300 μ L/min.

According to the embodiment of the invention, the mass spectrum detection conditions of the liquid chromatography-tandem mass spectrum system are as follows: the detection conditions of the characteristic peptide fragment of the A1 beta-casein are as follows: electrospray ionization (ESI) positive ion mode; ion source voltage: 3.5 kV; the flow rate of the sheath gas: 5 arb; auxiliary air flow rate: 20 arb; capillary tube temperature: 275 ℃ C, detection conditions of said characteristic peptide stretch of said A2 β -casein: ion source voltage: 3.6 kV; the flow rate of the sheath gas: 5 arb; auxiliary air flow rate: 0 arb; capillary tube temperature: 325 ℃.

According to an embodiment of the invention, the liquid chromatography mobile phase of the liquid chromatography-tandem mass spectrometry system: a: 0.1 vol% aqueous formic acid solution, B: 0.1% by volume formic acid in acetonitrile.

According to an embodiment of the invention, the liquid chromatography of the liquid chromatography-tandem mass spectrometry system is gradient elution.

According to an embodiment of the invention, the elution conditions of the gradient elution are 0-25 minutes, 5-55% B; 25-26 minutes, 55-60% B; 26-36 minutes, 60% B; 36-37 minutes, 60-95% B; 37-40 min, 95% B.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows the results of measurements of different cleavage conditions according to one embodiment of the present invention, wherein A is the effect of reaction time on signal intensity and B is the effect of thermolysin addition on signal intensity;

FIG. 2 shows a schematic of the ion flow for extracting characteristic peptide fragments according to an embodiment of the present invention, wherein A is A1 characteristic peptide fragment, B is A2 characteristic peptide fragment, A1 and A2 characteristic peptide fragments are extracted from m/z:615.34 and m/z:974.03 respectively;

FIG. 3 is a schematic primary mass spectrum of a characteristic peptide fragment according to an embodiment of the present invention, wherein A is A1 characteristic peptide fragment, B is A2 characteristic peptide fragment, and the inset is the isotopic distribution of the strongest peak;

FIG. 4 shows the effect of different ion source voltages on the signal intensities of the A1 and A2 signature peptides, wherein A is the A1 signature peptide and B is the A2 signature peptide, according to one embodiment of the present invention;

FIG. 5 shows the effect of sheath airflow and auxiliary airflow on the signal intensity of the A1 characteristic peptide fragment, B on the signal intensity of the A2 characteristic peptide fragment, C on the signal intensity of the A1 characteristic peptide fragment, and D on the signal intensity of the A2 characteristic peptide fragment according to one embodiment of the present invention;

FIG. 6 shows the effect of capillary temperature on the signal intensity of signature peptides, wherein A is A1 signature peptide and B is A2 signature peptide, according to one embodiment of the present invention;

FIG. 7 shows a CID spectrum and a diagram of information of daughter ions of different characteristic peptides, wherein A is A1 characteristic peptide fragment and B is A2 characteristic peptide fragment;

FIG. 8 shows CID spectra of different milk samples according to one embodiment of the present invention, wherein A, B, C and D correspond to milk samples Nos. 1-4, respectively;

FIG. 9 shows CID spectra of different milk samples according to one embodiment of the present invention, wherein A and B correspond to milk samples Nos. 5 and 6, respectively;

FIG. 10 shows ion chromatography schematic of A1 and A2 characteristic peptide extractions in milk according to one embodiment of the present invention;

FIG. 11 shows a graphical representation of the results of the relative amounts of A1 and A2 proteins in different milk samples according to one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

In the description of the present invention, the terms "longitudinal", "lateral", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, which are for convenience of description of the present invention only and do not require that the present invention must be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

According to one aspect of the invention, the invention provides a method for detecting the content of A1 beta-casein and A2 beta-casein in a dairy product. According to the method for detecting the contents of A1 beta-casein and A2 beta-casein in the dairy product, the thermolysin is adopted to carry out enzyme digestion on the protein of the milk or the dairy product, the enzyme digestion speed is high, the time is short, the impurities are few, the length and the composition difference of the characteristic peptide sections of the obtained A1 beta-casein and A2 beta-casein are obvious, the quality of the peptide sections is small, the A1 beta-casein and the A2 beta-casein are easy to separate and detect, and the detection sensitivity and the detection accuracy are high.

To facilitate an understanding of the method for measuring the content of a1 β -casein and a2 β -casein in milk and dairy products, the method is explained herein according to an embodiment of the present invention, and comprises:

s100 enzyme digestion treatment

According to the embodiment of the invention, thermolysin is used for carrying out enzyme digestion treatment on the milk or dairy product to be detected so as to obtain the enzyme digested protein liquid to be detected. Beta-casein fragments 66-76 and 59-76 containing amino acid position 67 are selected as characteristic peptide fragments by using thermolysin. Compared with two characteristic peptide fragments of which the length is 49 and only differs by one amino acid in the trypsin method, the characteristic peptide fragments of the embodiment of the invention have shorter length and larger difference, and are more favorable for liquid phase separation and mass spectrum detection.

Thermolysin (Thermolysin) is a thermostable extracellular metalloendopeptidase with lower specificity than trypsin, with consequent more cleavage sites and stronger reactivity, thus generating more short fragments when casein is cleaved. Thanks to these characteristics, the inventors have chosen thermolysin for the enzymatic cleavage with at least one of the following advantages compared to the use of trypsin:

(1) the thermolysin can keep good activity at a high temperature (60-80 ℃) due to good thermal stability, and the inventors can change the protein structure by increasing the temperature of the enzyme digestion system, so that a loose molecular structure more suitable for enzyme digestion is obtained, and the enzyme digestion efficiency can be improved without additionally adding denaturants such as acetonitrile and the like in the reaction system. The higher enzyme digestion efficiency shortens the time required by enzyme digestion, and fewer operation steps also reduce the possibility of introducing impurities, so that the detection is faster and more accurate.

(2) Compared with two very similar characteristic peptide fragments in a trypsin method, the length and the composition of the two characteristic peptide fragments obtained after the thermophilic protease is subjected to enzyme digestion are obviously different (table 1), so that the property difference is obvious, the liquid chromatogram separation difficulty is obviously reduced, the separation time can be shortened, and the detection period is further shortened.

(3) The characteristic peptide fragments generated after the thermophilic protease enzyme digestion of the A1 and A2 beta-casein are shorter in length and smaller in mass, so that clearer signals can be obtained during mass spectrum detection, and the sensitivity of the detection method can be improved.

Furthermore, the cleavage site for the thermolysin to cleave the protein follows the following rules: cleavage is carried out between any amino acid and leucine (L), phenylalanine (F), isoleucine (I), valine (L), methionine (M), alanine (A). There is only one exception: if the latter amino acid of the above amino acids is proline (P), cleavage does not occur.

The 67 th amino acids of the A1 and A2 proteins are histidine (H) and proline (P), respectively, and the enzyme cutting difference is generated at the different sites according to the enzyme cutting characteristics of thermolysin. After simulation calculation (Peptide Cutter), the characteristic Peptide fragment information containing the differential sites in the thermophilic protease method is shown in table 1.

TABLE 1 theoretical data for characteristic peptide stretches after thermolysin cleavage

Theoretical mass spectrum information of the characteristic peptide fragment is calculated, and primary mass spectrum data are shown in table 2.

TABLE 2 theoretical m/z data for two characteristic peptide fragments at different valency states

According to the embodiment of the invention, the mass ratio of the thermolysin to the protein in the milk or the milk product to be detected is 1: 10-25, preferably 1: 20. Therefore, on the basis of ensuring that the protein in the milk or dairy product to be detected is sufficiently enzyme-cut, the using amount of the thermolysin is reduced, the detection cost is reduced, and the interference of the protein on the detection is reduced, wherein when the mass ratio of the thermolysin to the protein in the milk or dairy product to be detected is 1:20, the effect is better.

According to the embodiment of the invention, the time of the enzyme digestion treatment is 3-6 hours. Therefore, the method is beneficial to full enzyme digestion of protein, effective enzyme digestion time is shortened, detection time is shortened, and detection efficiency is improved.

As the thermolysin can keep good activity under the condition of higher temperature, the inventor changes the protein structure by improving the temperature of an enzymolysis system, thereby obtaining a loose molecular structure more suitable for enzymolysis, and the enzymolysis efficiency can be improved without additionally adding denaturants such as acetonitrile and the like. According to an embodiment of the invention, the enzymatic cleavage is carried out at 50-70 ℃, preferably 60 ℃. Further, according to an embodiment of the present invention, the enzyme digestion treatment is performed under a condition of pH 8. Therefore, the enzyme digestion efficiency is high, the required time is short, the operation steps are simple, the possibility of introducing impurities is reduced, and the detection is faster and more accurate.

S200 detection analysis

According to the embodiment of the invention, a liquid chromatography-tandem mass spectrometry system is used for detecting the enzyme-cut protein solution to be detected, and based on the detection information of the characteristic peptide segment, the contents of A1 beta-casein and A2 beta-casein in the milk and the dairy product are obtained. Therefore, the liquid chromatography-tandem mass spectrometry system is used for detection, and the detection accuracy and sensitivity are high.

According to an embodiment of the present invention, the detection information of the characteristic peptide fragment is a mass-to-charge ratio of the characteristic peptide fragment. Therefore, the mass-to-charge ratio difference between the two is large, and mass spectrum detection is convenient.

Based on the characteristic peptide fragments described in table 1, according to the examples of the present invention, the mass-to-charge ratio of the characteristic peptide fragment first isotope of the a1 β -casein is 1229.66 ± 0.2, 615.34 ± 0.2, 410.56 ± 0.2. The mass-to-charge ratio of the second isotope of the characteristic peptide fragment of the A2 beta-casein is 1947.04 +/-0.2, 974.03 +/-0.2 and 649.68 +/-0.2.

According to the embodiment of the invention, the liquid chromatography detection conditions of the liquid chromatography-tandem mass spectrometry system are as follows: a chromatographic column: c18 column, flow rate: 300 μ L/min. Therefore, the separation of characteristic peptide fragments is convenient, and the separation degree of each peptide fragment is high.

Because the characteristic peptide segments of the thermolysin method are greatly different, detection parameters of two peptide segments need to be set respectively in the detection process to obtain the optimal signal intensity, according to the embodiment of the invention, the mass spectrum detection conditions of the liquid chromatogram-tandem mass spectrum system are as follows: the detection conditions of the characteristic peptide fragment of the A1 beta-casein are as follows: electrospray ionization (ESI) positive ion mode; ion source voltage: 3.5 kV; the flow rate of the sheath gas: 5 arb; auxiliary air flow rate: 20 arb; capillary tube temperature: 275 ℃ C, detection conditions of said characteristic peptide stretch of said A2 β -casein: ion source voltage: 3.6 kV; the flow rate of the sheath gas: 5 arb; auxiliary air flow rate: 0 arb; capillary tube temperature: 325 ℃. Therefore, the stability of detection is good, the detection signals of different characteristic peptide fragments are strong, and the sensitivity and the accuracy of detection are high.

According to an embodiment of the invention, the liquid chromatography mobile phase of the liquid chromatography-tandem mass spectrometry system: a: 0.1 vol% aqueous formic acid solution, B: 0.1% by volume formic acid in acetonitrile. Therefore, the mobile phase is beneficial to fully separating enzyme digestion products, and the matrix effect in mass spectrometry is reduced.

According to an embodiment of the invention, the liquid chromatography of the liquid chromatography-tandem mass spectrometry system is gradient elution. According to an embodiment of the invention, the elution conditions of the gradient elution are 0-25 minutes, 5-55% B; 25-26 minutes, 55-60% B; 26-36 minutes, 60% B; 36-37 minutes, 60-95% B; 37-40 min, 95% B. Therefore, the method is beneficial to separation of peptide fragments with different characteristics, has proper retention time and shortens detection time.

The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or apparatus used are conventional products which are commercially available, e.g. from Sigma, without reference to the manufacturer.

Example 1

In this embodiment, the detection conditions of the enzyme digestion treatment and the liquid chromatography tandem mass spectrometry system are optimized, and under the optimized conditions, the beta-casein in different milk samples is detected.

1. Experimental methods

(1) Optimization of enzyme digestion

(a) Enzyme cutting time: 5 portions of 0.5mg/mL bovine beta-casein standard solution (50mmol/L Tris-HCl, pH 8) are taken, and each portion is 1 mL. Preheating to 60 ℃, respectively adding 50 mu L of thermolysin aqueous solution (0.5mg/mL), centrifuging (1000r/min, 1min), transferring into a constant temperature incubator at 60 ℃, respectively culturing for 2, 4, 6, 8 and 22 hours, adding 20 mu L of formic acid solution (10%, v/v) after completing the culture to terminate enzyme digestion, centrifuging (10000r/min, 10min), and taking the supernatant for detection.

(b) Amount of thermolysin added: 5 parts of protein solution (same as above) were taken, and the ratio of enzyme: protein (w/w) ═ 1: 10; 1: 15; 1: 20; 1: 25; adding 100 mu L of the mixture at a ratio of 1: 30; 67 μ L; 50 mu L of the solution; 40 mu L of the solution; mu.L of thermolysin solution (0.5mg/mL) was reacted at 60 ℃ for 4 hours, followed by addition of formic acid solution as above after completion of the culture, centrifugation, and detection of the supernatant.

(2) Assay detection condition optimization

(a) Liquid phase gradient elution procedure and mass spectrometry parameter optimization

Since the composition difference of the two characteristic peptide fragments is obvious, the difference of the retention time of the two characteristic peptide fragments in the chromatogram is large, and the liquid phase separation time can be shortened. The gradient elution procedure was optimized using a mixed solution of synthetic A1 and A2 signature peptide standard (15. mu.g/mL) in mobile phase A of 0.1% formic acid in water (v/v) and mobile phase B of 0.1% formic acid in acetonitrile (v/v). Gradient elution procedure is shown in table 3, run-in: 10 μ L.

TABLE 3 liquid chromatography gradient elution procedure

Respectively preparing standard solutions of A1 and A2 characteristic peptides, wherein the concentration is 15 mug/mL; the solvents were 25% acetonitrile in water (v/v) and 35% acetonitrile in water (v/v), respectively. In an electrospray ionization (ESI) positive ion mode, the following four mass spectrum parameters are optimized in a direct injection mode: ion source voltage, sheath gas flow, auxiliary gas flow, capillary temperature. Voltage optimization range: 1.5-4.5kV, and the sampling interval is 0.1 kV; sheath airflow optimization range: 0-10arb, sampling interval 1 arb; auxiliary gas flow optimization range: 0-30arb, sampling interval 1 arb; the optimized range of the capillary temperature is as follows: 250 ℃ and 400 ℃ with a sampling interval of 25 ℃.

Secondary mass spectrometry uses a Collision Induced Dissociation (CID) mode to select a divalent A1 peptide (m/z: 615.34) and a divalent A2 peptide (m/z: 974.03) as targets in an Ion Trap (Ion Trap), window Width (ISO Width): 4, collision energies of 18eV and 24eV, respectively.

(3) Detection of A1 and A2 beta-casein in milk

After relevant parameters of enzyme digestion experiments, liquid phase elution procedures and mass spectrum detection are optimized, commercial milk samples (No. 1-4 common milk, No. 5-6A 2 milk) purchased from supermarkets are detected. Adding ultrapure water into 500. mu.L of solution to be detected in the same volume, preheating (60 ℃), adding 100. mu.L of thermolysin aqueous solution (0.5mg/mL), fully and uniformly mixing by vortex, centrifuging (1000r/min, 1min), incubating in an incubator at 60 ℃ for 4h, and adding 20. mu.L of formic acid solution (10%, v/v). After vortex mixing, centrifugation is carried out (10000r/min, 10min), and supernatant is taken for detection.

(4) Data processing

Liquid chromatography and mass spectrometry data self-contained using instrumentation

And (4) processing by software. In order to more accurately detect characteristic peptide signals of A1 and A2 beta-casein, data of a milk sample are collected from an ion extraction flow graph of a secondary mass spectrum, and control data are ion extraction flow graphs of secondary mass spectra of A1 and A2 characteristic peptide standard products.

The experimental conditions are optimized to obtain clearer detection signals, and the quality of the test conditions is judged by taking the mass-to-charge ratio (m/z) signal of the A2 characteristic peptide in the primary mass spectrum as a reference in the optimization process. The relative signal intensity of the ordinate in the optimization schematic diagram of all conditions is obtained by sorting the original data, the magnitude is simplified in a unified way, and the relative relation is not changed.

2. Experimental results and discussion

(1) Casein cleavage

(a) The enzyme digestion reaction time is as follows: the efficiency of the cleavage of casein with thermolysin at 60 ℃ is higher than with the trypsin/acetonitrile system, which results in less time required for the cleavage reaction. As can be seen from FIG. 1A, when the reaction time is increased within 4 hours, the signal intensity of the characteristic peptide fragment can be effectively increased, but after 4 hours, the signal intensity level is at the same level as 4 hours even if the reaction time is increased to 22 hours, which indicates that the thermophilic proteasome system can complete the enzyme digestion of the protein within about 4 hours. The enzyme digestion reaction is the step which takes the longest time in the whole set of detection method, and compared with the trypsin method, the enzyme digestion reaction is reduced by more than 30 percent, which is very significant for shortening the detection time and detecting in batches.

(b) Amount of thermolysin added: the amount of enzyme added affects the degree of cleavage of the protein, and the more the target peptide fragment is formed in the system, the more it is advantageous to detect it, as shown in FIG. 1B, when the amount is increased over 1:20, the signal intensity starts to be stable, and after that, the signal intensity of the peptide fragment with the desired characteristics is not increased by increasing the amount of enzyme added. In view of the ultimate control of assay cost and reduction of interference, thermolysin addition at 1:20 is suitable for the assay method of the present embodiments.

(2) Analysis of the detection conditions

(a) Retention time of characteristic peptide fragment standard and mass spectrum data

In the trypsin method test, because the two characteristic peptide fragments are very similar in composition, under the condition of using the same chromatographic column and mobile phase, a gradient program with the concentration change of the mobile phase B being 1 percent per minute and the total time being 50min is selected for separation. After the thermolysin is used, the two characteristic peptide segments are different in length and composition, and the properties of the two characteristic peptide segments are greatly different, so that the retention time in the gradient elution process has a more obvious difference. When a gradient elution procedure with a 2% change in concentration per minute, and a total time consumption of 25 minutes, was used, sufficient separation of the two characteristic peptide fragments could already be achieved, which resulted in a significantly shorter time consumption for liquid chromatographic separation than the trypsin method, with the results shown in fig. 2, where the retention times for the seed characteristic peptide fragments of a1 and a2 β -casein were 9 minutes and 15 minutes, respectively.

After liquid phase separation, the mixed solution of the standard substance enters mass spectrum for detection, as shown in fig. 3, and the detection result is completely consistent with the theoretical standard data shown in table 2. In the primary mass spectrum, the characteristic peptide fragments of A1 and A2 beta-casein can detect a plurality of different valence states, wherein bivalent A1 characteristic peptide ions (m/z: 615.34) and bivalent A2 characteristic peptide ions (m/z: 974.03) are the forms with the strongest signals.

(b) Mass spectral parameter comparison

Because the ion source is obviously influenced by the composition of the solvent during working, the solvent state of the two characteristic peptide fragments entering the ion source is simulated respectively to prepare a sample solution, and the solvent environment where the sample enters the ion source is reduced as much as possible.

When different parameters are compared, a larger parameter range is selected in order to ensure accuracy and regularity, selected points with too large signal intensity difference are eliminated in fig. 4-7, and the parameter points which can reflect regularity and obtain optimal signals are highlighted, and the results are as follows:

ion source voltage: as shown in FIG. 4, the optimum ion source voltages for the A1 and A2 signature peptides were 3.5kV and 3.6kV, respectively.

The flow rate of the sheath gas: as shown in fig. 5A and 5B, for both characteristic peptide fragments, the optimal intensity was obtained at a sheath airflow rate of 5arb, and a very significant intensity drop occurred after exceeding 5 arb.

Auxiliary air flow rate: as shown in FIGS. 5C and 5D, the auxiliary gas significantly promoted the increase in the A1 signature signal intensity, and the preferred signal was obtained at a flow rate of 20arb, but had a negative effect on the A2 signature, and the auxiliary gas was kept off when the A2 signature was detected.

Capillary temperature: as shown in FIG. 6, the capillary temperatures of the two characteristic peptide fragments are preferably 275 ℃ and 325 ℃, respectively. Unlike other transient-type mass spectrometry parameters, the stabilization time required when adjusting the capillary temperature is long, and the instrument temperature fluctuates around the target temperature during stabilization. If temperature parameters are respectively arranged for two characteristic peptide segments in one complete sample detection, the detector is unstable. If two peptide fragments are completely detected twice, the detection period is doubled. Therefore, when a sample is detected, the temperature of the capillary of the detector is kept stable at 300 ℃, although the optimal reaction condition is not strictly executed, the signal intensity can meet the detection requirement of the method, and the situation that the detection cannot be carried out due to insufficient signal intensity does not occur in primary and secondary mass spectrum detection.

Regarding the purge gas, the target signal intensity is reduced after the purge gas is turned on, and the purge gas is kept in a turned-off state during the test.

And (3) optimizing the CID condition: the optimization of CID conditions focuses on adjusting window width and collision energy. The window width determines whether the detector can identify the target under the selected mode, and when the window width is too small, the target ions cannot be identified, but when the window width is too large, irrelevant ions with similar mass are simultaneously crushed, and interference signals are increased. The size of the collision energy affects the degree of fragmentation of the parent ion, and in order to ensure the signal intensity of the daughter ion, the parent ion is completely fragmented as much as possible. After optimization, the window width of the characteristic peptide A1 is set to be 4, and the collision energy is as follows: 18 eV; the a2 signature peptide window width was set to 4, collision energy: 21 eV. Considering the longer length of the a2 peptide, it is expected that a greater collision energy is required.

The standard information of secondary mass spectrum ion of the characteristic peptide segment can be obtained while the CID condition is obtained, and each ion can be accurately identified after being compared with the theoretical standard data on the MS-product (figure 7), so that the comparison data can be provided for protein detection in subsequent milk or milk products.

3. Milk sample detection

In order to verify the practicability and stability of the detection method, the thermophilic protease method detection is carried out on the sample to be detected. In order to eliminate interference of similar impurities and the like, casein is extracted from all milk samples in an isoelectric precipitation mode, all characteristic peptide fragments are accurately identified in a tandem mass spectrum and are smashed as expected after enzyme digestion by thermolysin, and generated daughter ion signals can correspond to standard peptide fragments (figure 8). Compared with the standard product, the protein and enzymolysis products in the milk sample are much more complex, so that the recognition quantity of the pair ions is slightly less, but in the case, most of characteristic ion is still accurately recognized, and the A1 and A2 characteristic peptides can be proved to be detected in all common milk. When the milk 5 and the milk 6 are detected, the characteristic peptide signal of A1 is not recognized in the milk 5 sample. The signal intensity of daughter ions was already lower than that of background ions for the A1 signature peptide detected in sample No. 6 milk (FIG. 9). It was shown that the A1 signature peptide was present but in a very low amount in the milk sample No. 6.

Among the obtained daughter ion information of the A1 and A2 characteristic peptides, b9 and b16 daughter ions with the strongest signals are respectively selected, characteristic peptide fragments in all samples are extracted, and as shown in FIG. 10, the liquid chromatography retention time of the A1 and A2 characteristic peptides in all milk samples is consistent with that of a standard sample (FIG. 2), and the method has excellent stability.

The peak area information mainly represents the content information of characteristic peptides A1 and A2 in a sample, and the molecular weight information of the characteristic peptides needs to be substituted for conversion when the relative contents of A1 and A2 beta-casein in the sample are compared based on the extraction chromatographic peak area. Since the molecular weight ratio of the a1 and a2 peptides in the thermolysin method is about 1: 1.58, the relative contents of A1 and A2 proteins in the sample can be directly obtained by conversion according to the proportion coefficient according to the peak area data shown in FIG. 9, and the result is shown in FIG. 11, which shows that the contents of A1 protein in No. 1-4 milk samples are all higher than that of A2 protein, and the relative content is 2-4 times; in contrast, for both A2 milks, only the A2 protein signal was detected in the milk sample No. 5, while a trace amount of A1 protein was present in the milk sample No. 6. In addition, a standard curve is established by utilizing a standard substance of the characteristic peptide fragment, the content information of A1 and A2 peptides in a tested sample can be obtained, and the absolute content of A1 and A2 beta-casein can be obtained by calculating according to the molecular weight of the characteristic peptide fragment and the molecular weight of the protein because the characteristic peptide fragment and the protein have a relation of 1:1 molar ratio.

4. Conclusion of the experiment

According to the method for detecting A1 and A2 beta-casein in milk by using characteristic peptide fragments based on thermolysin, the A1 and A2 beta-casein are subjected to enzyme digestion, products are separated and detected by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS), and qualitative and semi-quantitative analysis of the A1 and A2 beta-casein in milk can be realized by identifying and determining the characteristic peptide fragments.

In the enzyme digestion process, the reaction temperature is increased to make the protein more suitable for enzyme digestion reaction in the solution, and no additional medicament is required to be added into the reaction system as an accelerant, so that the detection operation is simplified, and the possibility of introducing impurities is reduced. Thermophilic proteases are selected to cleave A1 and A2 beta-casein, since the commonly used proteases do not retain activity at higher temperatures. The optimized results for the digestion reaction were: reaction time, 4 hours; enzyme addition amount, 1:20 (w/w); the reaction temperature was 60 ℃.

After the protein is subjected to enzyme digestion by using thermolysin, beta-casein fragments 66-76 and 59-76 containing 67 amino acid positions are selected as characteristic peptide fragments. Compared with two characteristic peptide segments which have the same length of 49 and only differ by one amino acid in the trypsin method, the characteristic peptide segments in the chapter have shorter length and larger difference, and are more favorable for liquid phase separation and mass spectrum detection. After the optimization of the standard substance mixed solution, the concentration of the mobile phase B in the finally determined program is linearly increased from 5% to 55% in 25 minutes, so that the two characteristic peptides can be fully separated, and the separation time is only half of that of the trypsin method.

Related mass spectrum parameters are optimized respectively aiming at the two characteristic peptide segments, and finally, the detection condition of the A1 characteristic peptide is ion source voltage: 3.5 kV; the flow rate of the sheath gas: 5 arb; auxiliary air flow rate: 20 arb; capillary tube temperature: 275 ℃. The detection conditions of the A2 characteristic peptide are ion source voltage: 3.6 kV; the flow rate of the sheath gas: 5 arb; auxiliary air flow rate: 0 arb; capillary tube temperature: 325 ℃. Selecting bivalent A1 characteristic peptide ions (m/z: 615.34) and bivalent A2 characteristic peptide ions (m/z: 974.03) as parent ions in a tandem mass spectrum CID test, wherein the parent ions are respectively at the window width of 4; the test was carried out under the condition of a collision energy of 18/21 eV.

After the test conditions are optimized, the 6 milk samples are tested, and the results show that the enzyme digestion and liquid chromatography separation effects are good; mass spectral data are accurate (table 4); and the information of the daughter ions completely consistent with the theoretical standard data can be obtained in the CID test. In repeated experiments for different samples, good stability and reproducibility were exhibited.

TABLE 4 theoretical and actual information for characteristic peptides A1 and A2

The content of A1 protein is generally higher and is about 2-4 times of that of A2 protein in 4 types of common milk which is actually detected to have both A1 and A2 beta-casein. The two types of A2 milk only contain little or no A1 protein, and are in line with the characteristics of the commodity. After qualitative and semi-quantitative analysis of A1 and A2 beta-casein in milk, the conclusion same as that of the trypsin method is obtained, but the time required by thermolysin in enzyme digestion reaction and liquid phase separation is shorter, the operation is simpler and more convenient, and compared with the trypsin method, higher detection efficiency can be obtained.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for detecting the content of A1 beta-casein and A2 beta-casein in milk and dairy products is characterized by comprising the following steps:

performing enzyme digestion treatment on milk or dairy products to be detected by using thermolysin so as to obtain enzyme digested protein liquid to be detected; and

and detecting the enzyme-cut protein solution to be detected by using a liquid chromatography-tandem mass spectrometry system, and based on the detection information of the characteristic peptide fragment, so as to obtain the contents of A1 beta-casein and A2 beta-casein in the milk or the dairy product.

2. The method according to claim 1, wherein the mass ratio of the thermolysin to the protein in the milk or dairy product to be tested is 1: 10-25, preferably 1: 20.

3. The method of claim 1, wherein the time for the enzymatic cleavage is 3 to 6 hours.

4. The method according to claim 1, wherein said enzymatic cleavage is carried out at 50-70 ℃, preferably 60 ℃.

5. The method according to claim 1, wherein the amino acid sequence of the characteristic peptide stretch of a1 β -casein comprises SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence is shown in 1,

optionally, the amino acid sequence of the characteristic peptide stretch of the a2 β -casein comprises SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

6. The method of claim 1, wherein the detection information of the characteristic peptide fragment is the mass-to-charge ratio of the characteristic peptide fragment,

optionally, the mass to charge ratio of the characteristic peptide stretch first isotope of the A1 beta-casein is 1229.66 + -0.2, 615.34 + -0.2 or 410.56 + -0.2,

optionally, the mass to charge ratio of the characteristic peptide stretch first isotope of the a2 β -casein is 1947.04 ± 0.2, 974.03 ± 0.2, 649.68 ± 0.2.

7. The method according to claim 1, wherein the liquid chromatography detection conditions of the liquid chromatography tandem mass spectrometry system are as follows:

a chromatographic column: a C18 chromatography column;

flow rate: 100 μ L/min-500 μ L/min, preferably 300 μ L/min.

8. The method of claim 1, wherein the mass spectrometric detection conditions of the liquid chromatography-tandem mass spectrometry system are:

the detection conditions of the characteristic peptide fragment of the A1 beta-casein are as follows:

electrospray ionization (ESI) positive ion mode;

ion source voltage: 3.5 kV;

the flow rate of the sheath gas: 5 arb;

auxiliary air flow rate: 20 arb;

capillary tube temperature: at a temperature of 275 c,

the detection conditions of the characteristic peptide fragment of the A2 beta-casein are as follows:

ion source voltage: 3.6 kV;

the flow rate of the sheath gas: 5 arb;

auxiliary air flow rate: 0 arb;

capillary tube temperature: 325 ℃.

9. The method of claim 1, wherein the liquid chromatography mobile phase of the liquid chromatography-tandem mass spectrometry system: a: 0.1 vol% aqueous formic acid solution, B: 0.1% by volume formic acid in acetonitrile.

10. The method of claim 1, wherein the liquid chromatography of the liquid chromatography-tandem mass spectrometry system is gradient elution,

optionally, the gradient elution is at elution conditions of 0-25 minutes, 5-55% B; 25-26 minutes, 55-60% B; 26-36 minutes, 60% B; 36-37 minutes, 60-95% B; 37-40 min, 95% B.