Characteristic peptide fragment for identifying alpha-casein of cattle and sheep milk, qualitative identification method and application

Abstract

The invention belongs to the technical field of dairy product detection, and relates to a characteristic peptide fragment for identifying alpha-casein of cattle and sheep milk, a qualitative identification method and application. The characteristic peptide fragment comprises a characteristic peptide fragment of cow milk alpha-casein, and the characteristic peptide fragment of the cow milk alpha-casein is CAGCPHCPDHWMGYGDHCYYFSVEK or EYSTAHLSRTLTLVEQIK; the characteristic peptide fragment of the cow milk alpha-casein is CAGCPHCPDHWMGYGDHCYYFSVEK; the characteristic peptide fragment of the goat milk alpha-casein is SLCQETCCSPSCCQTTCCRTTCYR, and the characteristic peptide fragment of the goat milk alpha-casein is According to the method disclosed by the invention, the characteristic peptide fragments of cow milk alpha-casein and goat milk alpha-casein are screened by combining enzymolysis, high performance liquid chromatography and high-resolution mass spectrometry, and then the alpha-casein characteristic peptide fragments are used for qualitatively identifying cow milk and goat milk, so that the detection precision is high, and the accuracy of an identification result is high; and adulteration identification of cow milk and goat milk can be realized.

Description

Technical Field

[0001] The invention belongs to the technical field of dairy product detection and relates to a characteristic peptide segment for identifying bovine and goat milk α-casein, a qualitative identification method and an application thereof. Background Art

[0002] α-casein, one of the major casein isoforms in dairy products, accounts for 30%–40% of the total casein content. Its structure, function, and variability across different milk sources have profound implications for the processing properties, nutritional value, and bioactivity of dairy products. In recent years, with increasing consumer demand for specialty milks (such as goat or cow milk), research on the characterization of α-casein in milk from different species has become a focus of attention. This is because α-casein exhibits significant differences in its amino acid sequence, phosphorylation sites, and post-translational modifications across species. Consequently, interactions between α-casein and other caseins (β- and κ-casein) and calcium ions can directly impact the rheological, thermal, and digestive properties of dairy products. Furthermore, the high added value of the specialty milk market has also led to adulteration concerns, such as adulteration of goat milk with cow milk. Therefore, developing efficient identification technologies based on the species-specific characteristics of α-casein is crucial for ensuring the quality and safety of dairy products.

[0003] At present, the traditional methods for detecting α-casein in dairy products include electrophoresis and spectroscopy, but they have low sensitivity and anti-interference problems, which cannot meet the actual detection needs of cow's milk and goat's milk. Therefore, enzyme-linked immunosorbent assay, polymerase chain reaction technology and chromatography have emerged. In the existing technology, researchers used PCR technology to analyze α-casein in three native Egyptian sheep breeds. s1 - and α s2 -casein gene genetic polymorphisms, identifying the different gene sequences of the two α-casein isoforms; other researchers have used bovine α-casein monoclonal antibodies to establish indirect ELISA and indirect blocking ELISA detection methods to detect α-casein in goat milk; and others have used reversed-phase high-performance liquid chromatography (HPLC) to detect α-casein in goat milk, a method that can be completed within 24 minutes and has a relative standard deviation of less than 8%. However, due to the denaturation and inactivation of proteins during the processing of these specialty milks, HPLC methods are unable to detect the target protein, and ELISA methods can produce false positives, resulting in low precision and poor accuracy of α-casein detection, making it difficult to detect adulteration in goat milk.

[0004] Therefore, how to establish an accurate and efficient method to specifically identify α-casein in cow's milk and goat's milk and realize adulteration detection in goat's milk is a hot research issue. Summary of the Invention

[0005] In response to the technical problems in the existing detection of α-casein in bovine and goat milk products, such as the inability to detect the target protein and the occurrence of false positives due to protein denaturation and inactivation, resulting in low α-casein detection precision, poor accuracy of detection results, and difficulty in adulteration detection, the present invention provides a characteristic peptide segment, a qualitative identification method and application for identifying bovine and goat milk α-casein.

[0006] The present invention screens out characteristic peptide segments of α-casein in bovine and goat milk by combining enzymatic hydrolysis, high-performance liquid chromatography and high-resolution mass spectrometry, and then uses the characteristic peptide segments of α-casein to perform qualitative identification of bovine and goat milk, with high detection precision and high accuracy of identification results; and can also realize the identification of adulteration of bovine and goat milk.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for identifying characteristic peptide segments of bovine and goat milk α-casein, wherein the characteristic peptide segments include characteristic peptide segments of bovine milk α-casein and characteristic peptide segments of goat milk α-casein.

[0009] The characteristic peptide segment of bovine milk α-casein is: CAGCPHCPDHWMGYGDHCYYFSVEK or EYSTAHLSRTLTLVEQIK;

[0010] The characteristic peptide segment of goat milk α-casein is SLCQETCCSPSCCQTTCCRTTCYR.

[0011] A method for qualitatively identifying bovine and goat milk α-casein based on the characteristic peptide segment comprises the following steps:

[0012] S1. Extraction of α-casein

[0013] α-casein is extracted from the special milk to be tested by using urea differential precipitation method; the special milk is cow's milk or goat's milk;

[0014] S2, Enzymatic Hydrolysis

[0015] Adding trypsin to α-casein for enzymatic hydrolysis, and then inactivating the enzyme, cooling, centrifuging and filtering to obtain an α-casein enzymatic hydrolysate;

[0016] S3, ultra-high performance liquid chromatography-high resolution mass spectrometry detection

[0017] The α-casein hydrolysate obtained in step S2 is sequentially subjected to ultra-performance liquid chromatography-high-resolution mass spectrometry to obtain a spectrum;

[0018] S4. Qualitative identification

[0019] The spectrum obtained in step S3 is analyzed using workstation software; the specialty milk to be tested is qualitatively identified based on the characteristic peptide segments at the peak positions on the spectrum.

[0020] It is further defined that the specific process of extracting α-casein from special milk by urea differential precipitation method in step S1 is:

[0021] S1.1. Preparation of pure casein

[0022] The pH value of the special milk is adjusted to 4.0-5.0 with acetic acid, and the crude casein is obtained by standing and initial centrifugation; and then the pure casein is obtained by washing and re-centrifugation;

[0023] S1.2. Pure casein is dissolved in urea solution to obtain casein particles; the particles are then washed with urea solution, centrifuged and the precipitate is collected, and then dialyzed and dried to obtain powdered α-casein.

[0024] It is further defined that in step S1.1, the standing is to be treated in a refrigerator at 2-8°C overnight; the initial centrifugation is to be centrifuged at 2-8°C and 9500rpm-12000rpm for 20min-40min; the washing is carried out in sequence with pure water, ethanol and ethanol-petroleum ether; and the second centrifugation is to be centrifuged at 3800rpm-4500rpm for 5min-10min.

[0025] It is further defined that the specific process of enzymatic hydrolysis in step S2 is:

[0026] Water and NaOH solution are respectively added to powdered α-casein to a pH of 8.0-8.5, and ultrasonic treatment is performed until the concentration of α-casein is 1-10 mg / mL. Trypsin is added to a concentration of 4000-10000 U / g. Then, enzymatic hydrolysis is performed at 37°C and pH 8.0-8.5. The enzyme is inactivated by heating at 90°C-95°C for 5-15 minutes, and after cooling to room temperature, the solution is centrifuged at 4000-4500 rpm for 5-10 minutes. The supernatant is filtered through a 0.22 μm filter membrane to obtain an α-casein hydrolyzate.

[0027] It is further defined that in step S3, the conditions for ultra-high performance liquid chromatography are:

[0028] C18 chromatographic column, stationary phase particle size 5 μm, inner diameter 2.0-3.0 mm, column length 100-150 mm; column temperature: 30-35°C; flow rate 0.2-0.3 mL / min; injection volume 2-5 μL; mobile phase: A is 0.1-0.2% formic acid-water solution (V:V), B is acetonitrile, gradient elution, A linearly changes from 97% to 40% in 18-20 minutes, B linearly changes from 3% to 60%, within 1-2 minutes, A changes to 10%, B changes to 90%, maintain for 4-7 minutes, then return to the initial mobile phase, and equilibrate for 2-6 minutes.

[0029] It is further defined that the high-resolution mass spectrometry conditions in step S3 are:

[0030] Mass analyzer: Orbitrap; ion source: electrospray ion source (ESI); scan mode: positive ion mode under full scan mode (Full-MS); spray voltage: 3.2kV-3.8kV; lens voltage 55V; capillary temperature 300℃-350℃; auxiliary gas heating temperature 300℃-350℃; sheath gas pressure is 30-45arb, auxiliary gas pressure is 5arb-10arb; scan range: 200-1500m / z; primary mass spectrometry resolution R=70000FWHM; secondary mass spectrometry resolution R=13500FWHM; maximum dwell time is 50ms-100ms.

[0031] It is further defined that in step S4, the qualitative identification of the specialty milk to be tested is specifically as follows:

[0032] The parent ion m / z was 850.42289-850.42489, and the daughter ion m / z was 343.89255-343.89455 and 360.22196-360.22396, and the characteristic peptide CAGCPHCPDHWMGYGDHCYYFSVEK appeared, which was identified as bovine milk α-casein. At this time, the special milk was bovine milk;

[0033] The parent ion m / z was 602.34782-602.34982, and the product ion m / z was 416.00108-416.00308, 318.22269-318.22469, and the characteristic peptide EYSTAHLSRTLTLVEQIK appeared, which was identified as bovine milk α-casein. At this time, the special milk was bovine milk;

[0034] When the parent ion m / z is 1109.51914-1109.52114, the daughter ion m / z is 239.99964-240.00164, 808.22306-808.22506, and the characteristic peptide SLCQETCCSPSCCQTTCCRTTCYR appears, it is identified as goat milk α-casein, which indicates that the special milk is goat milk.

[0035] The characteristic peptide segment is used in the qualitative identification of bovine milk α-casein and / or goat milk α-casein.

[0036] The characteristic peptide segment is used to identify whether goat milk is mixed with cow milk.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. The present invention screens out characteristic α-casein peptides of cow's milk and goat's milk by combining enzymatic hydrolysis, high-performance liquid chromatography and high-resolution mass spectrometry, and then uses the characteristic α-casein peptides to perform qualitative identification of cow's milk and goat's milk, with high detection precision and high accuracy of identification results; the α-casein characteristic peptides are used to identify adulteration of cow's milk and goat's milk, and the identification results are highly reliable.

[0039] 2. The present invention improves the identification results by optimizing the heating time, heating temperature and storage days in the enzymatic hydrolysis. At the same time, it is verified that the response values ​​of α-casein and the characteristic peptide segment are in a defined relationship, with a correlation coefficient of 0.9983, a linear range of 0.5-15 mg / mL, a detection limit of 0.02 mg / mL, and a quantitative limit of 0.07 mg / mL. It can be seen that the qualitative identification method provided by the present invention has high accuracy and high sensitivity.

[0040] 3. The α-casein qualitative identification method provided by the present invention also has good reproducibility and stability, and is suitable for testing different batches of dairy products.

[0041] 4. The present invention was verified by taking the adulteration of goat milk with cow milk as an example. The detection rate of adulterated milk was 0.5%, indicating that it is feasible to identify the adulteration of goat milk with cow milk by using the screened characteristic peptide segments. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The chromatograms of characteristic peptides of α-casein from cow milk (a, b, c) and goat milk (d, e, f) hydrolyzed by trypsin.

[0043] Figure 2 The effect of heating temperature on α-casein;

[0044] Figure 3 The effect of heating time on α-casein;

[0045] Figure 4 The effect of storage days on α-casein;

[0046] Figure 5 The standard curve is for trypsin-digested bovine α-casein standard. DETAILED DESCRIPTION

[0047] The present invention will be described in further detail below with reference to the accompanying drawings and examples, but the embodiments of the present invention are not limited thereto. Other methods for preparing the compounds of the present invention are considered to be within the scope of the present invention by making some conventional modifications to the reaction conditions of the present invention.

[0048] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0049] Technologies, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, such technologies, methods, and equipment should be considered part of the specification.

[0050] It should also be understood that the specific embodiments described above are only used to explain the present invention, and the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention, should be covered by the scope of protection of the present invention.

[0051] The technical solution provided by the present invention is described in detail below.

[0052] It should be noted that, in the following examples, unless otherwise specified, the chemicals and reagents used are all conventional commercial products in the art.

[0053] It should be noted that in the following examples, unless otherwise specified, the operations used are all conventional operations; for example, the operating temperature is room temperature unless otherwise specified. The test methods are all standard test methods available in the art unless otherwise specified.

[0054] Example 1

[0055] The present invention provides a characteristic peptide segment for identifying bovine and goat milk α-casein, wherein the characteristic peptide segment includes a characteristic peptide segment of bovine milk α-casein and a characteristic peptide segment of goat milk α-casein. The characteristic peptide segment is screened and identified using the following method. Specifically, the following steps are included:

[0056] S1. α-casein extraction

[0057] S1.1. Preparation of pure casein

[0058] Take dairy products, then adjust the pH value to the target point value with 10% (volume fraction) acetic acid, place in a 4°C refrigerator overnight and centrifuge at 10,000 rpm at 20°C for 30 minutes. The resulting precipitate is crude casein. Wash the crude casein three times with pure water equal to the volume of the supernatant, wash once with 95% ethanol, and wash twice with ethanol-petroleum ether. Centrifuge at 4,000 rpm for 10 minutes each time and discard the upper liquid to obtain pure casein.

[0059] In this step, the dairy products are cow's milk and goat's milk, the target pH value of cow's milk is 4.6, and the target pH value of goat's milk is 4.1; the obtained pure caseins are cow's milk pure casein and goat's milk pure casein.

[0060] S1.2. Extraction of α-casein using urea differential precipitation method

[0061] The purified casein obtained above was dissolved in a 6.6M urea solution and diluted with water to a urea concentration of 4.63M. The casein particles were collected by centrifugation at 4000 rpm for 15 minutes. The casein particles were then redissolved in a 6.6M urea solution containing 0.18M NaCl, diluted with water to 4.63M, and centrifuged at 4000 rpm for 15 minutes to collect the casein particles. Finally, the casein was washed once in a 4.7M urea solution and centrifuged at 4000 rpm for 15 minutes to collect the precipitate, which is α-casein. The extracted α-casein was dialyzed in pure water for 24 hours and then dried in a vacuum drying oven at 60°C for 1 hour to obtain α-casein powder (or freeze-dried for 12 hours), which was stored in a refrigerator at 4°C.

[0062] In this step, the pure casein is pure casein from cow's milk and pure casein from goat's milk, and thus the obtained α-casein powders are α-casein powder from cow's milk and α-casein powder from goat's milk.

[0063] S2, Enzymatic Hydrolysis

[0064] Take the α-casein powder of step S1 respectively, its quality is in 10~100mg (accurate to 0.1mg), use ultrapure water to dissolve and add the NaOH solubilizer of 0.5mol / L, be settled to 10mL, add a certain volume of trypsin solution until the final concentration of trypsin is a certain value in 4000~10000U / g after ultrasonic 15min.At 37 ℃, pH=8.0 condition, respectively to two kinds of α-casein enzyme hydrolysis, then heat 10min enzyme inactivation at 95 ℃.After cooling to room temperature, 4000rpm centrifugal 10min, supernatant is crossed 0.22μm filter membrane, obtains α-casein enzyme hydrolyzate, is then used for ultra performance liquid chromatography-high resolution mass spectrometry detection.

[0065] Optimally, the species-specific process parameters for trypsin hydrolysis of α-casein are: the bovine milk system achieves optimal response at a substrate concentration of 5%, an enzyme dosage of 7800 U / g, and a hydrolysis time of 2 hours; the goat milk system requires a 1.5% substrate concentration and an enzyme activity of 8800 U / g for 5 hours. The bovine milk system exhibits significant short-term high efficiency, while the goat milk system relies on extended hydrolysis time to maximize peptide release. This difference in time dimension may be due to the fundamental differences in protein conformational stability between species.

[0066] S3, ultra-high performance liquid chromatography-high resolution mass spectrometry detection

[0067] The α-casein hydrolysate obtained in step S2 is sequentially subjected to ultra-performance liquid chromatography-high-resolution mass spectrometry to obtain a spectrum;

[0068] The ultra-high performance liquid chromatography test conditions are as follows: a C18 chromatographic column with a stationary phase particle size of 5 μm, an inner diameter of 2.0-3.0 mm, and a column length of 100-150 mm; column temperature: 30-35° C.; a flow rate of 0.2-0.3 mL / min; an injection volume of 2-5 μL; mobile phase: A is a 0.1-0.2% formic acid-water solution (V:V), B is acetonitrile, and gradient elution is performed, with A linearly changing from 97% to 40% within 18-20 minutes, and B linearly changing from 3% to 60%. Within 1-2 minutes, A changes to 10%, and B changes to 90%, and the mixture is maintained for 4-7 minutes, then restored to the initial mobile phase, and equilibrated for 2-6 minutes.

[0069] High-resolution mass spectrometry detection conditions: mass analyzer: Orbitrap; ion source: electrospray ion source (ESI); scan mode: positive ion mode under full-scan mode (Full-MS); spray voltage (Ion Sray Voltage): 3.8 kV; ion transfer tube temperature: 320°C; lens voltage: 55 V; sheath gas (Sheath Gas): 7 psi; auxiliary gas (Aux Gas): 5 psi; auxiliary gas temperature: 300°C; scan range: 200-1500 m / z; primary mass spectrometry resolution R = 70000 FWHM; secondary mass spectrometry resolution R = 13500 FWHM; maximum dwell time is 50-100 ms.

[0070] S4. Analysis of characteristic peptides of different milk α-caseins

[0071] After enzymatic hydrolysis, the α-casein hydrolysate passed through a 0.22 μm filter membrane was subjected to ultra-high performance liquid chromatography high-resolution mass spectrometry detection in step S3 and step S4, and the detection data spectra were analyzed using Qual Browser of Xcalibur 3.0.63 (Thermo Fisher Scientific, USA) software.

[0072] The raw data generated by mass spectrometry analysis of the trypsin-digested α-casein digest were searched using workstation software (MaxQuant software). The Uniprot database included a bovine protein library (Bos taurus, Taxonomy ID: 9913), a goat protein library (Goat, Taxonomy ID: 9925), and a Swiss-Prot complete protein library. The minimum peptide length was set to 3, the search was set to Trpsin / p, the maximum number of missed cleavage sites was set to 2, the fixed modification was set to Carbamidomethyl (C), the variable modifications were set to Acetyl (Protein N-term) and Oxidation (M), and the false-positive discovery rate (FDR) was set to 1%. All other parameters were set to default values.

[0073] Based on a unified analytical method, characteristic peptides produced by trypsin hydrolysis of α-casein were screened, such as Figure 1 As shown in the figure, after trypsin digestion, characteristic peptides appeared at m / z 1267.70215, 850.42389, and 602.34882 for bovine milk α-casein; and at m / z 1109.52014, 704.36218, and 643.38245 for goat milk α-casein. These characteristic peptides exhibited distinct chromatographic peaks only in samples of the corresponding species under their respective mass-to-charge ratios, demonstrating their species specificity.

[0074] Furthermore, the identified whey protein peptides were analyzed by secondary mass spectrometry using an established multiple reaction monitoring (MRM) method. Using collision-induced dissociation (CID), two product ions with good peak shape and high response were selected from each characteristic peptide for signal acquisition. To improve detection sensitivity, the system optimized two key mass spectrometry parameters: declustering voltage and collision energy. The final optimized conditions are shown in Table 1.

[0075] Table 1 MRM parameters of characteristic peptides after α-casein hydrolysis in different milks

[0076]

[0077] Based on the established mass spectrometry data analysis process, the UniProt database was integrated with MaxQuant software to systematically analyze the characteristic peptide sequences of trypsin-hydrolyzed bovine milk α-casein and goat milk α-casein as shown in Table 2.

[0078] Table 2 Summary of characteristic peptide information of trypsin-hydrolyzed whey protein

[0079]

[0080] As can be seen from Table 2, the UPLC-Q / Exactive method can successfully identify the characteristics of α-casein in cow's milk and goat's milk, and obtain the amino acid sequence. This has played a positive role in the subsequent study of the secondary structure of α-casein and the revelation of active sites or functional domains in α-casein. UPLC-Q / Exactive analysis shows that the method provided in this embodiment can achieve specific identification of characteristic peptides of two milk-derived α-caseins, and their amino acid sequence matching degrees are both higher than 98%, providing a molecular-level structural basis for species identification. The precise analysis of these sequence information not only provides key data support for the study of the structure-activity relationship of the secondary structure of α-casein, but also reveals the structural basis for the formation of its functional diversity through the positioning of active sites.

[0081] In the above embodiment 1, α-casein concentration is selected to replace in the range of 1~10mg / mL, and the final concentration of trypsin can be selected to replace in the range of 4000~10000U / g; The temperature of enzyme inactivation is selected to replace in the range of 90~95 ℃, the time of enzyme inactivation is selected to replace in the range of 5min~15min, the rotating speed of centrifugation is selected to replace in the range of 4000rpm~4500rpm, the time of centrifugation is selected to replace in the range of 5min~10min, the temperature of standing is selected to replace in the range of 2~8 ℃, the temperature of initial centrifugation is selected to replace in the range of 2~8 ℃, the rotating speed of initial centrifugation is selected to replace in the range of 9500rpm~12000rpm, and the time of initial centrifugation is selected to replace in the range of 20min~40min. In step S2, the pH of water and NaOH solution in enzymolysis can be selected to replace in the range of 8.0~8.5, and the pH of enzymolysis can be selected to replace in the range of 8.0~8.5.

[0082] In the above Example 1, the injection volume of the ultra-high performance liquid chromatography was selected and replaced within the range of 2 μL to 5 μL.

[0083] In the above Example 1, the spray voltage in the high-resolution mass spectrometer is selected and replaced within the range of 3.2kV-3.8kV, the capillary temperature is selected and replaced within the range of 300℃-350℃; the auxiliary gas heating temperature is selected and replaced within the range of 300℃-350℃; the sheath gas pressure is selected and replaced within the range of 30arb-45arb, and the auxiliary gas pressure is selected and replaced within the range of 5arb-10arb.

[0084] Example 2

[0085] This example mainly utilizes the characteristic peptide segments of α-casein in bovine and goat milk screened in Example 1 to qualitatively identify bovine and goat milk.

[0086] This embodiment provides a method for qualitative detection of cow's milk and goat's milk with reference to Example 1.

[0087] Five samples were collected from each of cow's and goat's milk, and these ten samples were unlabeled except for random numbering. Ten blind dairy samples were defatted and then enzymatically hydrolyzed. The resulting α-casein enzymatic hydrolysate was then analyzed using the UPLC-Q / Exactive method established in Example 1 to determine the source of each sample.

[0088] Ten blind samples were tested according to the method of Example 1. After analysis of the characteristic α-casein peptide, it was found that samples 1, 3, 6, 8, and 10 were cow's milk, and samples 2, 4, 5, 7, and 9 were goat's milk.

[0089] Example 3

[0090] This example mainly uses the characteristic peptide segments of α-casein in bovine and goat milk screened in Example 1 to identify adulterated bovine milk in goat milk.

[0091] The goat milk was artificially adulterated with cow milk, and the adulteration was identified using the UPLC-Q / Exactive method for characteristic identification of special milk α-casein established in Example 1.

[0092] Adulterated milk was obtained by adulterating cow's milk with goat's milk at ratios of 0.5%, 1.0%, 2.0%, 5%, 10%, 20%, and 50% (v / v). The adulterated milk was centrifuged at 5500 rpm for 15 minutes at 4°C. The supernatant fat was discarded, and the small amount of casein precipitated at the bottom was mixed to obtain skim milk. 1 mL of skim milk was diluted to 5 mL with purified water and then enzymatically hydrolyzed. The α-casein enzymatic hydrolyzate was analyzed using the UPLC-Q / Exactive method established in Example 1 to determine whether the goat's milk was adulterated.

[0093] The results of adulteration identification of specialty milk based on the characteristic peptide of α-casein are shown in Table 3. It can be seen from Table 3 that the detection ratio of adulterated milk is 0.5%, which shows that the detection method established in Example 1 is feasible.

[0094] Table 3 Identification results of adulterated specialty milk based on α-casein characteristic peptides

[0095] matrix Add milk source m / z Detection ratio (%, v / v) goat milk milk 1267.70215、850.42389、602.34882 0.5

[0096] In this example, goat milk was adulterated and then tested and analyzed to verify that the established method can be applied to identify adulterated cow milk in goat milk.

[0097] Furthermore, the factors influencing the characteristic peptide of α-casein in Example 1 were studied, and the accuracy of the identification method of the present invention was also verified.

[0098] 1. Experiment on factors affecting the characteristic peptide of special milk α-casein

[0099] The effects of enzymatic heating temperature, enzymatic heating time and storage days on the stability of characteristic peptides of α-casein in special milk (cow's milk and goat's milk) were investigated respectively.

[0100] First, the effects of different heating temperatures and heating times on the stability of characteristic peptides of α-casein were investigated. In the experiment, α-casein with a substrate concentration of 3% (w / v) was placed at room temperature and 40°C, 60°C, 80°C, and 90°C for 30 minutes and then enzymatically hydrolyzed for 3 hours.

[0101] Next, α-casein samples were heated at 60°C for 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h, followed by 3 h of enzymatic hydrolysis. The hydrolyzed solutions were analyzed by UPLC-Q / Exactive assay.

[0102] Finally, the α-casein samples were placed at room temperature for 1, 3, 7, and 15 days. The samples with a substrate concentration of 3% (w / v) were enzymatically hydrolyzed for 3 hours and then subjected to UPLC-Q / Exactive analysis to explore the effect of storage days on the stability of the characteristic peptide segments of α-casein.

[0103] See also Figure 2 The results of the effect of different heating temperatures on the stability of the α-casein characteristic peptide segment show that at 80°C and above, the response value of the α-casein characteristic peptide segment decreases slightly. This is because α-casein begins to denature at this temperature. This shows that protein denaturation has a certain impact on the response value of the characteristic peptide segment, but the overall impact is not significant. At 80°C, the response value of α-casein is the highest.

[0104] from Figure 3 The results of the effect of different heating times on the stability of the characteristic peptide of α-casein show that the response value of the characteristic peptide of α-casein decreases to a certain extent with the extension of heating time. Overall, the response value of the characteristic peptide of α-casein changes after different degrees of heat treatment. However, this effect does not have a significant impact on the stability of the characteristic peptide. Even when the protein is denatured, the characteristic peptide can still remain stable, indicating that the characteristic peptide of α-casein has a certain resistance to denaturation and can, to a certain extent, overcome the effects of external environmental factors on its structure and function. This provides an important reference for further exploring the application of the characteristic peptide of α-casein in food processing and storage, and also provides new ideas and directions for further research in related fields.

[0105] from Figure 4The results of the effect of different storage days on the stability of α-casein characteristic peptides show that the response values ​​of the characteristic peptides of α-casein in cow's milk and goat's milk decreased significantly in the first 7 days, but did not change significantly from the 7th to the 15th day. This may be because in the first 7 days, α-casein may be denatured due to environmental factors (such as temperature, pH value, etc.), resulting in changes in its spatial structure, exposing the characteristic peptides, thereby affecting their response values. In addition, the growth and reproduction of microorganisms may also cause protein degradation, further affecting the content and response value of the characteristic peptides. As the storage time increases, the denaturation and degradation of the protein gradually reach equilibrium, and the growth of microorganisms is also inhibited, so the response value of the characteristic peptides does not change significantly. In addition, the experiment found that on the 15th day, several dairy products had already deteriorated in terms of sensory perception, but their characteristic peptides could still be detected, indicating that the characteristic peptides screened in the early stage may have high structural stability and can resist chemical or physical changes during the deterioration process to a certain extent.

[0106] Studying the effect of storage time on the stability of characteristic peptides can help optimize the processing of bovine and goat milk. For example, during dairy processing, processes such as heat treatment and fermentation may cause changes in characteristic peptides. By studying these changes, optimal processing conditions can be determined to maintain or enhance the nutritional and functional properties of specialty milks.

[0107] 2. Validation of the detection method for the characteristic identification of α-casein in special milk

[0108] The established UPLC-Q / Exactive method for the characterization of specialty milk α-caseins was validated. Standard working solutions were used to evaluate and verify the method's linearity, precision, limit of quantification (LOQ), and limit of detection (LOD).

[0109] Linearity was assessed using calibration curves of α-casein standard working solutions with substrate concentrations of 0.5%, 1.0%, 3.0%, 5.0%, 10%, and 15% (w / v). A series of standard solutions were selected according to the experimental parameters described above. The calibration curve, regression equation, correlation coefficient, and linear range for trypsin-digested bovine α-casein were calculated with the standard concentration c as the abscissa and the characteristic peptide response value I as the ordinate. The limits of detection (LOD) and quantification (LOQ) were calculated using signal-to-noise ratios ≥10 and ≥3, respectively. Intra-batch precision was assessed by calculating the relative standard deviations (RSDs / %) of the α-casein hydrolysates from the same batch of bovine and goat milk, with substrate concentrations of 1.0%, 5.0%, and 10% (w / v). The inter-batch precision of the method was analyzed by injecting α-casein hydrolysate with substrate concentrations of 1.0%, 5.0%, and 10% (w / v) into five batches of bovine and goat milk and calculating the relative standard deviation (RSD / %).

[0110] (1) Method linear range, detection limit, and quantification limit

[0111] Linearity was assessed using calibration curves of bovine α-casein standard working solutions with substrate concentrations of 0.5%, 1.0%, 3.0%, 5.0%, 10%, and 15% (w / v). Calibration curves were generated with the standard concentration c as the abscissa and the characteristic peptide response value I as the ordinate. LOD and LOQ were calculated using signal-to-noise ratios of ≥10 and ≥3, respectively. The regression equation for trypsin-digested bovine α-casein was I = 1.26 × 10 7 c-1.14×10 6 , correlation coefficient (R 2 ) is 0.9983, the linear range is 0.5-15 mg / mL, the detection limit is 0.02 mg / mL, the quantification limit is 0.07 mg / mL, and the standard curve is as follows Figure 5 shown.

[0112] (2) Precision

[0113] The intra-batch precision and inter-batch precision of the detection method for characteristic peptides of α-casein hydrolyzed by trypsin were also verified according to the method of this experiment.

[0114] Taking bovine α-casein as an example, the results are shown in Table 4.

[0115] Table 4 Precision of the method (n=5)

[0116] Precision <![CDATA[t R (min)]]> <![CDATA[t R (%RSD)]]> Response value (%RSD) Within batch 13.83 0.54 1.78 Batch 13.83 0.32 6.32

[0117] As can be seen from Table 4, this method has good reproducibility and stability and is suitable for testing different batches of dairy products.

[0118] The above are several relatively preferred implementation methods of the preparation method of the present invention, but they cannot be used as limitations on the technical solutions protected by the present invention. Any replacement solutions obtained by ordinary technicians in this field without making creative work based on the technical ideas of the present invention should fall within the scope of protection of the present invention.

Claims

1. A characteristic peptide segment for identifying α-casein in bovine and goat milk, characterized in that: The characteristic peptide segments include the characteristic peptide segments of bovine milk α-casein and the characteristic peptide segments of goat milk α-casein. The characteristic peptide segments of bovine milk α-casein are: CAGCPHCPDHWMGYGDHCYYFSVEK or EYSTAHLSRTLTLVEQIK; The characteristic peptide segment of goat milk α-casein is SLCQETCCSPSCCQTTCCRTTCYR.

2. A method for qualitatively identifying bovine and goat milk α-casein based on the characteristic peptide segment according to claim 1, characterized in that: The following steps are involved: S1. Extraction of α-casein α-casein is extracted from the special milk to be tested by using urea differential precipitation method; the special milk is cow's milk or goat's milk; S2, Enzymatic Hydrolysis Adding trypsin to α-casein for enzymatic hydrolysis, and then inactivating the enzyme, cooling, centrifuging and filtering to obtain an α-casein enzymatic hydrolysate; S3, ultra-high performance liquid chromatography-high resolution mass spectrometry detection The α-casein hydrolysate obtained in step S2 is sequentially subjected to ultra-performance liquid chromatography-high-resolution mass spectrometry to obtain a spectrum; S4. Qualitative identification The spectrum obtained in step S3 is analyzed using workstation software; the specialty milk to be tested is qualitatively identified based on the characteristic peptide segments at the peak positions on the spectrum.

3. The method for qualitatively identifying bovine and goat milk α-casein by a characteristic peptide segment according to claim 2, characterized in that: The specific process of extracting α-casein from special milk by urea differential precipitation method in step S1 is: S1.

1. Preparation of pure casein The pH value of the special milk is adjusted to 4.0-5.0 with acetic acid, and the crude casein is obtained by standing and initial centrifugation; and then the pure casein is obtained by washing and re-centrifugation; S1.2, dissolving pure casein in urea solution to obtain casein particles; The product is then washed with a urea solution, centrifuged and the precipitate is collected, dialyzed and dried to obtain powdered α-casein.

4. The method for qualitatively identifying α-casein in special milk using characteristic peptide segments according to claim 3, characterized in that: In the step S1.1, the standing is performed in a refrigerator at 2-8°C overnight; the initial centrifugation is performed at 2-8°C and 9500-12000 rpm for 20-40 minutes; the washing is performed in sequence with pure water, ethanol, and ethanol-petroleum ether; and the second centrifugation is performed at 3800-4500 rpm for 5-10 minutes.

5. The method for qualitatively identifying α-casein in special milk using characteristic peptide segments according to claim 2, characterized in that: The specific process of enzymatic hydrolysis in step S2 is: Water and NaOH solution are respectively added to powdered α-casein to a pH of 8.0-8.5, and ultrasonic treatment is performed until the concentration of α-casein is 1-10 mg / mL. Trypsin is added to a concentration of 4000-10000 U / g; then enzymatic hydrolysis is performed at 36-38° C. and pH 8.0-8.5; the enzyme is inactivated by heating at 90-95° C. for 5-15 minutes, and after cooling to room temperature, the mixture is centrifuged at 4000-4500 rpm for 5-10 minutes, and the supernatant is filtered through a 0.22 μm filter membrane.

6. The method for qualitatively identifying α-casein in special milk using characteristic peptide segments according to claim 2, characterized in that: In step S3, the conditions for ultra-high performance liquid chromatography are: C18 chromatographic column, stationary phase particle size 5 μm, inner diameter 2.0-3.0 mm, column length 100-150 mm; column temperature: 30-35°C; flow rate 0.2-0.3 mL / min; injection volume 2-5 μL; mobile phase: A is 0.1-0.2% formic acid-water solution (V:V), B is acetonitrile, gradient elution, A linearly changes from 97% to 40% in 18-20 minutes, B linearly changes from 3% to 60%, within 1-2 minutes, A changes to 10%, B changes to 90%, maintain for 4-7 minutes, then return to the initial mobile phase, and equilibrate for 2-6 minutes.

7. The method for qualitatively identifying α-casein in special milk using characteristic peptide segments according to claim 2, characterized in that: The high-resolution mass spectrometry conditions in step S3 are: Mass analyzer: Orbitrap; Ion source: electrospray ion source (ESI); scan mode: positive ion mode under full scan mode (Full-MS); spray voltage: 3.2kV-3.8kV; lens voltage 55V; capillary temperature 300℃-350℃; auxiliary gas heating temperature 300℃-350℃; sheath gas pressure is 30arb~45arb, auxiliary gas pressure is 5arb~10arb; scan range: 200~1500m / z; primary mass spectrometry resolution R=70000FWHM; secondary mass spectrometry resolution R=13500FWHM; maximum dwell time is 50ms-100ms.

8. The method for qualitatively identifying α-casein in special milk using characteristic peptide segments according to claim 2, characterized in that: In step S4, the qualitative identification of the specialty milk to be tested is specifically as follows: The parent ion m / z was 850.42289-850.42489, and the daughter ion m / z was 343.89255-343.89455 and 360.22196-360.22396, and the characteristic peptide CAGCPHCPDHWMGYGDHCYYFSVEK appeared, which was identified as bovine milk α-casein. At this time, the special milk was bovine milk; The parent ion m / z was 602.34782-602.34982, and the product ion m / z was 416.00108-416.00308, 318.22269-318.22469, and the characteristic peptide EYSTAHLSRTLTLVEQIK appeared, which was identified as bovine milk α-casein. At this time, the special milk was bovine milk; When the parent ion m / z is 1109.51914-1109.52114, the daughter ion m / z is 239.99964-240.00164, 808.22306-808.22506, and the characteristic peptide SLCQETCCSPSCCQTTCCRTTCYR appears, it is identified as goat milk α-casein, which indicates that the special milk is goat milk.

9. Use of the characteristic peptide segment according to claim 1 in qualitative identification of bovine milk α-casein and / or goat milk α-casein.

10. Use of the characteristic peptide segment according to claim 1 in identifying whether goat milk is adulterated with cow milk.

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