Characteristic peptide fragment for identifying alpha-casein of camel 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 camel 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 camel milk alpha-casein is PLHYLTIINPKK. According to the method, the characteristic peptide fragment of the alpha-casein of the camel milk is screened by combining enzymolysis, high performance liquid chromatography and high-resolution mass spectrometry, and then the characteristic peptide fragment is used for qualitatively identifying the camel milk, so that the accuracy of an identification result is high; further, the identification of the adulterated cow milk in the camel milk is 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 alpha-casein in camel milk, a qualitative identification method and an application thereof. Background Art

[0002] α-casein is one of the main casein subtypes in dairy products, accounting for 30% to 40% of the total casein. s1 -Casein and α s2 -A family of polymorphic proteins composed of caseins, with a molecular weight of approximately 23 to 25 kDa and a highly phosphorylated characteristic. In dairy products, α-casein interacts with other caseins (β-, κ-casein) and calcium ions through a micellar structure to form a stable colloidal system, which directly affects the rheological properties, thermal stability and digestibility of dairy products. There are significant differences in the amino acid sequence, phosphorylation sites and post-translational modifications of α-casein in dairy products from different species, which leads to the diversity of its functional properties (such as gelation and emulsification) and biological activities (such as antibacterial and immunomodulatory). Therefore, studying the characteristics of α-casein in different specialty milks not only helps to understand the evolution and functional adaptability of milk proteins, but also provides a theoretical basis for the development of differentiated dairy products.

[0003] Camel milk is known to be rich in vitamin C and contains significant amounts of essential unsaturated fatty acids, iron, and B vitamins, making it a highly nutritious food. Existing research into the hypoallergenic properties of camel milk α-casein has emerged as a potential avenue for the development of functional dairy products, making the identification of α-casein in camel milk a key area of ​​research. Furthermore, in the dairy market, the high added value of camel milk, as it contains three times more vitamin C than cow's milk, has also led to adulteration, such as the use of cow's milk as camel milk. Therefore, based on the specific characteristics of α-casein, the differences in the characteristics of α-casein in camel and cow's milk have been analyzed, resulting in the development of an efficient camel milk identification technology, which is crucial for ensuring the quality and safety of camel dairy products.

[0004] Currently, the main methods for detecting α-casein in dairy products include enzyme-linked immunosorbent assay (ELISA) and chromatography. For example, patent document CN110018311A discloses an immunoassay kit and its application for detecting adulterated camel milk. This kit primarily uses an indirect ELISA method to qualitatively or quantitatively detect the content of bovine milk β-lactoglobulin, camel milk α-lactalbumin, bovine milk κ-casein, or camel milk αs1-casein in camel milk, thereby identifying whether the camel milk is adulterated. The results showed that the minimum detection limit for adulterated camel milk was 5%. However, due to protein denaturation and inactivation during dairy processing, chromatography methods such as HPLC are unable to detect the target protein, and ELISA methods can produce false positives, resulting in low detection precision for camel milk α-casein and bovine milk α-casein, poor test result accuracy, and difficulty in detecting adulteration.

[0005] Therefore, establishing an accurate and efficient method to specifically identify α-casein in cow's milk and camel's milk, and on this basis, to realize adulteration detection in camel's milk is an important research direction at present. Summary of the Invention

[0006] In view of the technical problems in the existing detection of α-casein in cow and camel 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 for identifying cow and camel milk α-casein, a qualitative identification method and application.

[0007] The present invention screens out characteristic peptide segments of α-casein in camel milk by combining enzymatic hydrolysis, high-performance liquid chromatography and high-resolution mass spectrometry, and then uses the characteristic peptide segments to perform qualitative identification of camel milk, with high accuracy of the identification result; thereby, identification of adulterated cow milk in camel milk is achieved.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A method for identifying characteristic peptide segments of α-casein in cow and camel milk, wherein the characteristic peptide segments include characteristic peptide segments of α-casein in cow milk and characteristic peptide segments of α-casein in camel milk.

[0010] The characteristic peptide segment of bovine milk α-casein is: CAGCPHCPDHWMGYGDHCYYFSVEK or EYSTAHLSRTLTLVEQIK;

[0011] The characteristic peptide of camel milk α-casein is PLHYLTIINPKK.

[0012] A method for qualitatively identifying camel milk α-casein based on the characteristic peptide segment, characterized by comprising the following steps:

[0013] S1. Extraction of α-casein

[0014] α-casein is extracted from the special milk to be tested by using a urea differential precipitation method; the special milk is cow's milk or camel's milk;

[0015] S2, Enzymatic Hydrolysis

[0016] Adding trypsin to α-casein for enzymatic hydrolysis, and then inactivating the enzyme, cooling, centrifuging and filtering to obtain an α-casein enzymatic hydrolysate;

[0017] S3, ultra-high performance liquid chromatography-high resolution mass spectrometry detection

[0018] The α-casein hydrolysate obtained in step S2 is sequentially subjected to ultra-performance liquid chromatography-high-resolution mass spectrometry to obtain a spectrum;

[0019] S4. Qualitative identification

[0020] The spectrum obtained in step S3 was analyzed using workstation software; the specialty milk to be tested was qualitatively identified based on the peak positions of characteristic peptide segments on the spectrum.

[0021] It is further defined that the specific process of extracting α-casein from special milk by urea differential precipitation method in step S1 is:

[0022] S1.1. Preparation of pure casein

[0023] The pH value of the specialty milk is adjusted to 4.4-4.7 with acetic acid, and the crude casein is obtained by standing and initial centrifugation; then the pure casein is obtained by washing and re-centrifugation;

[0024] S1.2. Pure casein is dissolved in a urea solution to obtain casein particles; the particles are then washed with a urea solution, centrifuged and the precipitate is collected, and then dialyzed and dried to obtain powdered α-casein.

[0025] 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 9000rpm-10000rpm for 20min-40min; washing is performed 3 times with pure water; and the second centrifugation is to be centrifuged at 4000rpm-4500rpm for 5min-10min.

[0026] It is further defined that the specific process of enzymatic hydrolysis in step S2 is:

[0027] 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 α-casein concentration is 1-15 mg / mL. Trypsin is added to a concentration of 4000-12000 U / g; then enzymatic hydrolysis is performed at 36-38°C and a pH of 8.0-8.5; heating is performed at 90-100°C for 10-15 minutes and formic acid is added to inactivate the enzyme. 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.

[0028] It is further defined that in step S3, the conditions for ultra-high performance liquid chromatography are:

[0029] C18 chromatographic column, fixed 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 a 0.1-0.2% formic acid-water solution (V:V) containing 4-5 mmol / L ammonium formate, B is acetonitrile, gradient elution, A linearly changes from 97% to 40% in 28-30 minutes, B linearly changes from 3% to 60%, within 0.5-1.5 minutes, A changes to 10%, B changes to 90%, maintain for 3-5 minutes, then return to the initial mobile phase, and equilibrate for 5-8 minutes.

[0030] It is further defined that the high-resolution mass spectrometry conditions in step S3 are:

[0031] Mass analyzer: Orbitrap; ion source: electrospray ion source ESI; scan mode: positive ion mode in full scan mode; spray voltage is 3.0kV-3.8kV; lens voltage is 55V; capillary temperature is 300℃-350℃; auxiliary gas heating temperature is 300℃-350℃; sheath gas pressure is 30arb~45arb, auxiliary gas pressure is 5arb~10arb; scan range: 200m / z~1500m / z; primary mass spectrometry resolution R=70000FWHM; secondary mass spectrometry resolution R=13500FWHM; maximum dwell time is 50ms-100ms.

[0032] It is further defined that in step S4, the qualitative identification of the specialty milk to be tested is specifically as follows:

[0033] When the parent ion m / z is 850.42289-850.42489 and the daughter ion m / z is 343.89255-343.89455 and 360.22196-360.22396, and the characteristic peptide CAGCPHCPDHWMGYGDHCYYFSVEK appears, it indicates that it is bovine milk α-casein. In this case, the special milk is bovine milk.

[0034] When the parent ion m / z is 602.34782-602.34982 and the product ion m / z is 416.00108-416.00308 and 318.22269-318.22469, and the characteristic peptide EYSTAHLSRTLTLVEQIK appears, it indicates bovine milk α-casein. In this case, the special milk is bovine milk.

[0035] When the parent ion m / z is 1046.06101-1046.06301 and the daughter ion m / z is 664.44907-664.45107 and 518.00126-518.00326, and the characteristic peptide PLHYLTIINPKK appears, it indicates camel milk α-casein. In this case, the special milk is camel milk.

[0036] The characteristic peptide segment is used in the qualitative identification of bovine milk α-casein and / or camel milk α-casein.

[0037] The characteristic peptide segment is used to identify adulterated cow milk in camel milk.

[0038] The characteristic peptide segment is used in the qualitative identification of bovine milk α-casein and / or camel milk α-casein.

[0039] Application of characteristic peptides in identifying adulterated cow milk in camel milk.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. The present invention screens out characteristic peptides of α-casein in cow and camel milk by combining enzymatic hydrolysis, high-performance liquid chromatography, and high-resolution mass spectrometry, and then uses the characteristic peptides to perform qualitative identification of cow's milk and camel milk, with high accuracy of the identification results. In addition, the characteristic peptides are used to identify adulteration of cow's milk and camel milk, with high reliability of the identification results.

[0042] 2. The present invention improves the accuracy of 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 sensitivity.

[0043] 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, with a wide range of applications.

[0044] 4. The present invention was verified by adulterating camel milk with cow's milk as an example. The detection rate of adulterated milk was 0.5%, indicating that it is feasible to use the screened characteristic peptides to realize the adulteration identification of camel milk with cow's milk, thereby realizing the accurate identification and detection of camel milk adulteration and ensuring the quality of camel milk. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The chromatograms of characteristic peptides of α-casein from trypsin-hydrolyzed cow milk (a, b, c) and camel milk (d, e, f) are shown;

[0046] Figure 2 The effect of heating temperature on α-casein;

[0047] Figure 3 The effect of heating time on α-casein;

[0048] Figure 4 The effect of storage days on α-casein;

[0049] Figure 5 The standard curve is for trypsin-digested bovine α-casein standard. DETAILED DESCRIPTION

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] The technical solution provided by the present invention is described in detail below.

[0055] 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.

[0056] 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.

[0057] Example 1

[0058] This embodiment provides a characteristic peptide segment for identifying α-casein from bovine and camel milk, wherein the characteristic peptide segment includes a characteristic peptide segment from bovine milk α-casein and a characteristic peptide segment from camel milk α-casein.

[0059] In this embodiment, the characteristic peptide segments of camel milk α-casein were screened out by the following method, which specifically includes the following steps:

[0060] S1. α-casein extraction

[0061] S1.1. Preparation of pure casein

[0062] 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 9800 rpm at 4°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, each time centrifuging at 4200 rpm for 10 minutes and discarding the upper liquid to obtain pure casein.

[0063] In this step, the dairy products are cow's milk and camel's milk, the target pH value of cow's milk is 4.6, and the target pH value of camel's milk is 4.4; the obtained pure caseins are cow's milk pure casein and camel's milk pure casein.

[0064] S1.2. Extraction of α-casein using urea differential precipitation method

[0065] The purified casein obtained above was dissolved in a 6.6M urea solution, diluted to 4.63M with water, and centrifuged at 4200 rpm for 10 minutes to collect the casein particles. It was then redissolved in a 6.6M urea solution containing 0.18M NaCl, diluted to 4.63M with water, and centrifuged at 4200 rpm for 10 minutes to collect the casein particles. Finally, it was washed once in a 4.7M urea solution and centrifuged at 4200 rpm for 10 minutes to collect the precipitate, which is α-casein. The extracted α-casein was dialyzed in pure water for 24 hours, then dried in a vacuum drying oven at 80°C for 1 hour to obtain α-casein powder (or freeze-dried for 12 hours), which was stored in a refrigerator at 4°C.

[0066] In this step, the pure casein is respectively cow's milk pure casein and camel's milk pure casein, and thus the obtained α-casein powders are respectively cow's milk α-casein powder and camel's milk α-casein powder.

[0067] S2, Enzymatic Hydrolysis

[0068] Weigh 150 mg (accurate to 0.1 mg) of the α-casein powder of step S1, dissolve it in ultrapure water and heat it to 40°C for solubilization, adjust the volume to 10 mL, and add a certain volume of trypsin solution after ultrasonication for 15 min until the final concentration of trypsin is reached. At 37°C, pH = 8.0, α-casein is enzymatically hydrolyzed, and then 200 μL of formic acid is added after heating at 90°C for 10 min to inactivate the enzyme. After cooling to room temperature, centrifuge at 4200 rpm for 5 min, and the supernatant is filtered through a 0.22 μm filter membrane to obtain an α-casein hydrolyzate, which is then used for ultra-performance liquid chromatography-high-resolution mass spectrometry detection.

[0069] Optimally, the species-specific process parameters for trypsin digestion of α-casein are: for the bovine milk system, the optimal response is achieved at a substrate concentration of 5%, an enzyme dosage of 7800 U / g, and a digestion time of 2 hours; for the camel milk system, a substrate concentration of 2.5% and an enzyme activity of 6300 U / g are required for 5 hours. The bovine milk system exhibits significant short-term high efficiency, while the camel milk system relies on extended digestion time to maximize peptide release. This difference in time dimension may be due to the fundamental differences in protein conformational stability between species.

[0070] S3, ultra-high performance liquid chromatography-high resolution mass spectrometry detection

[0071] The α-casein hydrolysate obtained in step S2 is sequentially subjected to ultra-performance liquid chromatography-high-resolution mass spectrometry to obtain a spectrum;

[0072] Ultra-high performance liquid chromatography test conditions are:

[0073] C18 chromatographic column, fixed 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 a 0.1-0.2% formic acid-water solution (V:V) containing 4-5 mmol / L ammonium formate, B is acetonitrile, gradient elution, A linearly changes from 97% to 40% in 28-30 minutes, B linearly changes from 3% to 60%, within 0.5-1.5 minutes, A changes to 10%, B changes to 90%, maintain for 3-5 minutes, then return to the initial mobile phase, and equilibrate for 5-8 minutes.

[0074] High-resolution mass spectrometry detection:

[0075] Mass spectrometry 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.

[0076] S4. Analysis of characteristic peptides of different milk α-casein

[0077] 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.

[0078] The raw data generated by mass spectrometry analysis of the trypsin-digested α-casein digest were searched using workstation software (MaxQuant software, version 2.5.2.0). The Uniprot database was used to search the bovine protein library (Bostaurus, Taxonomy ID: 9913), the camel protein library (Bactrian camel, Taxonomy ID: 9837), and the 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), the false-positive discovery rate (FDR) was set to 1%, and all other parameters were set to default values.

[0079] Based on a unified analytical method, characteristic peptides produced by trypsin hydrolysis of α-casein were screened, such as Figure 1As 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 1046.06201, 718.93469, and 700.42242 for camel 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.

[0080] Furthermore, the identified 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.

[0081] Table 1 MRM parameters of characteristic peptides after α-casein hydrolysis in different milks

[0082]

[0083] 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 camel milk α-casein as shown in Table 2.

[0084] Table 2 Summary of characteristic peptide information of trypsin-digested proteins

[0085]

[0086] As shown in Table 2, the UPLC-Q / Exactive method successfully characterized and identified α-casein in both cow and camel milk, and its amino acid sequence was obtained. This has played a positive role in subsequent studies of α-casein secondary structure and the identification of active sites or functional domains within α-casein.

[0087] UPLC-Q / Exactive analysis demonstrated that the method provided in this example can specifically identify characteristic peptides from two milk-derived α-caseins, with amino acid sequence matches exceeding 98%, providing a molecular-level structural basis for species identification. The precise analysis of this sequence information not only provides key data support for the study of the structure-activity relationship of α-casein secondary structure but also reveals the structural basis for its functional diversity through the mapping of active sites.

[0088] In the above Example 1, acetic acid is used to adjust the pH of the specialty milk to be within the range of 4.4 to 4.7.

[0089] In the above Example 1, the temperature of the refrigerator standstill is selected and replaced within the range of 2 to 8°C, the temperature of the initial centrifugation is selected and replaced within the range of 2 to 8°C, the speed of the initial centrifugation is selected and replaced within the range of 9000 rpm to 10000 rpm, the time of the initial centrifugation is selected and replaced within the range of 20 min to 40 min, the speed of the secondary centrifugation is selected and replaced within the range of 4000 rpm to 4200 rpm, and the time of the secondary centrifugation is selected and replaced within the range of 5 min to 10 min.

[0090] In the above Example 1, during the enzymatic hydrolysis process, the α-casein concentration is selected and replaced within the range of 1 to 15 mg / mL, the pH values ​​of water and NaOH solution are selected and replaced within the range of 8.0 to 8.5, and the final concentration of trypsin can be selected and replaced within the range of 4000 to 12000 U / g; the enzymatic hydrolysis temperature is selected and replaced within the range of 36 to 38° C., the enzymatic hydrolysis pH is selected and replaced within the range of 8.0 to 8.5, the enzyme inactivation temperature is selected and replaced within the range of 90 to 100° C., the enzyme inactivation time is selected and replaced within the range of 10 min to 15 min, the centrifugal speed is selected and replaced within the range of 4000 rpm to 4500 rpm, and the centrifugal time is selected and replaced within the range of 5 min to 10 min.

[0091] 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.

[0092] In the above Example 1, the spray voltage in the high-resolution mass spectrometer is selected and replaced within the range of 3.0 kV-3.8 kV, the capillary temperature is selected and replaced within the range of 300°C-350°C; the auxiliary gas heating temperature is selected and replaced within the range of 300°C-350°C; the sheath gas pressure is selected and replaced within the range of 30 to 45 arb, and the auxiliary gas pressure is selected and replaced within the range of 5 to 10 arb.

[0093] Example 2

[0094] Based on the characteristic peptides screened out in Example 1, this example provides a method for qualitatively identifying camel milk α-casein based on the characteristic peptides.

[0095] The method for qualitatively identifying camel milk α-casein based on characteristic peptide segments refers to Example 1.

[0096] Five samples were collected from each of cow's and camel's milk. These ten samples were unlabeled except for random numbering. Ten blind dairy samples were defatted and then enzymatically hydrolyzed. The resulting α-casein hydrolysates were analyzed using the UPLC-Q / Exactive method established in Example 1 to determine the origin of each sample.

[0097] Ten blind samples were tested according to the method of Example 1. After analysis of the characteristic peptide of α-casein, it was found that samples 1, 2, 4, 8, and 9 were cow's milk, and samples 3, 5, 6, 7, and 10 were camel milk.

[0098] Example 3

[0099] Based on the characteristic peptides screened out in Example 1, this example uses the characteristic peptides to identify adulteration of cow's milk and camel's milk.

[0100] Camel milk was artificially adulterated by adding cow milk into camel milk at a ratio of 0.5%, 1.0%, 2.0%, 5%, 10%, 20% and 50% (v / v) to obtain adulterated milk.

[0101] Adulteration identification was performed using the UPLC-Q / Exactive method for identifying the α-casein signature of specialty milk, established in Example 1. Specifically, the adulterated milk was centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant fat was discarded, and the small amount of casein precipitate at the bottom was mixed to obtain skim milk. 2 mL of skim milk was then diluted to 10 mL with pure water and enzymatically hydrolyzed. The α-casein hydrolyzate was then analyzed using the established UPLC-Q / Exactive method to determine whether the camel milk was adulterated with cow milk.

[0102] The results of adulteration identification of specialty milk based on the characteristic peptide of α-casein are shown in Table 3.

[0103] Table 3 Identification results of adulterated specialty milk based on α-casein characteristic peptides

[0104] matrix Add milk source m / z Detection ratio (%, v / v) camel milk milk 1267.70215、850.42389、602.34882 0.5

[0105] From Table 3, it can be seen that the detection rate of adulterated milk is 0.5%, which shows that the detection method established in Example 1 is feasible. It shows that the method provided in Example 1 can be applied to the identification of adulteration of camel milk.

[0106] In order to illustrate the technical advantages of the identification method of the present invention, the stability of the characteristic peptide segment of α-casein and the accuracy of the method of the present invention were further verified.

[0107] 1. Experiment on factors affecting the screening of α-casein characteristic peptides

[0108] The effects of enzymatic heating temperature, enzymatic heating time and storage days on the stability of α-casein characteristic peptides in special milk (cow's milk and camel's milk) were investigated respectively.

[0109] 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 was heated at room temperature and 40°C, 60°C, 80°C, and 90°C for 30 minutes and then enzymatically hydrolyzed for 5 hours.

[0110] Next, α-casein samples were heated at 80°C for 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h, followed by 5 h of enzymatic hydrolysis. The hydrolyzates were then analyzed using UPLC-Q / Exactive.

[0111] 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.

[0112] See also Figure 2 The effect of different heating temperatures on enzymatic peptides shows that at 80°C and higher, the response values ​​of α-casein characteristic peptides decrease slightly. This is because α-casein begins to denature at this temperature, indicating that protein denaturation has a certain effect on the response value of characteristic peptides, but generally speaking, the effect is not significant.

[0113] See also Figure 3 The effects of different enzymatic heating times on enzymatic peptides indicate that the response value of the characteristic α-casein peptide decreases with increasing enzymatic heating time. Overall, the response value of the characteristic α-casein peptide changes after varying degrees of heat treatment. However, this effect does not significantly affect the stability of the characteristic peptide. Even when the protein is denatured, the characteristic peptide remains stable, indicating that the characteristic α-casein peptide possesses a certain degree of 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 characteristic α-casein peptides in food processing and storage, and also offers new ideas and directions for further research in related fields.

[0114] from Figure 4The effect of storage days on characteristic peptides shows that the response values ​​of the characteristic peptides of α-casein in cow's milk and camel'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 microorganisms, light, etc.), resulting in changes in its spatial structure, exposing the characteristic peptides and thus affecting their response values. 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 values ​​of the characteristic peptides do 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.

[0115] Since heat treatment, fermentation and other processes may lead to changes in characteristic peptides during dairy processing, the effects of the above-mentioned enzymatic hydrolysis heating temperature, enzymatic hydrolysis heating time and storage days on characteristic peptides can help optimize the processing technology of cow's milk and camel milk, and determine the optimal processing conditions to maintain or enhance the nutritional and functional properties of cow's milk and camel milk.

[0116] 2. Validation of the identification method of characteristic peptides of bovine milk α-casein and camel milk α-casein

[0117] The UPLC-Q / Exactive method for identifying characteristic peptides of camel milk α-casein established in Example 1 was validated.

[0118] The linearity, precision, limit of quantification (LOQ), and limit of detection (LOD) of the analytical method were evaluated and validated using standard working solutions. Specifically, 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 standard curves, regression equations, correlation coefficients, and linear ranges for trypsin-digested bovine α-casein were generated, 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 of ≥10 and ≥3, respectively. Intra-batch precision was assessed by calculating the relative standard deviations (RSD / %) of the α-casein hydrolysates from the same batch of bovine and camel milk, with substrate concentrations of 1.0%, 5.0%, and 10% (w / v), with five consecutive injections. 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 cow's milk and camel's milk and calculating the relative standard deviation (RSD / %).

[0119] (1) Method linear range, detection limit, and quantification limit

[0120] The linearity was evaluated using a calibration curve of bovine α-casein standard working solution with substrate concentrations of 0.5%, 1.0%, 3.0%, 5.0%, 10% and 15% (w / v). The standard curves were obtained with the standard concentration c as the abscissa and the characteristic peptide response value I as the ordinate. The LOD and LOQ were calculated using a signal-to-noise ratio of ≥10 and ≥3, respectively.

[0121] The regression equation for trypsin hydrolysis of bovine α-casein is: I = 1.26 × 10 7 c-1.14×10 6 .

[0122] 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.

[0123] (2) Precision

[0124] 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.

[0125] Taking camel milk α-casein as an example, the results are shown in Table 4.

[0126] Table 4 Precision of the method (n=5)

[0127] Precision <![CDATA[t R (min)]]> <![CDATA[t R (%RSD)]]> Response value (%RSD) Within batch 9.25 0.57 2.68 Batch 9.25 0.92 7.37

[0128] As can be seen from Table 4, this method has good reproducibility and stability and is suitable for testing different batches of dairy products.

[0129] 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 for identifying α-casein in camel milk, characterized in that: The characteristic peptides include the characteristic peptides of bovine milk α-casein and camel milk α-casein. The characteristic peptide segment of bovine milk α-casein is: CAGCPHCPDHWMGYGDHCYYFSVEK or EYSTAHLSRTLTLVEQIK; The characteristic peptide of camel milk α-casein is PLHYLTIINPKK.

2. A method for qualitatively identifying α-casein in camel milk 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 a urea differential precipitation method; the special milk is cow's milk or camel'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 was analyzed using workstation software; the specialty milk to be tested was qualitatively identified based on the peak positions of characteristic peptide segments on the spectrum.

3. The method for qualitatively identifying camel milk α-casein by using characteristic peptide segments according to claim 1, 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 specialty milk is adjusted to 4.4-4.7 with acetic acid, and the crude casein is obtained by standing and initial centrifugation; 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 2, 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 9000-10000 rpm for 20-40 minutes; the washing is performed three times with pure water; and the secondary centrifugation is performed at 4000-4200 rpm for 5-10 minutes.

5. The method for qualitatively identifying α-casein in special milk using characteristic peptide segments according to claim 3, 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 α-casein concentration is 1-15 mg / mL. Trypsin is added to a concentration of 4000-12000 U / g; then, enzymatic hydrolysis is performed at 36-38°C and a pH of 8.0-8.5, and the mixture is heated at 90-100°C for 10-15 minutes and formic acid is added to inactivate the enzyme. 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 5, characterized in that: In step S3, the conditions for ultra-high performance liquid chromatography are: C18 chromatographic column, fixed 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 a 0.1-0.2% formic acid-water solution (V:V) containing 4-5 mmol / L ammonium formate, B is acetonitrile, gradient elution, A linearly changes from 97% to 40% in 28-30 minutes, B linearly changes from 3% to 60%, within 0.5-1.5 minutes, A changes to 10%, B changes to 90%, maintain for 3-5 minutes, then return to the initial mobile phase, and equilibrate for 5-8 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 in full scan mode; spray voltage is 3.0kV-3.8kV; lens voltage is 55V; capillary temperature is 300℃-350℃; auxiliary gas heating temperature is 300℃-350℃; sheath gas pressure is 30arb~45arb, and auxiliary gas pressure is 5arb~10arb; scan range: 200m / z~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: When the parent ion m / z is 850.42289-850.42489 and the daughter ion m / z is 343.89255-343.89455 and 360.22196-360.22396, and the characteristic peptide CAGCPHCPDHWMGYGDHCYYFSVEK appears, it indicates that it is bovine milk α-casein. In this case, the special milk is bovine milk. When the parent ion m / z is 602.34782-602.34982 and the product ion m / z is 416.00108-416.00308 and 318.22269-318.22469, and the characteristic peptide EYSTAHLSRTLTLVEQIK appears, it indicates bovine milk α-casein. In this case, the special milk is bovine milk. When the parent ion m / z is 1046.06101-1046.06301 and the daughter ion m / z is 664.44907-664.45107 and 518.00126-518.00326, and the characteristic peptide PLHYLTIINPKK appears, it indicates camel milk α-casein. In this case, the special milk is camel milk.

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

10. Use of the characteristic peptide segment according to claim 1 in identifying adulterated cow milk in camel milk.

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