Quality control method of corn oligopeptide and application thereof

Abstract

The invention provides a corn oligopeptide quality control method and application thereof, and relates to the technical field of oligopeptide quality control methods. The corn oligopeptide quality control method comprises the following steps: (A) preparing an alanine-tyrosine peptide standard substance solution and a corn oligopeptide sample solution to be detected; (B) performing component separation on the standard substance solution and the to-be-detected sample solution by using liquid chromatography to obtain an effluent component; and (C) carrying out mass spectrometric detection on an effluent component obtained after the standard substance and the sample to be detected are subjected to liquid chromatography separation, and carrying out qualitative and quantitative analysis on the alanine-tyrosine peptide according to mass spectrometric data. According to the method, alanine-tyrosine peptide with a high composition ratio in the corn oligopeptide is used as a characteristic peptide to establish a quality control method, a liquid chromatography-mass spectrometry analysis technology is adopted to carry out quality control and quality identification on a corn oligopeptide raw material, and compared with traditional evaluation modes such as peptide molecular weight distribution and hydrolysis degree, the quality control method has the advantages that the quality control method is established; the quality control and quality identification specificity is greatly enhanced, and the analysis efficiency is greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of oligopeptide quality control methods, in particular to a corn oligopeptide quality control method and application thereof. Background Art

[0002] Corn oligopeptides mainly refer to a low molecular weight mixture obtained after corn protein is degraded by enzyme catalysis, fermentation or strong acid. It has attracted much attention from scientific researchers and consumers because of its multiple physiological activities in sobering up, protecting the liver, lowering blood pressure, etc. At present, the quality analysis and control of corn oligopeptide raw materials and products are mainly carried out by monitoring the degree of hydrolysis, molecular weight distribution, amino acid composition, etc. of the enzymatic hydrolyzate. However, since the methods such as degree of hydrolysis, amino acids, and molecular weight can only indirectly or macroscopically describe the macroscopic changes or differences of corn oligopeptides, and cannot directly reflect the composition or changes of the enzymatic hydrolyzate at the polypeptide molecular level, the index homogeneity phenomenon is relatively serious between corn oligopeptides obtained by different processes and even oligopeptides from different plant raw materials, and it is difficult to effectively monitor the stability and reliability of raw material supply or production quality. In addition, the technical threshold for counterfeiting and shoddy raw materials is low, and vicious competition is prevalent, which has caused serious obstacles to the development of the industry.

[0003] In recent years, domestic researchers have found that corn protein hydrolysates generally contain a high level of alanine-tyrosine peptides (AY) through the separation and purification of corn protein hydrolysates. Therefore, some manufacturers have begun to use liquid chromatography to detect AY to strengthen the quality control of corn oligopeptides. However, due to the extremely complex composition of corn protein hydrolysates, which often contain hundreds, thousands or even tens of thousands of peptides, the separation capacity and separation degree of liquid chromatography are often significantly insufficient, and there are easily multiple co-eluting products, resulting in deviations in the test results, and the quality detection and specificity are obviously insufficient. In addition, the analysis time is relatively long, which is not conducive to the healthy and rapid development of the industry.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The first purpose of the present invention is to provide a corn oligopeptide quality control method based on alanine-tyrosine peptide detection and analysis, which mainly uses liquid chromatography-mass spectrometry analysis technology to determine the content of alanine-tyrosine peptide, thereby realizing quality identification and monitoring of corn oligopeptide raw materials from different sources and different batches.

[0006] The second object of the present invention is to provide a method for quality control of corn oligopeptides and its application in quality identification of corn oligopeptides.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] According to one aspect of the present invention, a quality control method for corn oligopeptides comprises:

[0009] (A) preparing alanine-tyrosine peptide standard solution;

[0010] and, a test sample solution of corn oligopeptide;

[0011] (B) separating the components of the standard solution and the sample solution by liquid chromatography to obtain effluent components;

[0012] (C) The effluent components of the standard and the sample to be tested after separation by liquid chromatography are subjected to mass spectrometry detection, and the alanine-tyrosine peptide is qualitatively and quantitatively analyzed based on the mass spectrometry data.

[0013] The present invention provides a method for quality control of corn oligopeptides. The method is established with alanine-tyrosine peptides having a relatively high composition ratio in corn oligopeptides as characteristic peptides, and liquid chromatography-mass spectrometry analysis technology is used to perform quality control and quality identification on corn oligopeptide raw materials. Compared with traditional evaluation methods such as peptide molecular weight distribution and hydrolysis degree, the specificity of quality control and quality identification is greatly enhanced, and the analysis efficiency is greatly improved.

[0014] In a preferred embodiment of the present invention, the purity of the alanine-tyrosine peptide in the alanine-tyrosine peptide standard solution is ≥95% and does not contain saline components.

[0015] Preferably, the above-mentioned alanine-tyrosine peptide is obtained by artificial chemical synthesis.

[0016] In a preferred embodiment of the present invention, the mass concentration of the alanine-tyrosine peptide standard solution is 0.5-10 μg / mL.

[0017] Preferably, the method for preparing the alanine-tyrosine peptide standard solution comprises: accurately weighing a certain amount of alanine-tyrosine peptide (abbreviated as AY) standard respectively, and using ultrapure water to prepare a series of standard solutions with a concentration range of 0.5 to 10 μg / mL.

[0018] In a preferred embodiment of the present invention, the method for preparing the corn oligopeptide test sample solution comprises:

[0019] The corn oligopeptide sample was dispersed in ultrapure water and homogenized, and then centrifuged to remove the insoluble precipitate; then an equal volume of organic solvent was added to stand for precipitation, and the precipitate was removed by centrifugation again, and the supernatant was taken and filtered to obtain the corn oligopeptide sample solution to be tested.

[0020] Preferably, the method for preparing the test sample solution of corn oligopeptide comprises: accurately weighing the test sample after grinding or not grinding, dispersing it in ultrapure water, using a high shear disperser to quickly homogenize, configuring it into a test sample solution of appropriate concentration, stirring or shaking at a constant temperature of 35-55°C for 10-30 minutes, centrifuging to remove insoluble precipitates, taking a certain amount of supernatant and adding an equal volume of organic solvent, standing for 5-10 minutes to precipitate macromolecular proteins and other substances, centrifuging again to remove the precipitate, taking the supernatant and passing it through a membrane to obtain the test sample solution of corn oligopeptide.

[0021] In the above preferred embodiment, the mass concentration of the corn oligopeptide sample solution to be tested is 10-4000 μg / mL.

[0022] In the above preferred embodiment, the organic solvent is methanol or acetonitrile;

[0023] In the above preferred embodiment, the filter membrane is a 0.22 μm organic filter membrane.

[0024] In a preferred embodiment of the present invention, the liquid chromatography in step (B) is a liquid chromatography with a pressure resistance of ≥ 600 bar;

[0025] The detection conditions of the liquid chromatography are:

[0026] The chromatographic analysis flow rate is 0.1-0.4 mL / min, the column temperature is 30°C; the inner diameter of the chromatographic column is 1.0 or 2.1 mm, the length is 50-100 mm, and the filler particle diameter is 1.8 μm;

[0027] The mobile phase consists of mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution containing 0.05-0.3% formic acid, and mobile phase B is an acetonitrile solution;

[0028] The elution gradient is: 0-1.00min 5.0% B, 1.00-1.50min 5.0-10.0% B, 1.50-7.50min 10.0-40.0% B, 7.50-8.50min 40.0-90.0% B, 8.50-10.00min 90% B, 10.00-10.50min 90.0-5.0% B, 10.50-12.00min, 5.0% B.

[0029] In a preferred embodiment of the present invention, the mass spectrometer in step (C) is at least one of a triple quadrupole mass spectrometer, a quadrupole-time-of-flight mass spectrometer or a quadrupole-ion trap mass spectrometer detector;

[0030] The mass spectrometer is equipped with an ESI electrospray ion source, and the ion source working mode is a positive ion mode; the mass spectrometer detection mode is an MRM multiple reaction ion monitoring mode, the primary precursor ion is set to 253.1m / z, the secondary fragmentation ion detection is set to 182.2 and 207.1m / z, and the scanning frequency is 50-1000Da / s; the secondary fragmentation energy of the AY precursor ion is 15eV.

[0031] As a preferred embodiment, the present application uses the above-mentioned mass spectrometry monitoring and analysis method to perform quality control and monitoring of corn oligopeptides from different sources and different batches of raw materials. The analysis time is only 12 minutes, which is efficient and fast. The target monitoring ion pairs have strong characteristic, good anti-interference ability, high accuracy, good reproducibility, simple operation and outstanding advantages.

[0032] In a preferred embodiment of the present invention, the method for qualitative analysis of alanine-tyrosine peptides in step (C) is:

[0033] The theoretical plate number of the standard is ≥8000, two secondary monitoring ions are detected in the sample to be tested at the same time and the signal-to-noise ratio of the chromatographic peak is greater than 3.

[0034] In a preferred embodiment of the present invention, the method for quantitative analysis of alanine-tyrosine peptides in step (C) is:

[0035] The characteristic ion pair 253.1 / 182.2 of alanine-tyrosine peptide was quantitatively analyzed by peak area using the external standard method.

[0036] It should be noted that the inventors extracted the parent ion of alanine-tyrosine peptide (AY) and fragmented it into a secondary mass spectrum for polypeptide structure identification. They found that the spectrum could not only match the AY polypeptide well, but also match the FS well at the same time. The difference between the two was mainly reflected in the mass-to-charge ratio ions (AY m / z 182.08; FS: m / z 120.08). The other mass-to-charge ratio ions such as m / z 207.1 and 235.1 were common characteristic fragment ions of the two polypeptides.

[0037] This shows that there are not only co-eluting products but also isomers (FS) at the AY peak time. Therefore, when performing quantitative detection of peptide AY, the ion pair (253.1 / 182.2) can be selected as the characteristic ion pair of AY, and the other ion pairs (253.1 / 207.1) and (253.1 / 235.1) can only be used as auxiliary qualitative ion pairs.

[0038] The present invention uses an ion pair (253.1 / 182.2) as the characteristic ion pair of AY. Compared with the original liquid chromatography method, the method greatly enhances the anti-interference of the co-eluting polypeptide and the specificity of the AY polypeptide detection. At the same time, it avoids the interference of the co-eluting isomer (FS) on the detection result. Therefore, in order to avoid the interference caused by isomers in the quality control detection of corn oligopeptides, the present application selects the ion pair (253.1 / 182.2) as the quantitative characteristic ion pair of alanine-tyrosine peptides.

[0039] According to one aspect of the present invention, an application of the above-mentioned corn oligopeptide quality control method in corn oligopeptide quality screening;

[0040] The quality control method of corn oligopeptides provided by the invention can be widely applied to the quality screening of corn oligopeptides.

[0041] In a preferred embodiment of the present invention, the quality screening of corn oligopeptides comprises: screening corn oligopeptide samples from different preparation methods or manufacturers;

[0042] Alternatively, quality monitoring can be performed on different batches of corn oligopeptide samples prepared using the same preparation method.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The present invention provides a method for quality control of corn oligopeptides. The method is established with alanine-tyrosine peptides having a relatively high composition ratio in corn oligopeptides as characteristic peptides, and liquid chromatography-mass spectrometry analysis technology is used to perform quality control and quality identification on corn oligopeptide raw materials. Compared with traditional evaluation methods such as peptide molecular weight distribution and hydrolysis degree, the specificity of quality control and quality identification is greatly enhanced, and the analysis efficiency is greatly improved.

[0045] In addition, it should be emphasized that the inventors extracted the parent ion of alanine-tyrosine peptide (AY) and fragmented it to identify the polypeptide structure. It was found that the spectrum can not only match the AY polypeptide well, but also match the FS well at the same time. This shows that there are not only co-eluting products at the AY peak time, but also isomers (FS). Therefore, in order to avoid the interference caused by isomers in the quality control detection of corn oligopeptides, this application selects ion pairs (253.1 / 182.2) as the quantitative characteristic ion pairs of alanine-tyrosine peptides.

[0046] The quality control method of corn oligopeptides provided by the invention can be widely applied to the quality screening of corn oligopeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0048] Figure 1a The primary ion peak monitoring diagram of the alanine-tyrosine peptide provided in Experimental Example 1 of the present invention;

[0049] Figure 1b The secondary fragment information diagram of the alanine-tyrosine peptide provided in Experimental Example 1 of the present invention;

[0050] Figure 1c The abundance change diagram of the target monitoring ion under different fragmentation energies (CE) of the alanine-tyrosine peptide provided in Experimental Example 1 of the present invention;

[0051] Figure 1d The abundance change diagram of the target monitoring ion under different declustering voltages of the alanine-tyrosine peptide provided in Experimental Example 1 of the present invention;

[0052] Figure 1e The elution change curve of the target monitoring ion of alanine-tyrosine peptide provided in Experimental Example 1 of the present invention with the retention time;

[0053] Figure 2a The primary mass spectrum at the retention time of the alanine-tyrosine peptide in sample A of Example 1 provided in Experimental Example 1 of the present invention;

[0054] Figure 2b The primary mass spectrum at the retention time of the alanine-tyrosine peptide in sample B of Example 1 provided in Experimental Example 1 of the present invention;

[0055] Figure 2c The primary mass spectrum at the retention time of alanine-tyrosine peptide in sample C of Example 1 provided in Experimental Example 1 of the present invention

[0056] Figure 3a The secondary mass spectrum of the parent ion 253.1177 in sample A of Example 1 provided in Experimental Example 1 of the present invention and the AY matching result diagram;

[0057] Figure 3b The secondary mass spectrum and FS matching result diagram of the parent ion 253.1177 in sample B of Example 1 provided in Experimental Example 1 of the present invention;

[0058] Figure 4 The relative composition ratio-length distribution diagram of polypeptide peak area of ​​different samples in Example 1 provided in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0059] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0060] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0061] Example 1 Determination of AY content in corn oligopeptides from different sources

[0062] A method for quality control of corn oligopeptides based on alanine-tyrosine peptide mass spectrometry analysis, the specific steps of the method are as follows:

[0063] (1) Preparation of standard products:

[0064] Accurately weigh a certain amount of Ala-Tyr peptide standard (purity>95%, desalted), and use ultrapure water to prepare a series of standard solutions with concentrations ranging from 0, 0.5, 1.5, 2.5, 3.5, and 4.5 μg / mL;

[0065] (2) Sample preparation:

[0066] Accurately weigh the ground sample to be tested, specifically:

[0067] Sample A, corn oligopeptide produced by Perfect (Guangdong) Daily Necessities Co., Ltd.;

[0068] Sample B, commercially available corn oligopeptide produced by Guangzhou Peptide Hui Biotechnology Co., Ltd.;

[0069] Sample C, corn oligopeptide produced by Banyan (Foshan) Technology Co., Ltd.

[0070] Each sample was dispersed in ultrapure water to prepare a sample solution of appropriate concentration (4000 μg / mL), stirred or shaken at constant temperature for 30 min at 50°C, centrifuged at 5000 rpm and 4°C for 10 min to remove insoluble precipitates, a certain amount of supernatant was added with an equal volume of acetonitrile, and allowed to stand for 10 min to precipitate macromolecular proteins and other substances, and centrifuged again at 5000 rpm and 4°C for 10 min to remove the precipitate, and the supernatant was filtered through a 0.22 μm organic filter membrane and analyzed by an analyzer;

[0071] (3) Liquid phase separation of samples:

[0072] The samples were separated by ultra-high pressure liquid chromatography (AB Company, USA) with a pressure resistance of 1000 bar. The mobile phase consisted of an aqueous formic acid solution (formic acid added in an amount of 0.1%, v / v, denoted as A) and an acetonitrile solution (formic acid added in an amount of 0.1%, v / v, denoted as B).

[0073] The elution gradient was: 0-1.00min 5.0% B, 1.00-1.50min 5.0-10.0% B, 1.50-7.50min 10.0-40.0% B, 7.50-8.50min 40.0-90.0% B, 8.50-10.00min 90% B, 10.00-10.50min 90.0-5.0% B, 10.50-12.00min, 5.0% B; the chromatographic column was ACQUITY UPLC HSS T3 (1.8 μm, 2.1×100 mm, The column temperature was 30° C. The chromatographic analysis flow rate was 0.4 ml / min.

[0074] (4) Mass spectrometry quantitative detection of samples:

[0075] The effluent components after chromatographic separation of the standard and the sample were ionized by an ESI electrospray ion source and then detected and analyzed by a triple quadrupole mass spectrometer detector (AB Company, USA); the ion source working mode was positive ion mode; the mass spectrometer detection mode was MRM multiple reaction ion monitoring mode, the primary precursor ion was set to 253.1m / z, the secondary fragmentation ion detection was set to 182.2 and 207.1m / z, and the scanning frequency was 50-1000Da / s; the secondary fragmentation energy of the AY precursor ion was 15eV.

[0076] (5) Qualitative and quantitative calculation and analysis of samples:

[0077] The characteristic peptide AY was qualitatively and quantitatively analyzed based on the mass spectrometry data.

[0078] in:

[0079] The basis for qualitative calculation is: the theoretical plate number of the standard is not less than 8000, two secondary monitoring ions are detected in the sample at the same time and the signal-to-noise ratio of the chromatographic peak is greater than 3;

[0080] The quantitative method is to use the external standard method to perform quantitative calculation and analysis on the characteristic ion pair 253.1 / 182.2 of the characteristic peptide AY based on the peak area.

[0081] Example 2 Determination of AY and dipeptide equivalents in different batches of corn oligopeptides

[0082] A method for quality control of corn oligopeptides based on alanine-tyrosine peptide mass spectrometry analysis, the specific steps of the method are as follows:

[0083] (1) Preparation of standard products:

[0084] Accurately weigh a certain amount of Ala-Tyr peptide synthetic standard (purity>95%, desalted), and use ultrapure water to prepare a series of standard solutions with concentrations ranging from 0, 0.5, 1.5, 2.5, 3.5, and 4.5 μg / mL;

[0085] (2) Sample preparation:

[0086] Accurately weigh the unground samples to be tested (samples 1 to 4 prepared in different batches by the same process, Perfect (Guangdong) Daily Necessities Co., Ltd.) and disperse them in ultrapure water to prepare a sample solution of appropriate concentration (400 μg / mL), stir or shake at a constant temperature of 45°C for 30 min, centrifuge at 8000 rpm and 10°C for 15 min to remove insoluble precipitates, take a certain amount of supernatant and add an equal volume of methanol, let it stand for 15 min to precipitate macromolecular proteins and other substances, centrifuge again at 8000 rpm and 10°C for 15 min to remove the precipitate, take the supernatant and pass it through a 0.22 μm organic filter membrane, and analyze it on an analyzer;

[0087] (3) Liquid phase separation of samples:

[0088] The samples were separated by ultra-high pressure liquid chromatography (AB Company, USA) with a pressure resistance of 1000 bar. The mobile phase consisted of an aqueous formic acid solution (formic acid addition amount 0.15%, v / v, recorded as A) and an acetonitrile solution (formic acid addition amount 0.15%, v / v, recorded as B). The elution gradient was: 0-1.00 min 5.0% B, 1.00-1.50 min 5.0-10.0% B, 1.50-7.50 min 10.0-40.0% B, 7.50-8.50 min 40.0-90.0% B, 8.50-10.00 min 90% B, 10.00-10.50 min 90.0-5.0% B, 10.50-12.00 min, 5.0% B. The chromatographic column was ACQUITY UPLC HSS T3(1.8μm, 1.0×100mm, The column temperature was 30° C. The chromatographic analysis flow rate was 0.05 ml / min.

[0089] (4) Mass spectrometry quantitative detection of samples:

[0090] The effluent components after chromatographic separation of the standard and the sample were ionized by an ESI electrospray ion source and then detected and analyzed by a triple quadrupole mass spectrometer detector (AB Company, USA); the ion source working mode was positive ion mode; the mass spectrometer detection mode was MRM multiple reaction ion monitoring mode, the primary precursor ion and secondary fragmentation ion detection were set to AY (primary ion 253.1m / z, secondary ions 182.2 and 207.1m / z), and the scanning frequency was 50-1000Da / s; the secondary fragmentation energy of the AY precursor ion was 15eV.

[0091] (5) Directional and quantitative calculation analysis of samples:

[0092] The characteristic peptide AY was qualitatively and quantitatively analyzed based on the mass spectrometry data.

[0093] The basis for qualitative calculation is: the theoretical plate number of the standard is not less than 8000, two secondary monitoring ions are detected in the sample at the same time and the signal-to-noise ratio of the chromatographic peak is greater than 3;

[0094] The quantitative method is to use the external standard method to perform quantitative calculation and analysis on the characteristic ion pair 253.1 / 182.2 of the characteristic peptide AY based on the peak area. The sample test results are shown in Table 1.

[0095] Table 1 Detection results of AY content in corn oligopeptides from different sources

[0096]

[0097] As can be seen from Table 2, the AY content monitoring in each repeated batch of corn oligopeptide raw materials in Example 2 is relatively stable, maintained at a level of 0.92-1.00% (the fluctuation of AY content in products between different batches is less than ±0.1%), reflecting that the method of the present invention can have a good quality control effect in different batches of products of the same raw material, which is of great significance to the production stability quality control and monitoring of corn oligopeptide raw materials.

[0098] (6) Sample quality control method and application: The above method was used to determine the AY content of the above samples. Quality control analysis was performed on corn oligopeptide raw materials from different batches of the same process. It was found that the fluctuation of AY content between different batches was less than ±0.1%.

[0099] (7) Evaluation of the reproducibility of the mass spectrometry quantitative detection method for AY:

[0100] The same concentration of sample 1 described in Example 2 was prepared, and the injection was repeated for three times in parallel, and the RSD value was detected to be 1.65%, reflecting that the method has good reproducibility; for a batch of samples of the same concentration described in Example 2, the content was determined at intervals of 0, 6, 18, 24, and 48 h, and the RSD value was 2.22%, which was less than 3%, reflecting that the method and the quantification of the target polypeptide have good stability within 48 h.

[0101] Experimental Example 1

[0102] The technical effect of the corn oligopeptide quality control method based on alanine-tyrosine peptide mass spectrometry analysis of the present application is analyzed.

[0103] (I) Analysis of ion fragmentation characteristics of AY peptide and optimization of MRM monitoring method:

[0104] The AY standard (concentration 4 μg / mL) was directly injected into the triple quadrupole mass spectrometer using a syringe pump at a flow rate of 10 μL / min. The changes in the abundance of primary and secondary ions and fragments with fragmentation energy were analyzed. The results are shown in Figure 1a to Figure 1e shown.

[0105] Figure 1a Primary ion peak monitoring diagram of alanine-tyrosine peptide provided for this experimental example.

[0106] Figure 1b Secondary fragmentation information diagram of the alanine-tyrosine peptide provided for this experimental example.

[0107] Figure 1c The abundance change diagram of the target monitoring ion under different fragmentation energies (CE) of the alanine-tyrosine peptide provided in this experimental example.

[0108] Figure 1d The abundance change diagram of the target monitoring ion under different declustering voltages for the alanine-tyrosine peptide provided in this experimental example.

[0109] Figure 1e The elution change curve of the target monitoring ion of alanine-tyrosine peptide provided in this experimental example versus retention time.

[0110] Depend on Figure 1a and Figure 1b It can be seen that by accurately searching the primary ion of AY polypeptide and then setting the fragmentation energy, two secondary characteristic ion clusters (182 and 207.1 m / z) were obtained. On this basis, the voltage and energy settings were optimized. Figure 1c and Figure 1d .

[0111] Depend on Figure 1c and Figure 1dIt can be seen that with the gradual increase of DP declustering voltage, the abundance of secondary ions shows an oscillating decrease and reaches a better value at 100V; with the increase of collision energy, the abundance of target secondary ions shows a trend of first increasing and then decreasing, reaching the optimal value at 15eV. This shows that the selection of declustering voltage of 100V and fragmentation energy of 15eV plays an important positive role in the monitoring of target peptides.

[0112] The MRM monitoring curves of the two ion pairs of AY standard are shown in Figure 1e ,Depend on Figure 1e It can be seen that the chromatographic peak emerges relatively quickly, presents a Gaussian chromatographic peak shape, is symmetrical, and has no shoulder peak, and the separation effect is good. The same concentration of AY standard was injected three times, and the peak area RSD value of the ion pair (253.1 / 182.2) was determined to be 1.78%, which is less than the requirement of the chromatographic analysis pharmacopoeia (2%), indicating that the method and instrument have good precision.

[0113] (II) Peptideomics analysis of corn oligopeptide samples:

[0114] The sample described in Example 1 was loaded onto an ultra-high pressure liquid chromatography-high resolution mass spectrometer (SCIEXX500R LC-QTOF, AB, USA) for peptide analysis. The ACQUITY UPLC HSS T3 (1.8 μm, 2.1×100 mm, Waters, USA) was used as the analytical chromatographic column. The mobile phase composition was the same as the quantitative analysis above. The elution method was: 0-6 min 10.0% B, 6-30 min 10.0-40.0% B, 30-35 min 40.0-90.0% B, 35-40.00 min 90.0% B, 40.00-42 min 90.0-5.0% B, 42-60.00 min 5.0% B, flow rate 0.20mL / min, column temperature 40℃, sample injection volume 2uL, mass spectrometry data enumeration and database search collaborative identification using PepOSX-Exhaust+Search engine, primary ion mass-to-charge ratio deviation 20ppm, secondary ion mass-to-charge ratio deviation 0.02Da, residue coverage threshold 50%, sequence library downloaded from Uniprot (search keyword "maize-reviewed"), ion extraction chromatogram mass-to-charge ratio deviation 20ppm, chromatographic peak identification method Gaussian identification, analysis of co-eluting products and isomers of enzymatic hydrolysate AY see Figure 2a to Figure 2c and Figure 3a to Figure 3b .

[0115] Figure 2a The primary mass spectrum at the retention time of the alanine-tyrosine peptide in sample A of Example 1 provided in this experimental example;

[0116] Figure 2bThe primary mass spectrum at the retention time of the alanine-tyrosine peptide in sample B of Example 1 provided in this experimental example;

[0117] Figure 2c The primary mass spectrum at the retention time of the alanine-tyrosine peptide in sample C of Example 1 provided in this experimental example

[0118] Figure 3a The secondary mass spectrum of the parent ion 253.1177 in sample A of Example 1 provided in this experimental example and the AY matching result diagram;

[0119] Figure 3b The secondary mass spectrum and FS matching result diagram of the parent ion 253.1177 in sample B of Example 1 provided in this experimental example.

[0120] (III) Identification and quality control analysis of corn oligopeptide raw materials from different sources:

[0121] The AY content of the sample described in Example 1 was determined according to QB / T4707-2014. The results are shown in Table 2.

[0122] Table 2 Determination results of AY in corn oligopeptides from different sources:

[0123]

[0124] Note: The AY mass spectrometry in the table is the result of Example 1; the AY chromatography is the result of the performance test in accordance with QB / T4707-2014; the dipeptide equivalent is the result of the peptide group analysis in Example 1.

[0125] As shown in Table 1, the differences between the AY content and dipeptide equivalent of corn oligopeptides from different sources are quite significant after mass spectrometry detection. These indicators can be used to effectively distinguish and monitor corn oligopeptide raw materials from different sources. In contrast, the differences between the AY content of corn oligopeptide raw materials from different sources detected by liquid chromatography are small, especially unable to distinguish between samples B and C. At the same time, the difference between sample A and samples B and C is also very small, and the raw material identification ability has certain limitations. In addition, it is worth noting that the AY content of the samples detected by chromatography is significantly higher than that obtained by mass spectrometry. The reason for the analysis may be that it is difficult for liquid chromatography to distinguish between polypeptide co-eluting products, resulting in a larger quantitative detection result. Since the composition of corn protein hydrolysate polypeptides is extremely complex, co-eluting products will cause greater interference to the target monitoring of the liquid chromatography ultraviolet detector, reducing the reliability of the method.

[0126] Further, by Figure 2aIt can be seen from the high-resolution mass spectrometry detection and analysis that there are many other polypeptides co-eluting ions when the AY ion peaks. These ions include 269.1130 (identification results show SY), 331.1975 (identification results show LAQ), 544.2714 (identification results show PAFLP), 432.2454 (identification results show LATQ), etc., which fully reflects the complexity and authenticity of the co-elution phenomenon of polypeptides in the enzymatic hydrolysate. This is the main reason why the AY content determined by liquid chromatography is higher than the mass spectrometry detection result.

[0127] In addition, the co-elution products of AY in samples from different sources (see Figure 2a to Figure 2c ), which is reflected in the complexity of the differences in the composition of the enzymatic hydrolysates of corn oligopeptides produced by different processes or manufacturers. This will cause serious interference to the detection of AY content based on liquid chromatography, and also fully demonstrates the creativity, advancement and practicality of the method of the present invention using mass spectrometry to detect AY dipeptide.

[0128] In addition, the inventors extracted the parent ion of AY and fragmented it to identify the peptide structure. They found that the spectrum can not only match the AY peptide well, but also match the FS well. The difference between the two is mainly reflected in the mass-to-charge ratio ions (AY m / z 182.08; FS: m / z 120.08). The other mass-to-charge ratio ions such as m / z 207.1 and 235.1 are common characteristic fragment ions of the two peptides. Figure 2a to Figure 2c , indicating that there are not only co-eluting products but also isomers (FS) at the AY peak time. Therefore, when performing quantitative detection of peptide AY, the ion pair (253.1 / 182.2) can be selected as the characteristic ion pair of AY, and the other ion pairs (253.1 / 207.1) and (253.1 / 235.1) can only be used as auxiliary qualitative ion pairs. In order to avoid interference caused by isomers, the ion pair (253.1 / 182.2) is selected as the better quantitative characteristic ion pair of AY.

[0129] Figure 3a The secondary mass spectrum of the parent ion 253.1177 in sample A of Example 1 provided in this experimental example and the AY matching result diagram; Figure 3b The secondary mass spectrum and FS matching result diagram of the parent ion 253.1177 in sample B of Example 1 provided in this experimental example.

[0130] (IV) Stability evaluation of mass spectrometry detection method

[0131] For a batch of samples with the same concentration described in Example 2, the injection was repeated for three times and the RSD value was analyzed to evaluate the reproducibility; for a batch of samples with the same concentration described in Example 2, the content was determined at intervals of 0, 6, 18, 24, and 48 h to evaluate the stability of the method.

[0132] (V) Molecular weight distribution analysis of corn oligopeptide samples

[0133] The peptide molecular weight distribution of the corn oligopeptide samples from different sources described in Example 1 was determined according to GB / T22492-2008 standard. The results are shown in Table 3.

[0134] Table 3 Analysis of molecular weight distribution of polypeptides from samples from different sources in Example 1

[0135]

[0136] As shown in Table 3, samples AC from different sources, due to the deep hydrolysis, accounted for more than 90% of the <1KDa part, and the distribution was close, with little difference between them, especially samples B and C were very close. Considering the peak drift phenomenon that may occur in the chromatographic separation process, such subtle differences will be difficult to effectively distinguish in the quality control analysis of the actual production practice process. This to a certain extent illustrates the limitations of using polypeptide molecular weight distribution in the quality control of corn oligopeptide raw materials.

[0137] (VI) Peptide length distribution of corn oligopeptides

[0138] The peptide length distribution of each sample in Example 1 was analyzed using high-resolution mass spectrometry-based peptide omics analysis technology as shown below: Figure 4 .

[0139] Figure 4 This is a diagram showing the relative composition ratio-length distribution of polypeptide peak areas of different samples in Example 1 provided in this experimental example.

[0140] Depend on Figure 4 It can be seen that different corn oligopeptides are mainly composed of 2-4 peptides, especially dipeptides. This is mainly related to the fact that corn oligopeptides usually use food-derived protein crude enzymes with a wide range of cleavage sites during the enzymatic preparation process. Due to the low purity of food proteases and a wide range of action sites, the enzymatic products are often dominated by short peptides. It is worth noting that AY also contributes about 1.1-1.7% to the relative peak area composition of polypeptides (the average number of polypeptide identifications in the three samples is 850, and more than 98% of the polypeptides account for less than 1.0% of the relative peak area composition), indicating that AY is one of the important characteristic polypeptides in corn oligopeptides, reflecting that the use of alanine-tyrosine peptides in this study to monitor corn oligopeptides has certain scientificity and effectiveness.

[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A quality control method for corn oligopeptides, characterized in that: The method comprises: (A) preparing alanine-tyrosine peptide standard solution; and, a test sample solution of corn oligopeptide; (B) separating the components of the standard solution and the sample solution by liquid chromatography to obtain effluent components; (C) The effluent components of the standard and the sample to be tested after separation by liquid chromatography are subjected to mass spectrometry detection, and the alanine-tyrosine peptide is qualitatively and quantitatively analyzed based on the mass spectrometry data.

2. The quality control method of corn oligopeptides according to claim 1, characterized in that: The purity of the alanine-tyrosine peptide in the alanine-tyrosine peptide standard solution is ≥95% and does not contain any salty components.

3. The quality control method of corn oligopeptides according to claim 1, characterized in that: The mass concentration of the alanine-tyrosine peptide standard solution is 0.5-10 μg / mL.

4. The quality control method of corn oligopeptides according to claim 1, characterized in that: The method for preparing the corn oligopeptide sample solution to be tested comprises: The corn oligopeptide sample is dispersed in ultrapure water and homogenized, and then centrifuged to remove the insoluble precipitate; then an equal volume of organic solvent is added to stand for precipitation, and the precipitate is removed by centrifugation again, and the supernatant is taken and filtered to obtain the corn oligopeptide sample solution to be tested; Preferably, the mass concentration of the corn oligopeptide sample solution to be tested is 10-4000 μg / mL; Preferably, the organic solvent is methanol or acetonitrile; Preferably, the filter membrane is a 0.22 μm organic filter membrane.

5. The quality control method of corn oligopeptides according to claim 1, characterized in that: The liquid chromatography in step (B) is a liquid chromatography with a pressure resistance of ≥ 600 bar; The detection conditions of the liquid chromatography are: The flow rate of the liquid chromatography is 0.1-0.4 mL / min, the column temperature is 30°C; the inner diameter of the chromatographic column is 1.0 or 2.1 mm, the length is 50-100 mm, and the filler particle diameter is 1.8 μm; The mobile phase consists of mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution containing 0.05-0.3% formic acid, and mobile phase B is an acetonitrile solution; The elution gradient was: 0-1.00 min 5.0% B, 1.00-1.50 min 5.0-10.0% B, 1.50-7.50 min 10.0-40.0% B, 7.50-8.50 min 40.0-90.0% B, 8.50-10.00 min 90% B, 10.00-10.50 min 90.0-5.0% B, 10.50-12.00 min, 5.0% B.

6. The quality control method of corn oligopeptides according to claim 1, characterized in that: The mass spectrometer in step (C) is at least one of a triple quadrupole mass spectrometer, a quadrupole-time-of-flight mass spectrometer or a quadrupole-ion trap mass spectrometer detector; The mass spectrometer is equipped with an ESI electrospray ion source, and the ion source working mode is a positive ion mode; the mass spectrometer detection mode is an MRM multiple reaction ion monitoring mode, the primary precursor ion is set to 253.1m / z, the secondary fragmentation ion detection is set to 182.2 and 207.1m / z, and the scanning frequency is 50-1000Da / s; the secondary fragmentation energy of the alanine-tyrosine peptide precursor ion is 15eV.

7. The quality control method of corn oligopeptides according to claim 1, characterized in that: The method for qualitative analysis of alanine-tyrosine peptides in step (C) is: The theoretical plate number of the standard is ≥8000, two secondary monitoring ions are detected in the sample to be tested at the same time and the signal-to-noise ratio of the chromatographic peak is greater than 3.

8. The quality control method of corn oligopeptides according to claim 1, characterized in that: The method for quantitatively analyzing the alanine-tyrosine peptide in step (C) is: The characteristic ion pair 253.1 / 182.2 of alanine-tyrosine peptide was quantitatively analyzed by peak area using the external standard method.

9. Use of the quality control method of corn oligopeptides according to any one of claims 1 to 8 in quality screening of corn oligopeptides.

10. The use according to claim 9, characterized in that: The corn oligopeptide quality screening includes: Identify corn oligopeptide samples from different preparation methods or manufacturers; Alternatively, quality monitoring can be performed on different batches of corn oligopeptide samples prepared using the same preparation method.

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