A biological peptide detection method

Abstract

The present invention discloses a biological polypeptide detection method, which belongs to the field of biological detection technology and comprises the following steps: taking muscle tissue of a sample, crushing it to obtain a homogenous sample for standby use; adding water to the homogenous sample, and after crude extraction, adding an adsorption resin, after static adsorption, filtering and collecting the adsorption resin, and using ethanol desorption treatment, filtering out the adsorption resin, and using the obtained filtrate as a sample solution for standby use; using UHPLC-MS / MS coupling method to detect the content of carnosine and anserine in the sample solution; wherein the adsorption resin is a polystyrene-divinylbenzene copolymer macroporous adsorption resin or a modified macroporous adsorption resin I, and the modified macroporous adsorption resin I adopts (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside to modify the polystyrene-divinylbenzene copolymer. The method of the present invention is simple to operate, has high accuracy and good reproducibility, and can improve the extraction effect of carnosine and anserine.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a biological polypeptide detection method (regarding carnosine and anserine). Background Art

[0002] Currently, there are few methods for detecting carnosine and anserine in China. According to literature, the main methods for their determination include high-performance liquid chromatography, ion-exchange chromatography, and capillary electrophoresis. While HPLC effectively measures carnosine and anserine, its use of a 210nm wavelength results in a high baseline and susceptibility to interference, and its accuracy remains to be determined.

[0003] Pretreatment techniques for carnosine and anserine primarily rely on ultrasonic extraction and derivatization of standard working solutions. However, these procedures are complex, and interference from oils and fats in the aquatic product matrix can affect the detection of these substances. The extraction method specified in NY / T 3946-2021, "Determination of Carnosine and Anserine in Animal-Derived Foods—High-Performance Liquid Chromatography," provides poor separation. GB / T 39101-2020, "Determination of Antimicrobial Activity of Polypeptides by Zone of Inhibition Method," and GB / T 39100-2020, "Determination of Antioxidant Activity of Polypeptides by DPPH and ABTS Method," are not applicable to the detection of carnosine and anserine in aquatic products, resulting in poor extraction results. Because aquatic product matrices contain a high concentration of impurities and oils, which affect the extraction of these substances, efficient and convenient extraction methods are needed. Summary of the Invention

[0004] The purpose of the present invention is to provide a biological polypeptide detection method (regarding carnosine and anserine) to improve the extraction rate of carnosine and anserine in aquatic products. The method is simple to operate, highly accurate and reproducible.

[0005] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0006] A method for detecting biological peptides comprises the following steps:

[0007] Muscle tissue of the sample was taken and mashed to obtain a homogenous sample for later use. Water was added to the homogenized sample and subjected to crude extraction. Adsorption resin was then added. After static adsorption, the adsorption resin was collected by filtration and desorbed with ethanol. After the adsorption resin was removed by filtration, the resulting filtrate was used as the sample solution for later use. The contents of carnosine and anserine in the sample solution were determined by UHPLC-MS / MS.

[0008] The adsorption resin is a polystyrene-divinylbenzene copolymer macroporous adsorption resin or a modified macroporous adsorption resin I, wherein the modified macroporous adsorption resin I is obtained by modifying the polystyrene-divinylbenzene copolymer with (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside.

[0009] Modified macroporous adsorption resin I, obtained by modifying polystyrene-divinylbenzene copolymer with (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside, significantly improves the adsorption of carnosine and anserine in samples, thereby helping to reduce detection errors. Furthermore, the desorption rate of the modified macroporous adsorption resin I is significantly higher than that of unmodified polystyrene-divinylbenzene copolymer macroporous adsorption resin.

[0010] Preferably, the conditions for crude extraction of the homogenized sample are as follows: placing the homogenized sample in deionized water at a mass volume ratio of 3-10 g:100-200 mL, vortexing for 1-5 minutes, placing it in an ultrasonic instrument for ultrasonic extraction for 20-60 minutes, and centrifuging at 5000-8000 r / min for 5-10 minutes.

[0011] Preferably, the mass ratio of the modified macroporous adsorption resin I to the homogeneous sample is 1-5:10.

[0012] Preferably, after the static adsorption is completed, the desorption treatment uses a 75% ethanol solution, and the mass volume ratio of the modified macroporous adsorption resin I to the ethanol solution is 0.5-2g:100mL.

[0013] Preferably, the desorption treatment conditions are on a constant temperature shaker at 40-55° C. and 120 r / min for 15-30 h.

[0014] Preferably, when the UHPLC-MS / MS method is used to detect the content of carnosine and anserine in the sample solution, the analysis conditions are:

[0015] Separation was performed using a Waters Acquity UPLC BEH Amide column (100 mm × 2.1 mm, 1.7 μm); column temperature was 35°C; mobile phase A consisted of 2 mmol / L ammonium acetate in 0.1% formic acid, and mobile phase B consisted of acetonitrile. The gradient elution program was as follows: initial 30% A; 0-3 min, 45% A; 3-4 min, 45% A; 4-4.5 min, 30% A; 4.5-5.5 min, 30% A. Injection volume: 10 μL; flow rate: 0.4 mL / min.

[0016] The mass spectrometer settings were as follows: electrospray ionization (ESI) source operated under positive ionization conditions and multiple reaction monitoring mode; capillary voltage: 3 kV; ion source temperature: 150 °C; desolvation temperature: 600 °C; cone gas flow rate: 150 L / h; and desolvation gas flow rate: 1000 L / h.

[0017] For carnosine, m / z 227.1>110 was selected as the quantitative ion pair, and m / z 227.1>156 was selected as the qualitative ion pair. For anserine, m / z 241.1>109 was selected as the quantitative ion pair, and m / z 227.1>170 was selected as the qualitative ion pair.

[0018] According to one aspect of the present invention, there is provided a method for preparing a modified macroporous adsorption resin, comprising the following steps:

[0019] S1. Preparation of chlorine balls: polystyrene-divinylbenzene copolymer, reacted with chloromethyl methyl ether in the presence of a catalyst to obtain chlorine balls;

[0020] S2. Preparation of amino resin: Add ethylenediamine to the chlorine ball obtained in S1 and react for 5-8h to obtain an amino resin;

[0021] S3. Add formaldehyde, (-)-catechin, and kaempferol-3-O-sophorobiose-7-O-glucoside to the amino resin obtained in S2, react for 4-8 hours, and filter to remove the filtrate to obtain modified macroporous adsorption resin I.

[0022] Preferably, in step S1, the polystyrene-divinylbenzene copolymer is pre-swelled with a swelling agent for 1-4 hours before reacting with chloromethyl methyl ether. The swelling agent is dichloromethane, and the mass volume ratio of the polystyrene-divinylbenzene copolymer to dichloromethane is 3-8 g:5-20 mL.

[0023] Preferably, in step S1, the molar ratio of polystyrene-divinylbenzene copolymer to chloromethyl methyl ether is 1:1.5-5.

[0024] Preferably, in step S1, the catalyst is one of SnCl4, ZnCl2, and TiCl4, the reaction is carried out at room temperature, and the molar ratio of the catalyst to the polystyrene-divinylbenzene copolymer is 1:4.5-15.

[0025] Preferably, in step S1, the polystyrene-divinylbenzene copolymer and chloromethyl methyl ether are first reacted at 3-10° C. for 1-4 hours, and then the temperature is raised to 20-35° C. for reaction for 4-10 hours.

[0026] Preferably, in step S1, the catalyst is added in portions to the polystyrene-divinylbenzene copolymer and chloromethyl methyl ether system under the reaction condition of 3-10°C.

[0027] Preferably, in step S2, the chlorine balls are pre-swelled with a swelling agent for 1-4 hours before reacting with ethylenediamine. The swelling agent is tetrahydrofuran, and the mass volume ratio of the chlorine balls to tetrahydrofuran is 3-8 g:5-20 mL.

[0028] Preferably, in step S2, the weight ratio of the chlorine balls to ethylenediamine is 1:5-10.

[0029] Preferably, in step S2, the reaction is carried out on a constant temperature shaker at 20-35°C with an oscillation frequency of 80-150 r / min.

[0030] Preferably, in step S3, the amino resin is pre-swelled with a swelling agent for 1-4 hours before reacting with (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside. The swelling agent is tetrahydrofuran, and the mass-to-volume ratio of amino resin to tetrahydrofuran is 3-8 g:5-20 mL.

[0031] Preferably, in step S3, the mass volume ratio of amino resin to formaldehyde is 10-30 g:10-100 mL, and the mass ratio of amino resin to (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside is 10-20:0.3-2:0.3-2.

[0032] Preferably, in step S3, the reaction is carried out on a constant temperature shaker at 20-35° C. with an oscillation frequency of 80-150 r / min.

[0033] Preferably, in step S3, the modified macroporous adsorption resin I is extracted with anhydrous ethanol for 4-8 hours before use, then washed with deionized water until neutral, and then shaken with 4% sodium hydroxide solution in a constant temperature shaker at 20-35°C and 80-150 r / min for 1-3 hours, and then washed again with deionized water until neutral, and dried in a vacuum drying oven at 45-60°C for 5-8 hours.

[0034] According to one embodiment of the present invention, when preparing the modified macroporous adsorption resin, kaempferol-3-O-p-coumaroyl rhamnosyl glucoside is added to the reaction system in step S2 while adding formaldehyde, (-)-catechin, and kaempferol-3-O-sophorobiose-7-O-glucoside.

[0035] Preferably, the mass ratio of the polystyrene-divinylbenzene copolymer to (-)-catechin, kaempferol-3-O-sophorobiose-7-O-glucoside, and kaempferol-3-O-p-coumaroyl rhamnosyl glucoside is 10-20:0.3-2:0.3-2:0.3-2.

[0036] The modified macroporous adsorption resin I prepared by combining (-)-catechin, kaempferol-3-O-sophorobiose-7-O-glucoside, and kaempferol-3-O-p-coumaryl rhamnosyl glucoside showed better adsorption efficiency for carnosine and anserine in the crude extract than that obtained by combining (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside alone. This may be because the combined use of (-)-catechin, kaempferol-3-O-sophorobiose-7-O-glucoside, and kaempferol-3-O-p-coumaryl rhamnosyl glucoside can further change the surface structure of the modified macroporous adsorption resin I, making it easier for it to bind to carnosine and anserine, thereby improving the extraction efficiency of carnosine and anserine from the homogenized sample.

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

[0038] 1. Modified macroporous polystyrene-divinylbenzene copolymer resin (PSDB) was modified with (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside to obtain modified macroporous resin I, which improved the extraction of carnosine and anserine from aquatic muscle tissue. This is likely due to the modification process, in which the addition of (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside altered the microstructure of the PSDB macroporous resin, increasing its specific surface area and thus enhancing its adsorption performance. Furthermore, the presence of phenolic groups and polyhydroxy structures in (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside facilitated the adsorption and binding of carnosine and anserine to modified macroporous resin I, thus enhancing the extraction of carnosine and anserine from aquatic muscle tissue.

[0039] 2. Modified macroporous adsorption resin I, obtained by modifying a polystyrene-divinylbenzene copolymer macroporous adsorption resin with (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside, significantly improved the recovery rate for the extraction of carnosine and anserine from aquatic muscle tissue. Modified macroporous adsorption resin I exhibited excellent adsorption and desorption properties for carnosine and anserine, thereby reducing detection errors. This may be because the addition of (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside during the modification process introduced polyhydroxy groups onto the resin surface, enhancing the resin's hydrophilicity and thus facilitating the desorption of carnosine and anserine adsorbed on the resin surface. In addition, the simultaneous use of (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside and the mutual influence between (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside during the reaction process may have further improved the spatial structure of the obtained modified macroporous adsorption resin I and may have promoted the formation of intramolecular hydrogen bonds and extramolecular hydrogen bonds, thereby facilitating the adsorption and desorption of carnosine and anserine.

[0040] 3. During the modification of a polystyrene-divinylbenzene copolymer macroporous adsorption resin, the combined use of kaempferol-3-O-p-coumaroyl rhamnosyl glucoside with (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside further enhanced the adsorption and desorption efficiency of the resulting modified macroporous adsorption resin I for carnosine and anserine. This is likely due to the complex molecular structures of kaempferol-3-O-sophorobiose-7-O-glucoside and kaempferol-3-O-p-coumaroyl rhamnosyl glucoside, which contain numerous phenolic and alcoholic hydroxyl groups. The active hydrogen atoms within the molecules are susceptible to reaction with aldehydes and amines. Furthermore, the molecular structure of kaempferol-3-O-p-coumaroyl rhamnosyl glucoside is even more complex, and the presence of olefinic bonds and carbonyl groups within the molecule also influences the microscopic properties of the resulting modified macroporous adsorption resin I, thereby enhancing its adsorption and desorption efficiency for carnosine and anserine. This may be because the mutual influence between (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside and / or kaempferol-3-O-p-coumaryl rhamnosyl glucoside during the reaction process may further improve the spatial structure of the obtained modified macroporous adsorption resin I, and may also promote the formation of intramolecular hydrogen bonds and extramolecular hydrogen bonds, thereby facilitating the adsorption and desorption of carnosine and anserine.

[0041] 4. In the process of separating and purifying carnosine and anserine from aquatic products, the crude extract was treated with the modified macroporous adsorption resin I described above. After adsorption and desorption, the resulting sample solution was subjected to UHPLC-MS / MS analysis. This method has high accuracy and good reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 chromatograms of carnosine and anserine standard solutions used in the test examples of the present invention;

[0043] Figure 2 The chromatogram is a 10-fold signal-to-noise ratio quantitative limit of the carnosine and anserine standard working solutions in the test examples of the present invention. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0045] Example 1

[0046] The content of carnosine and anserine in aquatic products was determined by high performance liquid chromatography tandem mass spectrometry. The operating steps and analytical conditions are as follows:

[0047] Sample pretreatment

[0048] After thawing the fresh frozen hairtail with ice water, the muscle tissue with bones removed was thoroughly and evenly crushed with a high-speed tissue crusher to obtain a homogeneous sample, which was then frozen and stored at -18°C for later use.

[0049] A homogenized sample was weighed and frozen in liquid nitrogen. The powder sample was placed in deionized water at a mass-to-volume ratio of 5 g:100 mL, vortexed for 2 min, placed in an ultrasonic instrument for ultrasonic extraction for 30 min, and centrifuged at 8000 r / min for 10 min to obtain a crude extract.

[0050] Add polystyrene-divinylbenzene copolymer macroporous adsorption resin to the crude extract at a mass ratio of 1:10. Oscillate on a thermostatic shaker at 30°C, 120 rpm for 24 hours for static adsorption. The macroporous adsorption resin is then collected by filtration and a 75% ethanol solution is added at a mass-to-volume ratio of 0.5 g:100 mL. Oscillate on a thermostatic shaker at 50°C, 120 rpm for 24 hours. After filtering out the macroporous adsorption resin, heat the resulting filtrate in an 80°C water bath for 2 hours to obtain the sample solution for later use.

[0051] The contents of carnosine and anserine in the obtained sample solution were determined by high performance liquid chromatography tandem mass spectrometry. The operating steps and analytical conditions are as follows:

[0052] Separation was performed using a Waters Acquity UPLC BEH Amide column (100 mm × 2.1 mm, 1.7 μm); column temperature was 35°C; mobile phase A consisted of 2 mmol / L ammonium acetate and 0.1% formic acid; mobile phase B consisted of acetonitrile. The gradient elution program was as follows: initial 30% A; 0-3 min, 45% A; 3-4 min, 45% A; 4-4.5 min, 30% A; 4.5-5.5 min, 30% A. Injection volume: 10 μL; flow rate: 0.4 mL / min.

[0053] The mass spectrometer settings were as follows: electrospray ionization (ESI) source operated under positive ionization conditions and multiple reaction monitoring mode; capillary voltage: 3 kV; ion source temperature: 150 °C; desolvation temperature: 600 °C; cone gas flow rate: 150 L / h; and desolvation gas flow rate: 1000 L / h.

[0054] For carnosine, m / z 227.1 > 110 was selected as the quantitative ion pair, and m / z 227.1 > 156 was selected as the qualitative ion pair. For anserine, m / z 241.1 > 109 was selected as the quantitative ion pair, and m / z 227.1 > 170 was selected as the qualitative ion pair. The ion pairs used in the multiple reaction monitoring mode are shown in Table 1.

[0055] Table 1 Selected reaction monitoring precursor ions, product ions, and collision voltages

[0056]

[0057] Example 2

[0058] This Example differs from Example 1 in that, during sample pretreatment, modified macroporous adsorption resin I is used in place of the macroporous adsorption resin. The mass ratio of modified macroporous adsorption resin I to homogenized sample is 1:10, and static adsorption is performed on a thermostatic shaker at 30°C and 120 r / min for 24 hours. The modified macroporous adsorption resin I is then collected by filtration, and 100 mL of a 25% ethanol solution is added thereto. The sample is shaken on a thermostatic shaker at 50°C and 120 r / min for 24 hours. After filtering out the modified macroporous adsorption resin I, the resulting filtrate is heated in an 80°C water bath for 2 hours to obtain a sample solution for later use.

[0059] Other conditions and steps are the same.

[0060] The preparation method of modified macroporous adsorption resin I is as follows:

[0061] S1. Preparation of chlorine spheres: Polystyrene-divinylbenzene copolymer was swelled in dichloromethane at a mass-to-volume ratio of 5g:10mL for 2h. Chloromethyl methyl ether was then added and the reaction was controlled at 4°C for 2h. During this time, SnCl4 was added dropwise four times. The temperature was then raised to 25°C and the reaction was continued for 6h. After the reaction, the microspheres were filtered and collected. The molar ratio of the white sphere, chloromethyl methyl ether, and SnCl4 was 1:2.5:8. The resulting microspheres were filtered and washed with deionized water, and then vacuum-dried at 60°C for 12h to obtain chlorine spheres.

[0062] S2. Preparation of amino resin: Tetrahydrofuran was added to the chlorine spheres obtained in S1 at a mass-to-volume ratio of 5 g:10 mL to allow swelling for 2 h. Ethylenediamine was then added and the mixture was shaken on a thermostatic shaker at 30°C, 120 rpm, for 6 h to introduce amino groups into the chlorine spheres. The reaction mixture was cooled to 25°C, filtered, washed with deionized water, and then dried to obtain the amino resin. The weight ratio of chlorine spheres to ethylenediamine was 1:8.

[0063] S3. Tetrahydrofuran was added to the amino resin obtained in S2 at a mass-to-volume ratio of 5 g:10 mL, and the mixture was swollen for 2 h. Formaldehyde, (-)-catechin, and kaempferol-3-O-sophorobiose-7-O-glucoside were then added. The mixture was shaken on a thermostatic shaker at 30°C and 120 rpm for 6 h. The filtrate was filtered to obtain modified macroporous adsorption resin I. The mass-to-volume ratio of amino resin to formaldehyde was 10 g:30 mL, and the mass ratio of amino resin to (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside was 10:1:1.

[0064] In addition, the obtained modified macroporous adsorption resin I was extracted with anhydrous ethanol for 6 hours before use, then washed with deionized water until neutral, and then treated with 4% sodium hydroxide solution in a constant temperature shaker at 30°C and oscillated at 120 r / min for 2 hours, and then washed with deionized water again until neutral, and dried in a vacuum drying oven at 60°C for 6 hours for use.

[0065] Example 3

[0066] The difference between this embodiment and embodiment 2 is that the mass ratio of modified macroporous adsorption resin I to homogeneous sample is 2:10. Other conditions and steps are the same.

[0067] Example 4

[0068] The difference between this embodiment and embodiment 2 is that the mass ratio of modified macroporous adsorption resin I to homogeneous sample is 3:10. Other conditions and steps are the same.

[0069] Example 5

[0070] Compared with Example 2, this embodiment differs in that: in the preparation method of modified macroporous adsorption resin I, in step S3, while adding formaldehyde and (-)-catechin, kaempferol-3-O-sophorobiose-7-O-glucoside to the reaction system of S2, kaempferol-3-O-p-coumaroyl rhamnosyl glucoside is added, and the mass ratio of amino resin to (-)-catechin, kaempferol-3-O-sophorobiose-7-O-glucoside, and kaempferol-3-O-p-coumaroyl rhamnosyl glucoside is 10:1:1:1.

[0071] Other conditions and steps are the same.

[0072] Example 6

[0073] Compared with Example 2, this embodiment differs in that: in the preparation method of the modified macroporous adsorption resin I, in step S3, while adding formaldehyde, (-)-catechin, and kaempferol-3-O-sophorobiose-7-O-glucoside to the reaction system of S2, kaempferol-3-O-p-coumaroyl rhamnosyl glucoside is added, and the mass ratio of the polystyrene-divinylbenzene copolymer to (-)-catechin, kaempferol-3-O-sophorobiose-7-O-glucoside, and kaempferol-3-O-p-coumaroyl rhamnosyl glucoside is 10:1:1:2.

[0074] Other conditions and steps are the same.

[0075] Comparative Example 1

[0076] This embodiment differs from Example 2 in that, in the preparation method of modified macroporous adsorption resin I, in step S3, kaempferol-3-O-sophorobiose-7-O-glucoside is replaced with an equal volume of (-)-catechin. Other conditions and steps are the same.

[0077] Comparative Example 2

[0078] This embodiment differs from Example 2 in that, in the preparation method of modified macroporous adsorption resin I, in step S3, (-)-catechin is replaced with an equal volume of kaempferol-3-O-sophorobiose-7-O-glucoside. Other conditions and steps are the same.

[0079] Test example

[0080] Adopt high performance liquid chromatography tandem mass spectrometry, measure the content of carnosine and anserine in carnosine and anserine standard solution, operating steps and analysis conditions are referring to Example 1. Carnosine and anserine standard solution preparation method is as follows:

[0081] A carnosine and anserine standard stock solution with a concentration of 100 μg / mL was prepared using carnosine and anserine standard powder; a carnosine and anserine standard intermediate solution with a concentration of 1.0 μg / mL was then prepared using the above carnosine and anserine standard stock solution; and a carnosine and anserine standard working solution with a concentration of 100 ng / mL was prepared using the above carnosine and anserine standard intermediate solution.

[0082] Under the above analytical conditions, the separation effect of carnosine and anserine in the carnosine and anserine standard solution was good, see Figure 1 .

[0083] Carnosine and anserine standard working solutions were serially diluted with deionized water to prepare a series of standard working solutions at concentrations of 1.00, 5.00, 10.0, 20.0, 50.0, and 100 ng / mL. The determinations were performed under the above analytical conditions. The limits of quantification for carnosine and anserine were calculated based on a 10-fold signal-to-noise ratio (S / N). The results are shown in Table 2. The limits of quantification for carnosine and anserine were both 10 μg / kg. The chromatograms of the 10-fold signal-to-noise ratio limits of quantification for carnosine and anserine are shown in Table 2. Figure 2 .

[0084] Table 2 Linear range and detection limit

[0085]

[0086] The contents of carnosine and anserine in the sample solutions obtained in Examples 1-6 and Comparative Examples 1-2 are shown in Table 3.

[0087] Table 3 Contents of carnosine and anserine in the sample solutions obtained in various examples and comparative examples (%)

[0088]

[0089] Referring to the data in Table 3, compared with Comparative Example 1, the contents of carnosine and anserine in the sample solutions obtained in Examples 2-6 and Comparative Examples 1-2 were greatly increased.

[0090] The carnosine contents in the sample solutions obtained in Examples 2-4 were increased by 42.8%, 157.1%, and 214.3%, respectively, compared to those in Example 1; and the anserine contents in the sample solutions obtained in Examples 2-4 were increased by 34.4%, 149.8%, and 191.1%, respectively, compared to those in Example 1. The carnosine contents in the sample solutions obtained in Comparative Examples 1-2 were increased by 14.3% and 28.6%, respectively, compared to those in Example 1; and the anserine contents in the sample solutions obtained in Comparative Examples 1-2 were increased by 12.3% and 20.4%, respectively, compared to those in Example 1.

[0091] It can be seen that within a certain dosage range, the dosage of modified macroporous adsorption resin I is positively correlated with the adsorption effect of carnosine and anserine in crude aquatic product extracts.

[0092] Compared to Example 1, when the amount of adsorption resin used was the same, the use of modified macroporous adsorption resin I (Example 2, Comparative Examples 1-2) significantly improved the adsorption efficiency of carnosine and anserine in crude aquatic product extracts. This indicates that the modified macroporous adsorption resin I, modified with (-)-catechin and / or kaempferol-3-O-sophorobiose-7-O-glucoside to treat homogenized samples of aquatic product muscle tissue, significantly improved the extraction efficiency of carnosine and anserine. This is likely due to the altered microstructure of the modified macroporous adsorption resin I compared to conventional macroporous adsorption resins, which enhances the adsorption efficiency of carnosine and anserine in crude aquatic product extracts. Furthermore, the desorption efficiency of carnosine and anserine was also enhanced during the desorption process.

[0093] Moreover, the contents of carnosine and anserine in the sample solution obtained in Example 2 were significantly higher than those in the sample solutions obtained in Comparative Examples 1-2. This shows that when the macroporous adsorption resin is modified, the modified macroporous adsorption resin I obtained by simultaneously using (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside has a significantly better adsorption effect on carnosine and anserine in the crude extract than using (-)-catechin or kaempferol-3-O-sophorobiose-7-O-glucoside alone. This may be because the surface structure of the modified macroporous adsorption resin I obtained by using (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside together is more easily bound to carnosine and anserine, thereby facilitating the improvement of the extraction effect of carnosine and anserine in the homogeneous sample.

[0094] In addition, the carnosine contents in the sample solutions obtained in Examples 5-6 were increased by 53.6% and 66.9% respectively compared with those in Example 2, and the anserine contents in the sample solutions obtained in Examples 5-6 were increased by 38.9% and 45.6% respectively compared with those in Example 1.

[0095] It can be seen that when the macroporous adsorption resin is modified, the modified macroporous adsorption resin I obtained by using (-)-catechin, kaempferol-3-O-sophorobiose-7-O-glucoside and kaempferol-3-O-p-coumaroylrhamnosyl glucoside at the same time has a significantly better adsorption effect on carnosine and anserine in the crude extract than that obtained by using (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside alone. This may be because the combined use of (-)-catechin, kaempferol-3-O-sophorobiose-7-O-glucoside and kaempferol-3-O-p-coumaroylrhamnosyl glucoside can further change the surface structure of the modified macroporous adsorption resin I, making it easier for it to bind to carnosine and anserine, thereby improving the extraction effect of carnosine and anserine in the homogeneous sample.

[0096] Spike recovery test

[0097] The sample solutions obtained in Examples 1-6 and Comparative Examples 1-2 were used to test the matrix effect (ME) of anserine at three gradient levels: low, medium, and high. Spiked recovery tests, including recovery rates and relative standard deviations (RSDs), were performed. The results are shown in Table 4. The ME of fresh, frozen hairtail samples was evaluated and calculated. The results showed that the ME of the sample solutions obtained in Examples 1-6 ranged from -12% to 10%. It is generally accepted that if the ME is above -20% and below 20%, signal enhancement or suppression is acceptable; otherwise, a strong matrix effect is present. The results indicate that the detection of anserine in the sample solutions obtained in Examples 1-6 is essentially free of matrix effect.

[0098] Table 4 Matrix effects, recoveries and relative standard deviations of spike recovery tests in fresh and frozen hairtail

[0099]

[0100] Referring to the data in Table 4, compared with Example 1, the recovery rates of the samples treated according to the technical solutions of Examples 2-6 and Comparative Examples 1-2 were all improved, and the recovery rates of the recovery solutions of Examples 2-6 were all above 85%. It can be seen that during the sample pretreatment process, the use of modified macroporous adsorption resin I to extract the homogenized sample of aquatic product muscle tissue can significantly improve the recovery rate, and the relative standard deviation of the spiked recovery rate is relatively low, which shows that this method has high accuracy and good reproducibility.

[0101] Among them, when the spiked amount was 10 μg / kg, the recoveries of Examples 2-4 were increased by 12.2%, 14.6%, and 17.1% relative to Example 1, respectively; the recoveries of Examples 5-6 were increased by 18.3% and 19.5% relative to Example 1, respectively; the recoveries of Comparative Examples 1-2 were increased by 4.9% and 8.5% relative to Example 1, respectively; when the spiked amount was 50 μg / kg, the recoveries of Examples 2-4 were increased by 15.6%, 16.9%, and 20.1% relative to Example 1, respectively; For Example 1, the recoveries increased by 23.4% and 24.7%, respectively; for Comparative Examples 1-2, the recoveries increased by 7.8% and 10.4%, respectively, compared to Example 1; at a spiked dose of 200 μg / kg, the recoveries for Examples 2-4 increased by 16.4%, 20.5%, and 24.6%, respectively, compared to Example 1; for Examples 5-6, the recoveries increased by 26.0% and 27.4%, respectively, compared to Example 1; and for Comparative Examples 1-2, the recoveries increased by 6.8% and 9.6%, respectively, compared to Example 1. Compared to the modified macroporous adsorption resin I obtained in Example 2, the recoveries of the modified macroporous adsorption resin I obtained in Comparative Examples 1-2 for extracting aquatic product muscle tissue decreased.

[0102] It can be seen that in the process of extracting carnosine and anserine from muscle tissue of aquatic products, the method of using modified macroporous adsorption resin I to treat the crude extract has high accuracy and good reproducibility; and under the same detection conditions and within a certain range, the more modified macroporous adsorption resin I is used, the better the accuracy and reproducibility of the method. Moreover, the recovery rate of the modified macroporous adsorption resin I obtained by using (-)-catechin, kaempferol-3-O-sophorobiose-7-O-glucoside and kaempferol-3-O-p-coumarylrhamnosyl glucoside simultaneously for extracting aquatic product muscle tissue was better than that of the modified macroporous adsorption resin I obtained by using only (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside; the recovery rate of the modified macroporous adsorption resin I obtained by using (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside simultaneously for extracting aquatic product muscle tissue was better than that of the modified macroporous adsorption resin I obtained by using only (-)-catechin or kaempferol-3-O-sophorobiose-7-O-glucoside.

[0103] This may be because (-)-catechin has a smaller molecular weight and simpler molecular structure than kaempferol-3-O-sophorobiose-7-O-glucoside and kaempferol-3-O-p-coumaryl rhamnosyl glucoside. Therefore, when (-)-catechin is used alone during the modification of the macroporous resin, its effect on the adsorption resin microstructure is relatively weak, resulting in relatively weak adsorption and desorption effects on carnosine and anserine. While kaempferol-3-O-sophorobiose-7-O-glucoside has a larger molecular weight and a relatively complex molecular structure, when used alone during the modification of the macroporous resin, the grafting rate of kaempferol-3-O-sophorobiose-7-O-glucoside onto the adsorption resin surface may be affected due to interference with the molecular spatial structure. The simultaneous use of (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside can circumvent the above problems. Moreover, when kaempferol-3-O-p-coumaryl rhamnosyl glucoside is further added, the adsorption and desorption effects of the obtained modified macroporous adsorption resin I on carnosine and anserine are enhanced.

[0104] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0105] The embodiments described above provide a detailed description of the technical solutions of the present invention. However, it should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications or substitutions that a person skilled in the art could readily conceive within the technical scope disclosed herein are intended to fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A method for detecting biological peptides, comprising the following steps: Muscle tissue of the sample was taken and mashed to obtain a homogenous sample for later use; water was added to the homogenous sample, and after crude extraction, adsorption resin was added. After static adsorption, the adsorption resin was collected by filtration and desorbed with ethanol. After filtering out the adsorption resin, the filtrate obtained was used as the sample solution for later use; the content of carnosine and anserine in the sample solution was determined by UHPLC-MS / MS; The adsorption resin is a modified macroporous adsorption resin I, which is obtained by modifying a polystyrene-divinylbenzene copolymer with (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside. The preparation method of the modified macroporous adsorption resin I comprises the following steps: S1. A polystyrene-divinylbenzene copolymer is reacted with chloromethyl methyl ether in the presence of a catalyst to obtain chlorine balls; S2. Add ethylenediamine to the chlorine ball obtained in S1 and react for 5-8 hours to obtain an amino resin; S3. Add formaldehyde, (-)-catechin, and kaempferol-3-O-sophorobiose-7-O-glucoside to the amino resin obtained in S2, react for 4-8 hours, and filter to remove the filtrate to obtain modified macroporous adsorption resin I.

2. The biological polypeptide detection method according to claim 1, characterized in that: The conditions for crude extraction of homogenized samples are as follows: place the homogenized sample in water at a mass-to-volume ratio of 3-10 g:100-200 mL, vortex for 1-5 minutes, perform ultrasonic extraction for 20-60 minutes, and centrifuge at 5000-8000 r / min for 5-10 minutes.

3. The biological polypeptide detection method according to claim 1, characterized in that: The mass ratio of modified macroporous adsorption resin I to homogeneous sample is 1-5:

10.

4. The biological polypeptide detection method according to claim 1, characterized in that: After the static adsorption was completed, 75% ethanol solution was used for desorption treatment, and the mass volume ratio of modified macroporous adsorption resin I to ethanol solution was 0.5-2g:100mL.

5. The biological polypeptide detection method according to claim 1, characterized in that: In step S1, the molar ratio of polystyrene-divinylbenzene copolymer to chloromethyl methyl ether is 1:1.5-5.

6. The biological polypeptide detection method according to claim 1, characterized in that: In step S2, the weight ratio of chlorine balls to ethylenediamine is 1:5-10; In step S2, the reaction is carried out on a constant temperature shaker at 20-35°C with an oscillation frequency of 80-150 r / min.

7. The biological polypeptide detection method according to claim 1, characterized in that: In step S3, the mass volume ratio of the amino resin to formaldehyde is 10-30 g:10-100 mL, and the mass ratio of the amino resin to (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside is 10-20:0.3-2:0.3-2.

8. The biological polypeptide detection method according to claim 1, characterized in that: In step S3, the obtained modified macroporous adsorption resin I is extracted with anhydrous ethanol for 4-8 hours, then washed with deionized water until neutral, and then shaken with 4% sodium hydroxide solution in a constant temperature shaker at 80-150 r / min at 20-35°C for 1-3 hours, and then washed again with deionized water until neutral, and dried in a vacuum drying oven at 45-60°C for 5-8 hours.

Images