Biological polypeptide detection method

Abstract

The invention discloses a biological polypeptide detection method, which belongs to the technical field of biological detection, and comprises the following steps: taking muscular tissues of a sample, and mashing to obtain a homogeneous sample for later use; adding water into the homogeneous sample, carrying out crude extraction, adding adsorption resin, carrying out static adsorption, filtering and collecting the adsorption resin, carrying out desorption treatment by adopting ethanol, and filtering out the adsorption resin to obtain filtrate as a sample solution for later use; detecting the content of carnosine and goose carnosine in the sample solution by adopting a UHPLC-MS / MS combined method; wherein the adsorption resin is polystyrene-divinylbenzene type copolymer macroporous adsorption resin or modified macroporous adsorption resin I, and the modified macroporous adsorption resin I is obtained by carrying out modification treatment on a polystyrene-divinylbenzene type copolymer by adopting (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside. The method is easy to operate, high in accuracy and good in reproducibility, and the extraction effect on carnosine and goose carnosine can be improved.

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] At present, there are few methods for detecting carnosine and anserine in China. According to literature reports, the main methods for determining carnosine and anserine are high performance liquid chromatography, ion exchange chromatography and capillary electrophoresis. High performance liquid chromatography effectively achieves the determination of carnosine and anserine, but this method uses a wavelength of 210nm for determination, which has a high baseline and is easily interfered with. The accuracy of the method still needs to be investigated.

[0003] The pretreatment technology of carnosine and anserine is mainly ultrasonic extraction, which uses the derivatization of standard working solutions, but the steps are complicated, and the oil interference in the matrix of aquatic products affects the content detection of carnosine and anserine. The extraction method in NY / T 3946-2021 "High Performance Liquid Chromatography Method for the Determination of Carnosine and Anserine in Animal-derived Foods" has poor separation effect, and GB / T 39101-2020 "Inhibition Zone Method for Determination of Antibacterial Activity of Polypeptides" and GB / T 39100-2020 "DPPH and ABTS Method for Determination of Antioxidant Activity of Polypeptides" are not suitable for the detection of carnosine and anserine in aquatic products, and the extraction effect is not good. Due to the large amount of impurities and oil in the matrix of aquatic products, the extraction effect of carnosine and anserine is affected, and it is necessary to establish an efficient and convenient extraction method. Summary of the invention

[0004] The purpose of the present invention is to provide a biological polypeptide detection method (about carnosine and anserine) to improve the extraction rate of carnosine and anserine in aquatic products. The method is simple to operate, high in accuracy and good in reproducibility.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A method for detecting biological peptides comprises the following steps: The muscle tissue of the sample is taken, and the homogenized sample is obtained after being crushed for later use; water is added to the homogenized sample, and after crude extraction, adsorption resin is added, and after static adsorption, the adsorption resin is collected by filtration, and ethanol is used for desorption treatment, and after the adsorption resin is filtered out, the obtained filtrate is used as the sample solution for later use; the contents of carnosine and anserine in the sample solution are detected by UHPLC-MS / MS coupling method.

[0006] 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.

[0007] The modified macroporous adsorption resin I obtained by modifying polystyrene-divinylbenzene copolymer with (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside can significantly improve the adsorption effect of carnosine and anserine in the sample, thereby helping to reduce the detection error. In addition, the desorption rate of the obtained modified macroporous adsorption resin I is significantly improved compared with that of the unmodified polystyrene-divinylbenzene copolymer macroporous adsorption resin.

[0008] Preferably, the conditions for crude extraction of the homogenized sample are: placing the homogenized sample in deionized water at a mass volume ratio of 3-10 g:100-200 mL, vortexing for 1-5 min, placing it in an ultrasonic instrument for ultrasonic extraction for 20-60 min, and centrifuging at 5000-8000 r / min for 5-10 min.

[0009] Preferably, the mass ratio of the modified macroporous adsorption resin I to the homogeneous sample is 1-5:10.

[0010] 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.

[0011] Preferably, the desorption treatment conditions are on a constant temperature shaker, 40-55° C., 120 r / min shaking for 15-30 h.

[0012] 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: A Waters Acquity UPLC BEH Amide column (100 mm×2.1 mm, 1.7 μm) was used for separation; column temperature: 35 °C; mobile phase A was 2 mmol / L ammonium acetate 0.1% formic acid solution, and mobile phase B was 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.

[0013] The mass spectrometer settings were as follows: electrospray ionization source (ESI) operated under positive ionization conditions, multiple reaction monitoring mode; capillary voltage: 3 kV; ion source temperature: 150 °C; desolvation temperature: 600 °C; cone gas flow: 150 L / h; desolvation gas flow: 1000 L / h.

[0014] 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.

[0015] According to one aspect of the present invention, there is provided a method for preparing a modified macroporous adsorption resin, comprising the following steps: S1. Preparation of chlorine balls: polystyrene-divinylbenzene copolymer, reacted with chloromethyl methyl ether in the presence of a catalyst to obtain chlorine balls; S2. Preparation of amino resin: Add ethylenediamine to the chlorine ball obtained in S1 and react for 5-8h 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, filter and remove the filtrate to obtain modified macroporous adsorption resin I.

[0016] Preferably, in step S1, the polystyrene-divinylbenzene copolymer is pre-swelled with a swelling agent for 1-4 hours before the reaction, and then reacted with chloromethyl methyl ether. The swelling agent is dichloromethane, and the mass volume ratio of the polystyrene-divinylbenzene copolymer to dichloromethane is 3-8g:5-20mL.

[0017] Preferably, in step S1, the molar ratio of the polystyrene-divinylbenzene copolymer to chloromethyl methyl ether is 1:1.5-5.

[0018] 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.

[0019] Preferably, in step S1, the polystyrene-divinylbenzene copolymer and chloromethyl methyl ether are first reacted at 3-10° C. for 1-4 h, and then the temperature is raised to 20-35° C. for reaction for 4-10 h.

[0020] Preferably, in step S1, the catalyst is added to the polystyrene-divinylbenzene copolymer and chloromethyl methyl ether system in portions under the reaction condition of 3-10°C.

[0021] Preferably, in step S2, the chlorine ball is pre-swelled with a swelling agent for 1-4 hours before the reaction, and then reacted with ethylenediamine. The swelling agent is tetrahydrofuran, and the mass volume ratio of the chlorine ball to tetrahydrofuran is 3-8g:5-20mL.

[0022] Preferably, in step S2, the weight ratio of chlorine balls to ethylenediamine is 1:5-10.

[0023] Preferably, in step S2, the reaction is carried out on a constant temperature shaker at 20-35°C and the shaking frequency is 80-150 r / min.

[0024] Preferably, in step S3, the amino resin is pre-swelled with a swelling agent for 1-4 hours before the reaction, and then reacted with (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside. The swelling agent is tetrahydrofuran, and the mass volume ratio of the amino resin to tetrahydrofuran is 3-8 g:5-20 mL.

[0025] Preferably, 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.

[0026] Preferably, in step S3, the reaction is carried out on a constant temperature shaker at 20-35°C and the shaking frequency is 80-150 r / min.

[0027] 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 at 80-150r / min for 1-3h, and then washed with deionized water again until neutral, and dried in a vacuum drying oven at 45-60°C for 5-8h.

[0028] 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-sopharobiose-7-O-glucoside.

[0029] 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.

[0030] The modified macroporous adsorption resin I obtained by using (-)-catechin, kaempferol-3-O-sophorobiose-7-O-glucoside and kaempferol-3-O-coumaroyl rhamnosyl glucoside at the same time had a better adsorption effect on carnosine and anserine in the crude extract than that using only (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside. This may be because the combined use of (-)-catechin, kaempferol-3-O-sophorobiose-7-O-glucoside and kaempferol-3-O-coumaroyl rhamnosyl glucoside can further change the surface structure of the modified macroporous adsorption resin I, making it easier to combine with carnosine and anserine, thereby helping to improve the extraction effect of carnosine and anserine in the homogenized sample.

[0031] Compared with the prior art, the present invention has the following beneficial effects: 1. The modified macroporous adsorption resin I was obtained by modifying the polystyrene-divinylbenzene copolymer macroporous adsorption resin with (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside, which can improve the extraction effect of carnosine and anserine in the muscle tissue of aquatic products. This may be because the use of (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside in the modification process changes the microstructure of the polystyrene-divinylbenzene copolymer macroporous adsorption resin, increases the specific surface area of ​​the resin, and thus helps to improve the adsorption performance of the resin. In addition, the presence of phenol groups and polyhydroxy structures in (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside makes it easier for the modified macroporous adsorption resin I to adsorb and bind to carnosine and anserine, which also helps to improve the extraction effect of carnosine and anserine in the muscle tissue of aquatic products.

[0032] 2. The modified macroporous adsorption resin I was obtained by modifying the polystyrene-divinylbenzene copolymer macroporous adsorption resin with (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside. The recovery rate of carnosine and anserine in the muscle tissue of aquatic products was greatly improved. The modified macroporous adsorption resin I had good adsorption and desorption effects on carnosine and anserine, thereby reducing the error in the detection process. This may be because the use of (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside in the modification process introduced polyhydroxy groups on the surface of the adsorption resin, which enhanced the hydrophilicity of the resin, making it easier for carnosine and anserine adsorbed on the resin surface to detach during the desorption process. 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 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.

[0033] 3. In the process of modifying the 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 helps to further improve the adsorption effect and desorption rate of the modified macroporous adsorption resin I for carnosine and anserine. This may be because the molecular structures of kaempferol-3-O-sophorobiose-7-O-glucoside and kaempferol-3-O-p-coumaroyl rhamnosyl glucoside are complex and contain a large number of phenolic hydroxyl groups and alcoholic hydroxyl groups. The active hydrogen atoms in the molecules are easy to react with aldehydes and amines. In addition, the molecular structure of kaempferol-3-O-p-coumaroyl rhamnosyl glucoside is more complex, and the olefinic bond, carbonyl and other structures in it also affect the microscopic results of the modified macroporous adsorption resin I, thereby improving its adsorption and desorption effects on carnosine and anserine. This may be because during the reaction process, (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside and / or kaempferol-3-O-p-coumaroyl rhamnosyl glucoside interact with each other, which may further improve the spatial structure of the modified macroporous adsorption resin I obtained, and may also promote the formation of intramolecular and extramolecular hydrogen bonds, thereby facilitating the adsorption and desorption of carnosine and anserine.

[0034] 4. In the process of separating and purifying carnosine and anserine in aquatic products, the crude extract was treated with the modified macroporous adsorption resin I mentioned above, and the obtained sample solution was subjected to UHPLC-MS / MS detection after adsorption and desorption. This method has high accuracy and good reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The chromatograms of the carnosine and anserine standard solution used in the test examples of the present invention are as follows; Figure 2 The chromatogram is a 10-fold signal-to-noise ratio quantitative limit of the standard working solution of carnosine and anserine in the test example of the present invention. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0037] Example 1 The content of carnosine and anserine in aquatic products was determined by high performance liquid chromatography tandem mass spectrometry. The operation steps and analysis conditions are as follows: Sample pretreatment After thawing the fresh frozen hairtail with ice water, the muscle tissue with bones removed was fully and evenly crushed with a high-speed tissue crusher to obtain a homogeneous sample, which was frozen and stored at -18°C for later use.

[0038] The homogenized sample was weighed and crushed by liquid nitrogen freezing. The powder sample was placed in deionized water at a mass 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.

[0039] Add polystyrene-divinylbenzene copolymer macroporous adsorption resin to the crude extract, the mass ratio of macroporous adsorption resin to homogenized sample is 1:10, and oscillate on a constant temperature shaker at 30°C and 120r / min for 24h for static adsorption. Then filter and collect the macroporous adsorption resin, add 75% ethanol solution to it at a mass volume ratio of 0.5g:100mL, and oscillate on a constant temperature shaker at 50°C and 120r / min for 24h. After filtering out the macroporous adsorption resin, heat the filtrate in a water bath at 80°C for 2h to obtain a sample solution for standby use.

[0040] 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 analysis conditions were as follows: A Waters Acquity UPLC BEH Amide column (100 mm×2.1 mm, 1.7 μm) was used for separation; column temperature: 35 °C; mobile phase A was 2 mmol / L ammonium acetate, 0.1% formic acid solution, and mobile phase B was 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.

[0041] The mass spectrometer settings were as follows: electrospray ionization source (ESI) operated under positive ionization conditions, multiple reaction monitoring mode; capillary voltage: 3 kV; ion source temperature: 150 °C; desolvation temperature: 600 °C; cone gas flow: 150 L / h; desolvation gas flow: 1000 L / h.

[0042] 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 in the multiple reaction monitoring mode are shown in Table 1.

[0043] Table 1 Selected reaction monitoring precursor ions, product ions and collision voltages

[0044] Example 2 Compared with Example 1, this embodiment is different in that: during the sample pretreatment process, modified macroporous adsorption resin I is used to replace the macroporous adsorption resin, the mass ratio of modified macroporous adsorption resin I to the homogenized sample is 1:10, and static adsorption is performed on a constant temperature shaker at 30°C and 120r / min for 24h. Then, the modified macroporous adsorption resin I is collected by filtration, and 100mL of 25% ethanol solution is added thereto, and the modified macroporous adsorption resin I is filtered out, and the filtrate is heated in a water bath at 80°C for 2h to obtain a sample solution for standby use.

[0045] Other conditions and steps are the same.

[0046] The preparation method of modified macroporous adsorption resin I is as follows: S1. Preparation of chlorine balls: polystyrene-divinylbenzene copolymer was swollen in dichloromethane at a mass volume ratio of 5g:10mL for 2h, then chloromethyl methyl ether was added, the temperature was controlled at 4°C for 2h, SnCl4 was added dropwise four times during the reaction, and then the temperature was raised to 25°C for 6h. After the reaction, the microspheres were filtered and collected. Among them, the molar ratio of white ball, chloromethyl methyl ether and SnCl4 was 1:2.5:8. The obtained microspheres were filtered and washed with deionized water and vacuum dried at 60°C for 12h to obtain chlorine balls.

[0047] S2. Preparation of amino resin: tetrahydrofuran was added to the chlorine ball obtained in S1 at a mass volume ratio of 5g:10mL to swell for 2h, ethylenediamine was added thereto, and the mixture was shaken on a constant temperature shaker at 30°C and 120r / min for 6h to introduce amine groups into the chlorine ball. After the reactant was cooled to 25°C, it was filtered with deionized water, washed and dried to obtain amino resin. The weight ratio of chlorine ball to ethylenediamine was 1:8.

[0048] S3. Tetrahydrofuran was added to the amino resin obtained in S2 at a mass volume ratio of 5 g:10 mL to swell for 2 h, and formaldehyde and (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside were added thereto, and the mixture was shaken on a constant temperature shaker at 30°C and 120 r / min for 6 h, and the filtrate was filtered to remove the filtrate to obtain modified macroporous adsorption resin I. The mass 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.

[0049] In addition, the obtained modified macroporous adsorption resin I was extracted with anhydrous ethanol for 6 hours before use, then washed with deionized water to neutrality, and then treated with 4% sodium hydroxide solution in a constant temperature shaker at 30°C, oscillated at 120 r / min for 2 hours, washed again with deionized water to neutrality, and dried in a vacuum drying oven at 60°C for 6 hours for use.

[0050] Example 3 Compared with Example 2, this example is different in that the mass ratio of the modified macroporous adsorption resin I to the homogeneous sample is 2:10. Other conditions and steps are the same.

[0051] Example 4 Compared with Example 2, this example is different in that the mass ratio of the modified macroporous adsorption resin I to the homogeneous sample is 3:10. Other conditions and steps are the same.

[0052] Example 5 Compared with Example 2, the present embodiment is different in that: in the preparation method of modified macroporous adsorption resin I, in step S3, while adding formaldehyde and (-)-catechin and kaempferol-3-O-sophobiose-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-sophobiose-7-O-glucoside and kaempferol-3-O-p-coumaroyl rhamnosyl glucoside is 10:1:1:1.

[0053] Other conditions and steps are the same.

[0054] Example 6 Compared with Example 2, the present embodiment is different in that: in the preparation method of modified macroporous adsorption resin I, in step S3, while adding formaldehyde and (-)-catechin and kaempferol-3-O-sophobiose-7-O-glucoside to the reaction system of S2, kaempferol-3-O-p-coumaroyl rhamnosyl glucoside is added, and the mass ratio of polystyrene-divinylbenzene copolymer to (-)-catechin, kaempferol-3-O-sophobiose-7-O-glucoside and kaempferol-3-O-p-coumaroyl rhamnosyl glucoside is 10:1:1:2.

[0055] Other conditions and steps are the same.

[0056] Comparative Example 1 Compared with Example 2, this example is different 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.

[0057] Comparative Example 2 Compared with Example 2, this example is different 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.

[0058] Test example Adopt high performance liquid chromatography tandem mass spectrometry, measure the content of carnosine and anserine in carnosine and anserine standard solution, operation steps and analysis conditions refer to embodiment 1. Carnosine and anserine standard solution preparation method is as follows: Carnosine and anserine standard powder was used to prepare a carnosine and anserine standard stock solution with a concentration of 100 μg / mL; the above carnosine and anserine standard stock solution was then used to prepare a carnosine and anserine standard intermediate solution with a concentration of 1.0 μg / mL; the above carnosine and anserine standard intermediate solution was used to prepare a carnosine and anserine standard working solution with a concentration of 100 ng / mL.

[0059] Under the above analytical conditions, the separation effect of carnosine and anserine in the standard solution of carnosine and anserine was good, see Figure 1 .

[0060] The carnosine and anserine standard working solutions were diluted with deionized water to a series of standard working solutions of 1.00, 5.00, 10.0, 20.0, 50.0, and 100 ng / mL. The determination was carried out under the above analytical conditions. The quantification limits of carnosine and anserine were calculated according to the 10-fold signal-to-noise ratio S / N. The results are shown in Table 2. The quantification limits of carnosine and anserine are both 10 μg / kg. The chromatograms of the 10-fold signal-to-noise ratio quantification limits of carnosine and anserine are shown in Figure 2 .

[0061] Table 2 Linear range and detection limit

[0062] See Table 3 for the contents of carnosine and anserine in the sample solutions obtained in Examples 1-6 and Comparative Examples 1-2.

[0063] Table 3 Contents of carnosine and anserine in the sample solutions obtained in various examples and comparative examples (%)

[0064] 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.

[0065] Among them, the carnosine contents in the sample solutions obtained in Examples 2-4 were increased by 42.8%, 157.1%, and 214.3% respectively compared with 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 with 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 with 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 with those in Example 1.

[0066] 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 extracts of aquatic products.

[0067] Compared with Example 1, when the amount of adsorption resin used is the same, the use of modified macroporous adsorption resin I (Example 2, Comparative Example 1-2) can significantly improve the adsorption effect of carnosine and anserine in the crude extract of aquatic products. It can be seen that the use of (-)-catechin and / or kaempferol-3-O-sophorobiose-7-O-glucoside to modify the polystyrene-divinylbenzene type copolymer macroporous adsorption resin, and the obtained modified macroporous adsorption resin I to treat the homogenized sample of aquatic product muscle tissue can greatly improve the extraction effect of carnosine and anserine. This may be because the modified macroporous adsorption resin I obtained by the modification treatment has a changed microstructure compared with the ordinary macroporous adsorption resin, which improves the adsorption effect of carnosine and anserine in the crude extract of aquatic products, and in the desorption process, the desorption effect of carnosine and anserine is also improved.

[0068] Moreover, the contents of carnosine and anserine in the sample solution obtained in Example 2 were significantly higher than those in the sample solution obtained in Comparative Examples 1-2. It can be seen that when the macroporous adsorption resin is modified, the modified macroporous adsorption resin I obtained by using (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside at the same time has a significantly better adsorption effect on carnosine and anserine in the crude extract than using (-)-catechin alone 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 in combination is easier to combine with carnosine and anserine, thereby facilitating the improvement of the extraction effect of carnosine and anserine in the homogeneous sample.

[0069] 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.

[0070] 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-coumaroyl rhamnosyl glucoside at the same time has a significantly better adsorption effect on carnosine and anserine in the crude extract than that using only (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside. This may be because the use of (-)-catechin, kaempferol-3-O-sophorobiose-7-O-glucoside and kaempferol-3-O-coumaroyl rhamnosyl glucoside can further change the surface structure of the modified macroporous adsorption resin I, making it easier to combine with carnosine and anserine, thereby helping to improve the extraction effect of carnosine and anserine in the homogenized sample.

[0071] Spike recovery test The sample solutions obtained in Examples 1-6 and Comparative Examples 1-2 were used to conduct the matrix effect (ME) of anserine at three gradient levels of low, medium and high, spike recovery test, recovery rate and relative standard deviation (RSD), and the results are shown in Table 4. The ME of the fresh frozen hairtail samples was evaluated and calculated, and the results showed that the ME of the sample solutions obtained in Examples 1-6 ranged from -12% to 10%. It is generally believed that if the ME is higher than -20% and lower than 20%, signal enhancement or suppression is acceptable, otherwise there is a strong matrix effect. From the results, the detection of anserine in the sample solutions obtained in Examples 1-6 has basically no matrix effect.

[0072] Table 4 Matrix effects, recoveries and relative standard deviations of spike recovery tests in fresh and frozen hairtail

[0073] Referring to the data in Table 4, compared with Example 1, the recovery rates of samples treated according to the technical solutions of Examples 2-6 and Comparative Examples 1-2 were improved, and the recovery rates of Examples 2-6 were all above 85%. It can be seen that in 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.

[0074] 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% respectively relative to Example 1; the recoveries of Examples 5-6 were increased by 18.3% and 19.5% respectively relative to Example 1; the recoveries of Comparative Examples 1-2 were increased by 4.9% and 8.5% respectively relative to Example 1; when the spiked amount was 50 μg / kg, the recoveries of Examples 2-4 were increased by 15.6%, 16.9%, and 20.1% respectively relative to Example 1; For Example 1, the recovery rates were increased by 23.4% and 24.7% respectively; the recovery rates of Comparative Examples 1-2 were increased by 7.8% and 10.4% respectively compared with Example 1; when the spiked amount was 200 μg / kg, the recovery rates of Examples 2-4 were increased by 16.4%, 20.5% and 24.6% respectively compared with Example 1; the recovery rates of Examples 5-6 were increased by 26.0% and 27.4% respectively compared with Example 1; the recovery rates of Comparative Examples 1-2 were increased by 6.8% and 9.6% respectively compared with Example 1. Compared with the modified macroporous adsorption resin I obtained in Example 2, the recovery rates of the experimental schemes for extracting aquatic product muscle tissue using the modified macroporous adsorption resin I obtained in Comparative Examples 1-2 were all reduced.

[0075] 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, 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-coumaroyl rhamnosyl glucoside simultaneously for extracting aquatic product muscle tissue is 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 is better than that of the modified macroporous adsorption resin I obtained by using (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside alone.

[0076] This may be because (-)-catechin has a small molecular weight and a simple molecular structure compared to kaempferol-3-O-sophorobiose-7-O-glucoside and kaempferol-3-O-p-coumaroyl rhamnosyl glucoside. In the process of modifying the macroporous resin, if (-)-catechin is used alone, the effect on the microstructure of the adsorption resin is relatively weak, and the adsorption and desorption effects on carnosine and anserine are relatively weak. Although kaempferol-3-O-sophorobiose-7-O-glucoside has a large molecular weight and a relatively complex molecular structure, in the process of modifying the macroporous resin, if kaempferol-3-O-sophorobiose-7-O-glucoside is used alone, the grafting rate of kaempferol-3-O-sophorobiose-7-O-glucoside on the surface of the adsorption resin may be affected due to the interference of the molecular spatial structure. The simultaneous use of (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside can circumvent the above problems, and when kaempferol-3-O-p-coumaroyl rhamnosyl glucoside is further added, the adsorption and desorption effects of the obtained modified macroporous adsorption resin I on carnosine and anserine are enhanced.

[0077] 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.

[0078] The above-described embodiments provide a detailed description of the technical solution of the present invention. It should be understood that the above-described embodiments are only specific embodiments of the present invention and are not intended to limit the present invention. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A method for detecting biological peptides, comprising the following steps: Take the muscle tissue of the sample, mash it to obtain a homogenous sample, and set it aside; add water to the homogenous sample, and after rough extraction, add adsorption resin, after static adsorption, filter and collect the adsorption resin, and use ethanol for desorption treatment, filter out the adsorption resin, and use the filtrate obtained as the sample solution for use; use UHPLC-MS / MS combined method to detect the content of carnosine and anserine in the sample solution; 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.

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

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, the desorption treatment used 75% ethanol solution, and the mass volume ratio of the modified macroporous adsorption resin I to the ethanol solution was 0.5-2g:100mL.

5. A method for preparing a modified macroporous adsorption resin, comprising 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, filter and remove the filtrate to obtain modified macroporous adsorption resin I.

6. The method for preparing the modified macroporous adsorption resin according to claim 5, characterized in that: In step S1, the molar ratio of the polystyrene-divinylbenzene copolymer to chloromethyl methyl ether is 1:1.5-5.

7. The method for preparing the modified macroporous adsorption resin according to claim 5, 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.

8. The method for preparing the modified macroporous adsorption resin according to claim 6, 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.

9. The method for preparing the modified macroporous adsorption resin according to claim 5, 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 20-35°C, 80-150r / min for 1-3 hours, and then washed with deionized water again until neutral, and dried in a vacuum drying oven at 45-60°C for 5-8 hours.

10. The modified macroporous adsorption resin obtained according to any one of the preparation methods of claims 5 to 9, characterized in that: The modified macroporous adsorption resin I is obtained by modifying a polystyrene-divinylbenzene copolymer with (-)-catechin and kaempferol-3-O-sophorobiose-7-O-glucoside.

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