Method for detecting content of goose carnosine in animal-derived peptide product

Abstract

The invention discloses a method for detecting the content of goose carnosine in an animal-derived peptide product, and belongs to the technical field of small molecule peptide detection. The detection method comprises the following steps: preparing a control sample and a sample to be tested, carrying out HPLC detection, drawing a standard curve, and calculating the mass percentage content of the goose carnosine in the sample to be tested according to the standard curve, the method comprises the following steps: preparing a to-be-detected solution by using mobile phases (a mobile phase A and a mobile phase B are mixed in equal volume, the mobile phase A is methanol, the mobile phase B is a 60mM phosphate buffer solution with the pH value of 6.0), and filtering the to-be-detected solution by using a filter membrane for later use; the method has the beneficial effects that the detection method provided by the invention has the advantages that the operation is simple, a sample does not need to be derived, the goose carnosine separation effect is relatively good, the chromatographic peak is relatively symmetrical, and the content of the goose carnosine in the animal source peptide product can be rapidly and reliably detected.

Description

Technical Field

[0001] The invention relates to a method for detecting the content of anserine, in particular to a method for detecting the content of anserine in an animal-derived peptide product, and belongs to the technical field of small molecule peptide detection. Background Art

[0002] Tuna peptide is a small molecule peptide product made from tuna meat, which is extracted and purified by bio-protease hydrolysis. Tuna peptide has attracted wide attention due to its easy absorption, high activity, antioxidant, anti-fatigue, and uric acid lowering characteristics. Its main active ingredient is anserine (β-alanyl-1-methyl L-histidine).

[0003] (1) Anti-fatigue: Anserine is a highly stable water-soluble dipeptide. The imidazole ring in its structure determines that it has good pH buffering capacity, which can neutralize lactic acid in the body and relieve muscle fatigue after exercise.

[0004] (2) Antioxidant: Under physiological conditions, the molecular conformation of anserine is in the shape of a bent pliers, which can embed a variety of free radicals in its molecular cavity to form stable adducts, thus showing a strong antioxidant effect.

[0005] (3) Lowering uric acid: The imidazole group in the structure of anserine can inhibit uric acid production and promote uric acid excretion by regulating the enzyme. In addition, since the imidazole group has proton buffering capacity, anserine can stabilize the pH value of urine, increase the solubility of uric acid and accelerate uric acid excretion.

[0006] The content of anserine in tuna peptides is the focus of everyone's attention. The content of anserine in different varieties of tuna varies greatly; even for the same variety of tuna, the content of anserine in different parts (such as back meat, belly meat, head meat, dark meat, fish bones, viscera, etc.) varies greatly; in addition, tuna contains carnosinase, which can cause anserine to decompose, so differences in fishing and storage conditions will also lead to significant differences in anserine content.

[0007] At present, there is no relevant national standard for the detection method of anserine content, and the detection research is mainly focused on the detection of anserine content in animal-derived muscle tissue, while there is little research on the detection of anserine content in animal-derived peptide products. The national standard NY / T 3946 "High-performance liquid chromatography method for the determination of carnosine and anserine in animal-derived foods" is only applicable to the determination of carnosine and anserine content in animal-derived muscle tissue (anserine is a water-soluble dipeptide that exists freely in muscle tissue, so the water extraction method is used to extract anserine from muscle tissue. The muscle tissue water extract is mainly anserine, and also contains a small amount of water-soluble free amino acids and nucleotides, etc. These water-soluble free amino acids and nucleotides have less interference with the detection results of anserine), and is not applicable to peptide products prepared by biological enzymatic hydrolysis of animal-derived muscle tissue, because peptide products are mainly various small molecule peptides, the types of peptides are complex and diverse, and some small molecule peptides will react with the derivatization reagents in this method, causing serious interference to the detection.

[0008] Tuna peptide products on the market vary greatly in quality, so it is necessary to establish a rapid and reliable method for detecting anserine in tuna peptides. Summary of the invention

[0009] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a method for detecting the content of anserine in animal-derived peptide products, which is simple to operate, does not require sample derivatization, and can quickly and reliably detect the content of anserine in animal-derived peptide products.

[0010] In order to achieve the above object, the present invention adopts the following technical solution: The method for detecting the content of anserine in an animal-derived peptide product comprises the following steps: (1) Preparation of control samples: accurately weigh anserine standard, prepare anserine standard solutions of different concentrations with mobile phase, filter through a filter membrane and set aside, wherein the mobile phase is a mixture of mobile phase A and mobile phase B in a volume ratio of 1:1, mobile phase A is methanol, and mobile phase B is a 60 mM, pH = 6.0 phosphate buffer; (2) Preparation of test samples: Weigh the sample to be tested, prepare a test solution of appropriate concentration with mobile phase, filter it through a filter membrane and set it aside for use, wherein the mobile phase is a mixture of mobile phase A and mobile phase B in a volume ratio of 1:1, mobile phase A is methanol, and mobile phase B is 60 mM phosphate buffer with a pH of 6.0; (3) Performing HPLC detection: Performing HPLC detection on the anserine standard solution and the test solution respectively; (4) Draw a standard curve: Draw a standard curve with the concentration of anserine standard solution as the horizontal axis and the peak area of ​​HPLC as the vertical axis; (5) Calculate the mass percentage of anserine in the test sample: Calculate the concentration of anserine from the standard curve based on the peak area, and then calculate the mass percentage of anserine in the test sample.

[0011] Preferably, in step (1), the concentration of the anserine standard solution is in the range of 100 μg / mL-500 μg / mL.

[0012] Preferably, in step (2), the concentration of the test solution is in the range of 100 μg / mL-500 μg / mL.

[0013] Preferably, in step (3), the chromatographic conditions are: Column: Gemsil NH 2 , 250mm×4.6mm, 5µm; Mobile phase: Mobile phase A is methanol; mobile phase B is 60mM, pH=6.0 phosphate buffer; Elution conditions: 0-15 min, 40%→55% mobile phase A, 60%→45% mobile phase B; 15-35 min, 55% mobile phase A, 45% mobile phase B; Detection wavelength: 210nm; Detection temperature: 27℃; Injection volume: 20µL; Detection flow rate: 0.8mL / min.

[0014] Preferably, in step (5), the formula for calculating the mass percentage of anserine in the test sample is:

[0015] In the formula, W is the mass percentage of anserine in the test sample (%); C is the concentration of anserine obtained from the standard curve, in μg / mL; V is the fixed volume of the test sample solution, in mL; m is the sampling mass of the test sample, in g; 10 -6 is the factor for converting μg to g.

[0016] The benefits of the present invention are as follows: the detection method provided by the present invention has the advantages of simple operation and no need for sample derivatization, and the anserine separation effect is good, the chromatographic peaks are relatively symmetrical, and the content of anserine in animal-derived peptide products can be quickly and reliably detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the standard curve of anserine standard; Figure 2 is the HPLC chart of anserine standard solution with a concentration of 100 μg / mL; Figure 3 is the HPLC chart of the first solution to be tested; Figure 4 is the HPLC chart of the second solution to be tested; Figure 5 This is the HPLC chart of the third solution to be tested. DETAILED DESCRIPTION

[0018] Taking tuna peptide as an example of an animal-derived peptide product, the present invention is specifically introduced below in conjunction with the accompanying drawings and specific embodiments.

[0019] 1. Detection method The method for detecting the content of anserine in an animal-derived peptide product provided by the present invention specifically comprises the following steps: 1. Prepare samples (1) Control sample Accurately weigh 0.0125 g of anserine standard into a 25 mL volumetric flask, and dilute to volume with mobile phase (mobile phase A: mobile phase B = 1:1, v / v) to prepare a stock solution with a concentration of 500 μg / mL.

[0020] Accurately measure the above stock solution and use the mobile phase (mobile phase A: mobile phase B = 1:1, v / v) to prepare anserine standard solutions of different concentrations, namely 100μg / mL, 200μg / mL, 300μg / mL, 400μg / mL, and 500μg / mL. Filter through a 0.2µm filter membrane (polytetrafluoroethylene or nylon) and set aside.

[0021] (2) Test samples Weigh 3 samples to be tested, each weighing about 500 mg, and place them in beakers respectively. Dissolve them with mobile phase (mobile phase A: mobile phase B = 1:1, v / v), then transfer them to a 50 mL volumetric flask, and make up to a suitable concentration (within the range of 100 μg / mL-500 μg / mL) of the test solution with mobile phase (mobile phase A: mobile phase B = 1:1, v / v). Filter through a 0.2 μm filter membrane (polytetrafluoroethylene or nylon) and set aside.

[0022] Mobile phase A: methanol.

[0023] Mobile phase B: 60 mM phosphate buffer (monopotassium phosphate, disodium hydrogen phosphate), pH = 6.0.

[0024] 2. Determine the chromatographic conditions (1) Investigate the effect of different mobile phase systems on the separation of anserine in samples The effects of four mobile phase systems, methanol-phosphate buffer, methanol-water, acetonitrile-phosphate buffer and acetonitrile-water, on the separation of anserine in the sample were investigated respectively.

[0025] The results showed that when the mobile phase contained acetonitrile, the tuna peptide sample would produce a large amount of precipitation, affecting the detection results, so acetonitrile was not considered; both the methanol-phosphate buffer and methanol-water mobile phase systems could separate anserine from other substances in tuna peptides, among which the separation effect of methanol-phosphate buffer was better than that of methanol-water, so methanol-phosphate buffer was selected as the mobile phase system.

[0026] (2) Investigate the effect of specific mobile phase parameters on the separation of anserine in samples The effects of the concentration, pH value and mixing ratio of mobile phase A (methanol) and mobile phase B (phosphate buffer) on the separation of anserine in the sample were investigated.

[0027] The results showed that when the concentration of mobile phase B (phosphate buffer) was too low, the anserine peak was prone to asymmetric peak shape and tailing phenomenon; when the pH value of mobile phase B (phosphate buffer) was too high or too low, anserine could not be completely separated from the impurity peaks; when the mixing ratio of mobile phase A (methanol) and mobile phase B (phosphate buffer) was low, anserine could not be completely separated from the impurity peaks, and when the mixing ratio was high, the separation degree between anserine and the impurity peaks increased, but the retention value of anserine became larger; when the concentration of mobile phase B (phosphate buffer) was 60mmol / L, pH=6.0, and the mixing ratio of mobile phase A (methanol) and mobile phase B (phosphate buffer) was 55:45 (v / v), the chromatographic peaks of anserine in tuna peptides were well separated, the chromatographic peaks were relatively symmetrical, and its peak area could be accurately determined for quantitative analysis.

[0028] (3) Determine elution conditions and other chromatographic conditions Detection instrument: high performance liquid chromatography; Column: Gemsil NH 2 (250mm×4.6mm, 5µm); Mobile phase: Mobile phase A is methanol; mobile phase B is 60mM, pH=6.0 phosphate buffer (monopotassium phosphate, disodium hydrogen phosphate); Elution conditions: 0-15 min, 40%→55% mobile phase A, 60%→45% mobile phase B; 15-35 min, 55% mobile phase A, 45% mobile phase B; Detection wavelength: 210nm; Detection temperature: 27℃; Injection volume: 20µL; Detection flow rate: 0.8mL / min 3. Draw the standard curve HPLC detection was performed on five different concentrations of anserine standard solutions (100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL). The HPLC profile of the 100 μg / mL anserine standard solution is shown in Figure 2 .

[0029] The concentration of anserine standard solution was used as the horizontal axis and the peak area of ​​HPLC was used as the vertical axis to draw a standard curve. The results are shown in Figure 1 .

[0030] Depend on Figure 1 It can be seen that when the concentration of anserine is in the range of 100-500µg / mL, the data has a good linear relationship, and the regression equation is y= 35.607x+296.05, R 2 =0.9997.

[0031] 4. Calculate the mass percentage of anserine in the test sample The three solutions to be tested were subjected to HPLC detection. The HPLC graphs of the three solutions to be tested are shown in Figure 3 , Figure 4 and Figure 5 .

[0032] The concentration of anserine was obtained from the standard curve according to the peak area, and then the mass percentage of anserine in each test sample was calculated according to the following formula:

[0033] In the formula, W is the mass percentage of anserine in the test sample (%); C is the concentration of anserine obtained from the standard curve, in μg / mL; V is the fixed volume of the test sample solution, in mL; m is the sampling mass of the test sample, in g; 10 -6 is the factor for converting μg to g.

[0034] After calculation, the mass percentages of anserine in the three test samples were 10.06%, 6.86% and 4.01% respectively.

[0035] 2. Investigate sample recovery, sample stability and chromatographic peak symmetry 1. Sample recovery rate Accurately weigh 5 portions of anserine standard, about 5 mg each, and use mobile phase (mobile phase A: mobile phase B = 1:1, v / v) to make up to 50 mL. After filtering through a 0.2 µm filter membrane (polytetrafluoroethylene or nylon), perform HPLC detection and record the peak area. Calculate the anserine content based on the peak area and the standard curve. The calculation results are shown in Table 1.

[0036] Table 1 Sample recovery rate

[0037] It can be seen from Table 1 that the sample recovery rate is in the range of 97.0-103.0%, with an average value of 99.43%. The test results are accurate and reliable.

[0038] 2. Sample stability Accurately weigh 11.3 mg of anserine standard, dilute to 25 mL with mobile phase (mobile phase A: mobile phase B = 1:1, v / v), filter through a 0.2 µm filter membrane (polytetrafluoroethylene or nylon) and perform HPLC detection. Detect once every 2 hours. Samples are numbered 0, 1, 2, 3, and 4 in sequence, and the peak areas are recorded. The results are shown in Table 2.

[0039] Table 2 Sample stability

[0040] It can be seen from Table 2 that anserine has good stability in the mobile phase (mobile phase A: mobile phase B = 1:1, v / v) and can be stably stored for at least 8 hours.

[0041] 3. Chromatographic peak symmetry Figure 2 This is the HPLC chart of anserine standard solution with a concentration of 100 μg / mL.

[0042] Figure 3 , Figure 4 and Figure 5 The HPLC charts of three test solutions are shown respectively.

[0043] Depend on Figure 2 , Figure 3 , Figure 4 and Figure 5 It can be seen that the detection method provided by the present invention has a good separation effect of anserine, and the chromatographic peak is relatively symmetrical.

[0044] In summary, the detection method provided by the present invention can quickly and reliably detect the content of anserine in animal-derived peptide products.

[0045] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for detecting the content of anserine in animal-derived peptide products, characterized in that: The following steps are involved: (1) Preparation of control samples: accurately weigh anserine standard, prepare anserine standard solutions of different concentrations with mobile phase, filter through a filter membrane and set aside, wherein the mobile phase is a mixture of mobile phase A and mobile phase B in a volume ratio of 1:1, mobile phase A is methanol, and mobile phase B is a 60 mM, pH = 6.0 phosphate buffer; (2) Preparation of test samples: Weigh the sample to be tested, prepare a test solution of appropriate concentration with mobile phase, filter it through a filter membrane and set it aside for use, wherein the mobile phase is a mixture of mobile phase A and mobile phase B in a volume ratio of 1:1, mobile phase A is methanol, and mobile phase B is 60 mM phosphate buffer with a pH of 6.0; (3) Performing HPLC detection: Performing HPLC detection on the anserine standard solution and the test solution respectively; (4) Draw a standard curve: Draw a standard curve with the concentration of anserine standard solution as the horizontal axis and the peak area of ​​HPLC as the vertical axis; (5) Calculate the mass percentage of anserine in the test sample: Calculate the concentration of anserine from the standard curve based on the peak area, and then calculate the mass percentage of anserine in the test sample.

2. The method according to claim 1, characterized in that In step (1), the concentration of the anserine standard solution is in the range of 100 μg / mL-500 μg / mL.

3. The method according to claim 1, characterized in that In step (2), the concentration of the test solution is in the range of 100 μg / mL-500 μg / mL.

4. The method according to claim 1, characterized in that: In step (3), the chromatographic conditions are: Chromatographic column: Gemsil NH2, 250*4.6mm, 5µm; Mobile phase: Mobile phase A is methanol; mobile phase B is 60mM, pH=6.0 phosphate buffer; Elution conditions: 0-15 min, 40%→55% mobile phase A, 60%→45% mobile phase B; 15-35 min, 55% mobile phase A, 45% mobile phase B; Detection wavelength: 210nm; Detection temperature: 27℃; Injection volume: 20µL; Detection flow rate: 0.8mL / min.

5. The method according to claim 1, characterized in that In step (5), the formula for calculating the mass percentage of anserine in the test sample is: ； In the formula, W is the mass percentage of anserine in the test sample (%); C is the concentration of anserine obtained from the standard curve, in μg / mL; V is the fixed volume of the test sample solution, in mL; m is the sampling mass of the test sample, in g; 10 -6 is the factor for converting μg to g.

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