A method for determining meat content using guanidine hydrochloride pretreatment

Abstract

The application belongs to the technical field of food detection, and particularly relates to a method for determining meat content by using guanidine hydrochloride pretreatment. The method for determining meat content by using guanidine hydrochloride pretreatment provided by the application mixes meat samples with guanidine hydrochloride buffer extraction solution to obtain meat total protein supernatant, and sequentially reacts the meat total protein supernatant with dithiothreitol solution, iodine acetamide solution and trypsin solution to obtain enzymolysis peptide segment solution; LC-MS / MS method is used to detect peak area, and according to the peak area and meat content standard curve, the content of meat in the sample is obtained. The guanidine hydrochloride used in the application can more completely destroy the spatial structure and interaction of proteins in meat products, and the provided method can improve the detection accuracy and sensitivity of the meat content in meat products.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a method for determining meat content using guanidine hydrochloride pretreatment. Background Technology

[0002] The market still sees the phenomenon of using low-priced raw materials to replace high-priced raw materials, but these are not accurately listed in the ingredient list. The order of ingredients in the ingredient list does not represent the true content, resulting in the actual price of the product being lower than the market price. This hinders the products of compliant meat product companies in the market, making them less competitive, and in the long run, it affects the survival and development of these companies.

[0003] Traditional detection methods use a high-concentration urea and thiourea mixed buffer solution to extract and analyze proteins in meat products; however, existing methods have low accuracy in detecting meat content in complex meat products and cannot meet market demands. Currently, there is a lack of accurate quantitative detection methods for the true meat content in meat products. Summary of the Invention

[0004] The purpose of this invention is to provide a method for pretreating meat products with guanidine hydrochloride and determining the meat content in the meat products. The method provided by this invention can improve the accuracy and sensitivity of detecting the meat content in meat products.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A method for pretreatment with guanidine hydrochloride includes the following steps: Meat samples were mixed with a guanidine hydrochloride buffer extract for protein extraction to obtain a total meat protein supernatant; the guanidine hydrochloride buffer extract included guanidine hydrochloride, tris(hydroxymethyl)aminomethane hydrochloride and dithiothreitol. The supernatant of total meat protein was mixed with dithiothreitol solution and a reduction reaction was carried out to obtain denatured meat protein solution A. Under light-protected conditions, the denatured meat protein solution A was mixed with an iodoacetamide solution and subjected to an alkylation reaction to obtain denatured meat protein solution B. The denatured meat protein solution B, tris(hydroxymethyl)aminomethane hydrochloride solution, and trypsin solution were mixed and subjected to enzymatic hydrolysis to obtain an enzymatically hydrolyzed peptide solution.

[0006] Preferably, the concentration of guanidine hydrochloride in the guanidine hydrochloride buffer extract is 5-6 mol / L, the concentration of tris(hydroxymethyl)aminomethane hydrochloride is 0.1-0.2 mol / L, and the concentration of dithiothreitol is 10-15 mmol / L; the pH value of the guanidine hydrochloride buffer extract is 8.0-8.5.

[0007] Preferably, the ratio of the meat sample to the guanidine hydrochloride buffer extract is 2-4 g: 20-40 mL; the reaction temperature is 90-100℃ and the time is 20-40 min; the centrifugation speed is 10000-12000 r / min and the time is 15-20 min.

[0008] Preferably, the concentration of the dithiothreitol solution is 80-120 mmol / L; the volume ratio of the meat total protein supernatant to the dithiothreitol solution is 200-240:30-50.

[0009] Preferably, the reduction reaction is carried out at a temperature of 50-60°C for 1-2 hours, and the reduction reaction is carried out under oscillation conditions.

[0010] Preferably, the concentration of the iodoacetamide solution is 80~120 mmol / L; the volume ratio of the denatured meat protein solution A to the iodoacetamide solution is 200:30~50.

[0011] Preferably, the alkylation reaction is carried out at room temperature for 20-40 minutes.

[0012] Preferably, the concentration of the tris(hydroxymethyl)aminomethane hydrochloride solution is 20-30 mmol / L, the pH value is 8.5, and the concentration of the trypsin solution is 3-8 mg / mL; the volume ratio of the denatured meat protein solution B, the tris(hydroxymethyl)aminomethane hydrochloride solution, and the trypsin solution is 200:1800-2000:20-30.

[0013] Preferably, the enzymatic hydrolysis reaction is carried out at a temperature of 35-37°C for 8-16 hours.

[0014] This invention provides a method for detecting the meat content in meat products, comprising the following steps: The peak area of ​​the enzymatically hydrolyzed peptide solution was detected by LC-MS / MS. The meat content in the sample was obtained by plotting a standard curve based on the peak area of ​​the species-specific quantitative peptide and the meat content. The meat content standard curve is a linear regression equation between the meat content and the peak area of ​​the species-specific quantitative peptide. The enzymatically hydrolyzed peptide solution is the enzymatically hydrolyzed peptide solution obtained by the method described in the above technical solution.

[0015] The method for pretreatment of meat products using guanidine hydrochloride provided by this invention uses guanidine hydrochloride as a strong protein denaturant, which can more thoroughly destroy the spatial structure and interactions of proteins in meat products, and efficiently dissolve various characteristic proteins of meat, including myofibrillar proteins and sarcoplasmic proteins. This avoids the problem of insufficient extraction of some poorly soluble proteins in the traditional urea-thiourea system, making the quantitative analysis results of meat more closely closer to the true values, and effectively improving the sensitivity and reliability of the detection.

[0016] Compared to traditional protein extraction methods (urea-thiourea system), this invention uses guanidine hydrochloride extraction, which can more accurately determine the meat content in meat products. By commissioning companies to produce meat products with known pork content, the accuracy of different protein extraction methods was compared and analyzed. The results show that the extraction method of this invention has an accuracy of over 80%, especially demonstrating stronger adaptability in complex meat product matrices (such as samples with protein denaturation during complex processing), further ensuring the accuracy and stability of pork content determination. Attached Figure Description

[0017] Figure 1 A standard curve of pork content plotted for Example 1; Figure 2 The chromatogram of the quantitative ions in Example 1; Figure 3 This is an overlay chromatogram of quantitative and qualitative ions from Example 1. Detailed Implementation

[0018] This invention provides a method for pretreatment using guanidine hydrochloride, comprising the following steps: Meat samples were mixed with a guanidine hydrochloride buffer extract for protein extraction to obtain a total meat protein supernatant; the guanidine hydrochloride buffer extract included guanidine hydrochloride, tris(hydroxymethyl)aminomethane hydrochloride and dithiothreitol. The supernatant of total meat protein was mixed with dithiothreitol solution and a reduction reaction was carried out to obtain denatured meat protein solution A. Under light-protected conditions, the denatured meat protein solution A was mixed with an iodoacetamide solution and subjected to an alkylation reaction to obtain denatured meat protein solution B. The denatured meat protein solution B, tris(hydroxymethyl)aminomethane hydrochloride solution, and trypsin solution were mixed and subjected to enzymatic hydrolysis to obtain an enzymatically hydrolyzed peptide solution.

[0019] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0020] This invention involves mixing a meat sample with a guanidine hydrochloride buffer extract, reacting the mixture, and centrifuging to obtain a supernatant of total meat protein. The guanidine hydrochloride buffer extract comprises guanidine hydrochloride, Tris-HCl, and dithiothreitol. In one embodiment, the meat sample includes pork. The concentration of guanidine hydrochloride in the buffer extract is 5-6 mol / L, the concentration of Tris-HCl is 0.1-0.2 mol / L, and the concentration of dithiothreitol is 10-15 mmol / L. The pH of the guanidine hydrochloride buffer extract is 8.0-8.5.

[0021] The guanidine hydrochloride used in this invention is a strong protein denaturant that can more thoroughly destroy the spatial structure and interactions of proteins in meat products and efficiently dissolve various characteristic meat proteins, including myofibrillar proteins and sarcoplasmic proteins. Tris(hydroxymethyl)aminomethane hydrochloride provides a stable weakly alkaline buffer environment to maintain the pH stability of the extract and subsequent reaction system. Dithiothreitol is used to reduce the disulfide bonds (–S–S–) in proteins to obtain two free thiol groups (–SH), which further promotes protein denaturation and makes the protein more soluble in the extract.

[0022] In one embodiment of the present invention, the ratio of the meat sample to the guanidine hydrochloride buffer extract is 2-4 g: 20-40 mL, specifically 2 g: 20 mL; the reaction temperature is 90-100°C, specifically 95°C, and the reaction time is 20-40 min, specifically 30 min; the reaction preferably includes: allowing the mixture of the meat sample and the guanidine hydrochloride buffer extract to stand, vortexing every 10 min, with each vortex lasting 30 s; the centrifugation speed is 10000-12000 r / min, and the centrifugation time is 15-20 min.

[0023] This invention uses a guanidine hydrochloride buffer extract to extract proteins from meat samples. Guanidine hydrochloride, as a strong protein denaturant, can more thoroughly destroy the spatial structure and interactions of proteins in meat products, making the proteins easier to dissolve in the extraction solution.

[0024] After obtaining the supernatant of total meat protein, this invention mixes the supernatant with a dithiothreitol solution and performs a reduction reaction to obtain denatured meat protein solution A. In one embodiment of this invention, the concentration of the dithiothreitol solution is 80-120 mmol / L, specifically 100 mmol / L; the volume ratio of the total meat protein supernatant to the dithiothreitol solution is 200-240:30-50, specifically 200:30. In another embodiment of this invention, the reduction reaction is carried out at a temperature of 50-60°C, specifically 56°C, for 1-2 hours, under shaking conditions.

[0025] This invention adds dithiothreitol to the supernatant of total meat protein. Dithiothreitol reduces the disulfide bonds (–S–S–) in the protein to two free thiol groups (–SH), making the protein completely linearized and thus exposing the enzyme cleavage sites.

[0026] After obtaining denatured meat protein solution A, the present invention, under light-protected conditions, mixes denatured meat protein solution A with iodoacetamide solution to carry out an alkylation reaction to obtain denatured meat protein solution B. In one embodiment of the present invention, the concentration of the iodoacetamide solution is 80-120 mmol / L, specifically 100 mmol / L; the volume ratio of denatured meat protein solution A to iodoacetamide solution is 200:30-50, specifically 200:30; the alkylation reaction is carried out at room temperature for 20-40 min, specifically 30 min.

[0027] This invention uses iodoacetamide to alkylate the free thiol groups (–SH) reduced from the protein in denatured meat protein solution A, permanently blocking the thiol groups and preventing the reformation of disulfide bonds. The iodoacetamide is photosensitive and needs to be reacted in the dark to prevent photolysis of the iodoacetamide and ensure the specificity and completeness of the alkylation reaction.

[0028] After obtaining denatured meat protein solution B, the present invention mixes the denatured meat protein solution B, tris(hydroxymethyl)aminomethane hydrochloride solution, and trypsin solution to perform an enzymatic hydrolysis reaction to obtain an enzymatically hydrolyzed peptide solution. In one embodiment of the present invention, the concentration of the Tris-HCl solution is 20-30 mmol / L, specifically 25 mmol / L; the concentration of the trypsin solution is 3-8 mg / mL, specifically 5 mg / mL; the volume ratio of the denatured meat protein solution B, Tris-HCl solution, and trypsin solution is 200:1800-2000:20-30, specifically 200:1800:20. In another embodiment of the present invention, the enzymatic hydrolysis reaction is carried out at a temperature of 35-37°C for an overnight time, or more specifically, for 8-16 hours.

[0029] This invention utilizes trypsin to hydrolyze denatured and alkylated total pork protein solution B into polypeptide fragments. Trypsin is a commonly used protease in proteomics analysis, possessing specific cleavage sites, specifically C-terminal peptide bonds at the carboxyl terminus of lysine (Lys or K) and arginine (Arg or R) residues. It exhibits good cleavage stability, facilitating subsequent protein identification and quantitative polypeptide analysis. This invention employs Tris-HCl solution to provide a suitable buffer environment for the trypsin hydrolysis reaction, diluting the concentration of the guanidine hydrochloride extraction solution and ensuring the activity of the trypsin.

[0030] As one embodiment of the present invention, the enzymatic hydrolysis reaction further includes: post-processing the obtained enzymatic hydrolysis system; the post-processing includes terminating the reaction by adding acid, purifying the reaction system by solid-phase extraction, and collecting the eluent to obtain an enzymatically hydrolyzed peptide solution; the terminating reaction by adding acid includes adding acid to the enzymatic hydrolysis system to make the pH of the enzymatic hydrolysis system < 2 to terminate the enzymatic hydrolysis reaction, the acid including a 5% formic acid solution; the solid-phase extraction purification includes desalting the reaction system by passing it through an HLB column; the HLB column is activated sequentially with acetonitrile and a 0.1% formic acid aqueous solution, the reaction system is loaded onto the column, and the column is washed sequentially with water and a 0.1% formic acid aqueous solution, and then eluted with a mixed solution of 2 mL of acetonitrile and a 0.1% formic acid aqueous solution (the volume ratio of acetonitrile to the 0.1% formic acid aqueous solution is 1:1), and the eluent is collected; the eluent is filtered through a 0.22 μm filter membrane.

[0031] This invention provides a method for detecting the meat content in meat products, comprising the following steps: The peak area of ​​the enzymatically hydrolyzed peptide solution was detected by LC-MS / MS. The meat content in the sample was obtained by plotting a standard curve based on the peak area of ​​the species-specific quantitative peptide and the meat content. The meat content standard curve is a linear regression equation between the meat content and the peak area of ​​the species-specific quantitative peptide. The enzymatically hydrolyzed peptide solution is the enzymatically hydrolyzed peptide solution obtained by the method described in the above technical solution.

[0032] In one embodiment of the present invention, the LC-MS / MS method is performed using a Thermo LC-MS / MS mass spectrometer; the conditions for the LC-MS / MS method are as follows: chromatographic conditions: Hypersil GOLD C18 column, flow rate 0.3 mL / min; column temperature 40℃, injection volume 2 µL; mobile phase A is FA / H2O with a volume percentage of 0.1%, and mobile phase B is FA / ACN with a volume percentage of 0.1%; the elution gradient of the liquid chromatography is: 0~0.2 min, 3%~10% B; 0.2~8 min, 10%~40% B; 8~9 min, 40%~95% B; 9~10 min, 95% B; 10~10.1 min, 95%~3% B; 10.1~12.5 min, 3% B. Mass spectrometry conditions: spray voltage 3500V; sheath gas 40Arb; auxiliary gas 15Arb; ion transmission tube temperature 275℃; ion source nebulization temperature 350℃; transfer line temperature 275℃; ion source temperature 350℃; acquisition period 0.5s; collision gas 1.5mTorr; Q1 resolution 0.7, Q3 resolution 0.7.

[0033] Knowing that the characteristic polypeptide sequence in pork is GGPLTAAYR, this invention uses LC-MS / MS to determine the mass-to-charge ratio of the parent ion and the daughter ion; then, gradient standard solutions are prepared using pure pork and detected separately to obtain the chromatographic peak areas at the mass-to-charge ratio values ​​of the parent ion and the daughter ion, and then a concentration-peak area curve is plotted to obtain the meat content standard curve.

[0034] To further illustrate the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments thereof. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0035] Example 1 Weigh 2g of pork sample and add 20mL of a guanidine hydrochloride buffer extract with a pH of 8.5. The guanidine hydrochloride buffer extract contains 0.1mol / L Tris-HCl, 6mol / L guanidine hydrochloride, and 10mmol / L dithiothreitol. React at 95℃ for 30min, vortexing for 30s every 10min. Centrifuge the resulting reaction solution at 12000r / min for 20min to obtain a supernatant of total meat protein. Take 200μL of this supernatant and add 30μL of a 100mmol / L dithiothreitol solution. Reduce the mixture at 56℃ with shaking for 1h. Cool to room temperature and add 30μL of a 100mmol / L iodoacetamide solution under light-protected conditions for alkylation reaction for 30min. Continue the reaction... 1.8 mL of 25 mmol / L Tris-HCl solution (pH 8.5) was added to the system, followed by 20 μL of 5 mg / mL trypsin solution. The reaction was carried out overnight at 37°C. After the reaction was completed, 5% formic acid solution was added to adjust the pH to <2 to terminate the reaction, yielding the enzymatic hydrolysate. The hydrolysate was desalted using an HLB column. Specifically, the HLB column was first activated sequentially with acetonitrile and then with a 0.1% formic acid aqueous solution. The hydrolysate was then loaded onto the column, followed by rinsing with water and then with a 0.1% formic acid aqueous solution. Finally, the column was eluted with 2 mL of acetonitrile and a 0.1% formic acid aqueous solution (the volume ratio of acetonitrile to 0.1% formic acid aqueous solution was 1:1). The collected eluent was filtered through a 0.22 μm filter membrane to obtain a pork enzymatic hydrolysate peptide solution.

[0036] Establishment of a standard curve for pork content: Weigh 2g of pork and react according to the steps described above to obtain a supernatant of total protein from the meat. Take 2μL, 10μL, 20μL, 50μL, 100μL, and 200μL respectively, and bring the volume to 200μL with guanidine hydrochloride buffer. Subsequent steps are the same as described above. A standard curve of meat content and peak area of ​​species-specific quantitative peptides is obtained. Figure 1 A standard curve of pork content was plotted for Example 1; based on Figure 1 It can be seen that the linear regression equation of the standard curve for meat content is Y=1.24e 4 X-1.259e 3 ;R 2 =0.9966.

[0037] Thermo LC-MS / MS mass spectrometer was used to collect and quantify peptides using the LC-MS / MS method. The detection conditions are as follows: Chromatographic conditions: Hypersil GOLD C18 column, flow rate 0.3 mL / min; column temperature 40℃, injection volume 2 µL; mobile phase A was 0.1% FA / H2O (v / v), and mobile phase B was 0.1% FA / ACN (v / v); the elution gradient for the liquid chromatography was: 0–0.2 min, 3%–10% B; 0.2–8 min, 10%–40% B; 8–9 min, 40%–95% B; 9–10 min, 95% B; 10–10.1 min, 95%–3% B; 10.1–12.5 min, 3% B.

[0038] Mass spectrometry conditions: spray voltage 3500V; sheath gas 40Arb; auxiliary gas 15Arb; ion transmission tube temperature 275℃; ion source nebulization temperature 350℃; transfer line temperature 275℃; ion source temperature 350℃; acquisition period 0.5s; collision gas 1.5mTorr; Q1 resolution 0.7, Q3 resolution 0.7.

[0039] The polypeptide information of the pork is shown in Table 1.

[0040] Table 1. Characteristic polypeptide information of pork

[0041] Daughter ions include quantitative ions and qualitative ions. Figure 2 The chromatogram of the quantitative ions in Example 1; Figure 3 This is an overlay chromatogram of quantitative and qualitative ions from Example 1.

[0042] Comparative Example 1 Weigh 0.5g of pork sample and add 5mL of buffered salt extract with a pH of 8.0. The buffered salt extract consists of 0.05mol / L Tris-HCl, 6mol / L urea, and 2mol / L thiourea. Grind the sample under liquid nitrogen for 45s. Centrifuge the resulting reaction solution at 15000r / min for 10min to obtain the supernatant. Take 300μL of the supernatant and add 10μL of 50mmol / L dithiothreitol solution. Incubate the reaction at 56℃ with shaking for 1h. After cooling to room temperature, add 20μL of 100mmol / L iodoacetamide solution under light-protected conditions and perform an alkylation reaction for 30min. Add 100μL of a solution of 50mmol / L dithiothreitol to the above reaction system. A 50 mmol / L NH4HCO3 solution was prepared, followed by the addition of 20 μg of trypsin. The reaction was carried out at 37 °C for 12 h. After the reaction was completed, 10% acetic acid solution was added to adjust the pH to <2 to terminate the reaction. 1 mL of 0.1% acetic acid aqueous solution was added to obtain the enzymatic hydrolysate. The enzymatic hydrolysate was desalted using an HLB column. Specifically, the HLB column was first activated sequentially with acetonitrile and 0.1% acetic acid aqueous solution. The enzymatic hydrolysate was then loaded onto the column, followed by rinsing with water and 0.5% acetic acid aqueous solution. Finally, elution was performed with 1 mL of 60% acetonitrile and 0.5% acetic acid aqueous solution. The collected eluent was filtered through a 0.22 μm filter membrane to obtain the pork enzymatic hydrolysate peptide solution.

[0043] Subsequent operations are the same as in Example 1.

[0044] Test Example 1 We commissioned a meat processing company to prepare meat products with a known pork content. The specific parameters are shown in Table 2.

[0045] Table 2. Different types of meat products with known pork content and their pork content.

[0046] The pork content of the meat products in Table 2 was determined using the methods of Example 1 and Comparative Example 1, respectively, and the results are shown in Table 3.

[0047] Table 3. Detection of pork content in meat products in Example 1 and Comparative Example 1.

[0048] As can be seen from the results in Table 3, the method for determining meat content in meat products using guanidine hydrochloride pretreatment provided by the present invention yields more accurate results.

[0049] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, those skilled in the art will recognize that, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for pretreatment with guanidine hydrochloride, comprising the following steps: Meat samples were mixed with a buffer containing guanidine hydrochloride for protein extraction to obtain a supernatant of total meat protein. The guanidine hydrochloride buffer extract includes guanidine hydrochloride, tris(hydroxymethyl)aminomethane hydrochloride, and dithiothreitol; The supernatant of total meat protein was mixed with dithiothreitol solution and a reduction reaction was carried out to obtain denatured meat protein solution A. Under light-protected conditions, the denatured meat protein solution A was mixed with an iodoacetamide solution and subjected to an alkylation reaction to obtain denatured meat protein solution B. The denatured meat protein solution B, tris(hydroxymethyl)aminomethane hydrochloride solution, and trypsin solution were mixed and subjected to enzymatic hydrolysis to obtain an enzymatically hydrolyzed peptide solution.

2. The method according to claim 1, characterized in that, The guanidine hydrochloride buffer extract contains guanidine hydrochloride at a concentration of 5-6 mol / L, tris(hydroxymethyl)aminomethane hydrochloride at a concentration of 0.1-0.2 mol / L, and dithiothreitol at a concentration of 10-15 mmol / L; the pH of the guanidine hydrochloride buffer extract is 8.0-8.

5.

3. The method according to claim 2, characterized in that, The ratio of meat sample to guanidine hydrochloride buffer extract is 2-4 g: 20-40 mL; the reaction temperature is 90-100℃ and the time is 20-40 min; the centrifugation speed is 10000-12000 r / min and the time is 15-20 min.

4. The method according to claim 1, characterized in that, The concentration of the dithiothreitol solution is 80-120 mmol / L; the volume ratio of the meat total protein supernatant to the dithiothreitol solution is 200-240:30-50.

5. The method according to claim 1, characterized in that, The reduction reaction is carried out at a temperature of 50-60°C for 1-2 hours under oscillating conditions.

6. The method according to claim 1, characterized in that, The concentration of the iodoacetamide solution is 80~120 mmol / L; the volume ratio of the denatured meat protein solution A to the iodoacetamide solution is 200:30~50.

7. The method according to claim 1, characterized in that, The alkylation reaction was carried out at room temperature for 20-40 minutes.

8. The method according to claim 1, characterized in that, The concentration of the tris(hydroxymethyl)aminomethane hydrochloride solution is 20-30 mmol / L, the pH value is 8.5, and the concentration of the trypsin solution is 3-8 mg / mL; the volume ratio of the denatured meat protein solution B, the tris(hydroxymethyl)aminomethane hydrochloride solution and the trypsin solution is 200:1800-2000:20-30.

9. The method according to claim 1, characterized in that, The enzymatic hydrolysis reaction is carried out at a temperature of 35-37°C for 8-16 hours.

10. A method for detecting the meat content in meat products, comprising the following steps: The peak area of ​​the enzymatically hydrolyzed peptide solution was detected by LC-MS / MS. The meat content in the sample was obtained by plotting a standard curve based on the peak area of ​​the species-specific quantitative peptide and the meat content. The meat content standard curve is a linear regression equation between the meat content and the peak area of ​​the species-specific quantitative peptide. The enzymatically hydrolyzed peptide solution is the enzymatically hydrolyzed peptide solution obtained by the method according to any one of claims 1 to 9.

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