Method for extracting and detecting free amino acids in meat

Abstract

The invention provides a method for extracting and detecting free amino acids in meat. The method comprises the following steps: cutting a meat sample into blocks, freeze-drying, crushing and sieving to prepare meat powder; weighing meat powder, adding distilled water, uniformly mixing by vortex, performing water-bath constant-temperature oscillation extraction, and centrifuging to obtain a crude extract; adding sulfosalicylic acid precipitated protein into the crude extracting solution, centrifuging again, taking supernate, and adjusting the pH value with diluted hydrochloric acid to obtain an extracting solution; and filtering the extracting solution by a filter membrane, and detecting by a full-automatic amino acid analyzer. The method takes distilled water as an extraction solvent, efficiently extracts the free amino acid through water bath oscillation, is simple and convenient to operate, high in extraction efficiency and good in stability, is suitable for free amino acid detection of various meat samples such as chicken, beef and pork, and is high in practicability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for extracting and detecting free amino acids in meat. Background Technology

[0002] Free amino acids are amino acids that exist in a free state in the body. They are obtained through protein degradation, dietary intake, and other metabolic pathways and can be directly absorbed by the human body. As the basic building blocks of proteins, they are essential for maintaining normal metabolism and physiological functions. Free amino acids can be divided into essential amino acids and non-essential amino acids. The nutritional value of food proteins mainly depends on the composition and ratio of their essential amino acids. Many studies have shown that the content of free amino acids in meat affects meat tenderness, pH, and other meat quality characteristics. Therefore, the types and contents of free amino acids are important factors affecting the quality of meat products.

[0003] In addition, free amino acids are important flavor compounds. Based on their flavor characteristics, free amino acids can be mainly divided into three types: umami, sweet, and bitter. Aspartic acid (Asp) and glutamic acid (Glu) are common umami amino acids that can enhance the umami flavor of meat. Sweet amino acids include serine (Ser), glycine (Gly), threonine (Thr), and alanine (Ala), which increase the sweetness of meat and are crucial for the overall flavor balance. Bitter amino acids include valine (Val), methionine (Met), isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), lysine (Lys), arginine (Arg), and histidine (His). While their content is usually relatively low, they can still affect the overall taste of meat.

[0004] Currently, solvent extraction methods for the pretreatment of free amino acids mainly include water bath constant temperature shaking extraction, high-pressure hot water extraction, ultrasonic-assisted extraction, and ethanol reflux. Common extraction solvents for free amino acids in food include water, hydrochloric acid, trichloroacetic acid, and ethanol. Different extraction solvents may affect the extraction efficiency of free amino acids.

[0005] Currently, most methods for the extraction and detection of free amino acids are based on tea and fruits and vegetables, while there are few reports on methods for the extraction and detection of free amino acids in meat. Due to the huge differences in tissue morphology and type between animals and plants, it is necessary to provide a method for the extraction and detection of free amino acids in meat. Summary of the Invention

[0006] The purpose of this invention is to provide a method for the extraction and detection of free amino acids from meat.

[0007] To achieve the above-mentioned objectives, the present invention aims to provide the following technical solution:

[0008] A method for extracting and detecting free amino acids from meat samples includes operations such as freeze-drying and pulverizing, constant-temperature shaking in a water bath, protein precipitation, and filtration. The steps are as follows:

[0009] (1) After pretreatment, the meat sample is dried, pulverized, and sieved to obtain meat powder;

[0010] (2) Mix meat powder and extraction solvent at a ratio of 1g:(10-50)ml, extract by vortexing and constant temperature water bath shaking, and centrifuge to obtain crude extract;

[0011] (3) Add sulfosalicylic acid to the crude extract to precipitate the protein, and centrifuge to separate the supernatant;

[0012] (4) Adjust the pH of the supernatant to 1-2 with dilute hydrochloric acid, filter it and then use an amino acid analyzer for detection.

[0013] Specifically, it includes the following steps:

[0014] (1) After removing the tendons and fat from the fresh meat sample, cut it into small pieces, freeze-dry the meat sample thoroughly using a vacuum freeze dryer, grind it in a grinder, and sieve it to obtain meat powder for later use.

[0015] (2) Weigh 1g of spare meat powder, add 25ml of extraction solvent, vortex for 10s to mix thoroughly, then shake in a water bath at a constant temperature. After taking it out, invert it 20 times to fully extract the free amino acids in the sample. Centrifuge to obtain crude extract.

[0016] (3) Add 10% sulfosalicylic acid to the crude extract, mix by inverting, centrifuge, and separate the supernatant. The supernatant is the extract.

[0017] (4) Add 0.02M dilute hydrochloric acid to the extract to adjust the pH to 1-2, then filter it through a 0.22μm pinhole filter membrane and transfer it to a sample bottle for testing using a fully automated amino acid analyzer.

[0018] (5) The detection method is set to the physiological liquid analysis method in the L-8900 high-speed amino acid analyzer.

[0019] Preferably, the freeze-dried meat sample in step (1) above is a meat sample that has been thoroughly freeze-dried by a vacuum freeze dryer and has been sieved through a mesh size of 16 to 24.

[0020] Preferably, the extraction solvent in step (2) above is one of water, 15% trichloroacetic acid solution, 0.1M hydrochloric acid solution, or 4% sulfosalicylic acid solution.

[0021] Preferably, the temperature conditions for water bath oscillation extraction in step (2) above are 0-50℃ and the oscillation time is 5-30min.

[0022] The centrifugation described in steps (2) and (3) above is controlled at 4°C, with a centrifugation speed of 4000 r / min and a centrifugation time of 15 min.

[0023] More preferably, the solvent extracted in step (2) above is water.

[0024] More preferably, the temperature conditions for water bath oscillation extraction in step (2) above are 40°C and the oscillation time is 25 min.

[0025] Furthermore, the amino acid analyzer described in step (5) above uses an ion exchange column with a specification of 4.6×60mm and a packing material of 3μm particle cation exchange resin, and the separation column temperature is 57℃.

[0026] Furthermore, the detection wavelengths of the visible spectrophotometer of the amino acid analyzer described in step (5) above are 570 nm and 440 nm.

[0027] Acid extraction is currently the most commonly used extraction method. However, acids may disrupt peptide bonds in proteins, causing some proteins to hydrolyze into free amino acids, interfering with the detection of amino acids actually present in the free state in the sample. Ethanol extraction can produce significant errors, possibly because the side chains of amino acids affect their solubility in a mixture of ethanol and water, leading to inaccurate amino acid determination results. Amino acids are generally readily soluble in water and do not damage the protein structure, resulting in stable extraction. Furthermore, acid extraction and organic solvent extraction are more expensive and less polluting than water extraction.

[0028] This invention uses distilled water as the extraction solvent and employs a water bath constant-temperature oscillation method to extract free amino acids. An amino acid analyzer is used for detection. This method is convenient and simple to operate, has high extraction efficiency, and stable extraction results. It has advantages such as short operation time, low material consumption, and low pollution. Furthermore, it can be applied to the extraction and detection of free amino acids in meats such as chicken, beef, and pork, demonstrating great practicality. Attached Figure Description

[0029] Figure 1 The following is a flowchart illustrating the implementation process of this invention. Detailed Implementation

[0030] The present invention will be further explained and described below with reference to the accompanying drawings in the embodiments of the present invention. This example is only used to specifically illustrate the method and is not intended to limit the scope of application of the present invention.

[0031] Let's take fresh chicken as an example for a detailed explanation.

[0032] Example 1

[0033] like Figure 1 As shown, Figure 1 This is a flowchart of a first embodiment of the present invention applicable to the extraction and detection of free amino acids in meat samples.

[0034] 1. Pre-treatment of chicken

[0035] After removing large pieces of tendons and fat from fresh chicken, cut it into small pieces, place it in a glass dish, and freeze-dry it in a vacuum freeze dryer. After it is thoroughly dried, grind it in a grinder, sieve it, and set the sieved chicken powder aside.

[0036] 2. The extraction solvent is distilled water.

[0037] 3. Extraction of free amino acids

[0038] (1) After removing large pieces of tendons and fat from fresh chicken, cut it into small pieces, place it in a glass dish, put it in a vacuum freeze dryer for freeze drying, and then crush it through a 16-24 mesh sieve.

[0039] (2) Weigh 1g of the above chicken powder (accurate to 0.0001g), add 25ml of extraction solvent, vortex for 10s to mix thoroughly, then shake in a water bath at a constant temperature. After taking it out, invert it 20 times to fully extract the free amino acids. Then put it into a high-speed refrigerated centrifuge and centrifuge at 4000r / min for 15min at 4℃. Take 2ml of supernatant into a 5ml centrifuge tube to obtain the crude extract.

[0040] (3) Add 2 ml of 10% sulfosalicylic acid to a 5 ml centrifuge tube containing crude extract to precipitate the protein, and centrifuge again at 4000 r / min for 15 min at 4℃, and separate 2 ml of supernatant.

[0041] (4) Add 2 ml of 0.02 M dilute hydrochloric acid to the centrifuge tube containing the supernatant to adjust the pH to 1-2, mix thoroughly to obtain the extract. Use a 1 ml syringe to draw up the extract, filter it through a 0.22 μm needle membrane, and transfer it to a sample vial for analysis using a fully automated amino acid analyzer.

[0042] 4. Detection of free amino acids

[0043] (1) Take 2 ml of a standard solution of amino acid mixture containing physiological fluid, consisting of AN and B (produced by Wako Pure Chemical Industries, Ltd., Japan, 5 ml content), and dilute it with 0.02 M hydrochloric acid to a 50 ml solution, i.e., a standard sample containing 2 nmol of amino acids in 20 μl solution, for instrument calibration. Prepare physiological analysis elution buffers B1-B6, R3, R1 ninhydrin, and R2 ninhydrin buffers (analytical conditions are shown in Table 1) according to the instrument instructions, and perform gradient elution using a cation exchange column. The eluted amino acids react with the ninhydrin reaction solution at 135 °C to generate a blue-purple substance detectable at 570 nm and 440 nm in a spectrophotometer. The system calculates the concentration of each amino acid based on the standard curve and the sample response.

[0044] (2) The L-8900 automatic amino acid analyzer generates chromatograms and data reports containing the concentrations of each amino acid, and analyzes the amino acid concentrations in the sample. During the data analysis process, signal correction, noise filtering, and peak identification are usually required to ensure the accuracy and reliability of the results.

[0045] Table 1: Conditions for Physiological Fluid Analysis

[0046]

[0047]

[0048] 5. Orthogonal experimental design

[0049] Table 2 Factors and Levels

[0050] level Screening mesh count (mesh) Extraction time (min) Extraction temperature (°C) 1 16 5 0 2 18 15 20 3 20 25 40 4 24 30 50

[0051] Table 3. Orthogonal experimental design and results

[0052]

[0053]

[0054] Table 3 shows that the factors affecting the content of free amino acids, in descending order of importance, are: extraction temperature > sieve mesh size > extraction time. The optimal extraction process is A2B3C3, which involves pulverizing the amino acid through an 18-mesh sieve, extracting for 25 minutes, and extracting at 40℃.

[0055] 6. Verification Experiment

[0056] Table 4 Results of Optimal Process Validation Tests

[0057]

[0058] Note: Different letters (a, b, c) in the same column indicate significant differences (P<0.05), while the same letter indicates no significant difference.

[0059] Extraction was performed using the optimized process, which involved passing the meat sample through an 18-mesh sieve, with an extraction time of 25 minutes and an extraction temperature of 40℃. The average content of the three replicates was 2392.51 μg / g (see Table 4). The total extracted amount was significantly higher than that of other similar groups, indicating that the optimization results are reliable.

[0060] Example 2: Comparison of extraction with different solvents

[0061] The extraction method of the first embodiment was used, except that the extraction solvent was changed (15% trichloroacetic acid TCA, 0.1M hydrochloric acid HCl, distilled water, 4% sulfosalicylic acid SSA). The extraction experimental results of different solvents are shown in Table 5-7.

[0062] Table 5: Test Results Table

[0063] Amino acids (μg / g) 15% TCA 0.1M HCl 4% SSA distilled water Taurine <![CDATA[281.63±18.72 bc ]]> <![CDATA[271.92±10.89 c ]]> <![CDATA[318.93±20.97 a ]]> <![CDATA[298.40±13.74 b ]]> Aspartic acid Asp <![CDATA[0±0 b ]]> <![CDATA[45.78±11.47 a ]]> <![CDATA[45.96±8.99 a ]]> <![CDATA[48.39±12.49 a ]]> Threonine Thr 187.95±74.39 188.35±81.37 203.54±94.38 205.49±93.92 Serine 467.17±54.95 461.09±56.95 469.56±34.46 486.04±38.45 Alanine (Ala) <![CDATA[493.95±22.65 b ]]> <![CDATA[503.81±17.16 b ]]> <![CDATA[539.34±21.11 a ]]> <![CDATA[546.26±7.26 a ]]> Glutamic acid (Glu) 196.51±51.83 207.81±27.11 232.51±19.42 238.73±22.58 Cysteine 0±0 0±0 0±0 0±0 Glycine 103.29±7.07 103.01±9.58 103.13±7.88 105.08±6.28 Valine 60.27±11.06 62.99±10.97 67.73±11.62 70.24±11.38 Methionine (Met) <![CDATA[14.92±1.40 b ]]> <![CDATA[16.96±1.86 ab ]]> <![CDATA[23.32±9.89 a ]]> <![CDATA[20.62±6.6 ab ]]> Isoleucine Ile <![CDATA[35.08±3.85 c ]]> <![CDATA[38.64±2.81 bc ]]> <![CDATA[40.31±2.64 ab ]]> <![CDATA[43.01±3.05 a ]]> Leucine Leu <![CDATA[62.48±11.65 b ]]> <![CDATA[64.03±5.92 ab ]]> <![CDATA[72.09±10.01 ab ]]> <![CDATA[75.32±7.72 a ]]> Tyrosine Tyr 53.98±10.87 55.01±15.03 58.79±14.28 63.67±15.70 Phenylalanine (Phe) <![CDATA[43.05±7.26 b ]]> <![CDATA[48.58±6.75 ab ]]> <![CDATA[51.35±6.82 ab ]]> <![CDATA[55.24±6.66 a ]]> Lysine 83.59±36.90 84.17±29.71 88.58±30.28 88.60±27.13 Arginine (Arg) 98.08±29.24 106.77±30.08 105.88±29.10 106.49±21.84

[0064] Note: Different letters (a, b, c) in the same row indicate significant differences (P<0.05), while the same letters indicate no significant differences.

[0065] Table 6: Test Results Table—Classified by Essential and Non-essential Amino Acids

[0066]

[0067]

[0068] Same as above.

[0069] Table 7: Test Results Table—Classified by Amino Acid Flavor Characteristics

[0070]

[0071] Same as above.

[0072] As shown in Table 5, except for the undetectable aspartic acid when trichloroacetic acid was used as the extraction solvent, there was no difference in the types of free amino acids extracted by the four extraction solvents, with a total of 15 amino acids detected. Among them, the content of 12 amino acids was higher when distilled water was used as the extraction solvent than in the other three treatments.

[0073] As shown in Table 6, the total amount of free amino acids extracted by different extractants varied. When distilled water was used as the extractant, the content of essential amino acids, non-essential amino acids, and total free amino acids was significantly higher than that of 0.1M hydrochloric acid solution and 15% TCA solution (P<0.05), while there was no significant difference compared with 4% SSA extraction.

[0074] As shown in Table 7, the content of flavor amino acids extracted by different extractants varies. When distilled water is used as the extractant, the extraction efficiency of umami, sweet and bitter free amino acids is the highest. Among them, the extraction content of umami and sweet amino acids is significantly higher than that of 0.1M hydrochloric acid solution and 15% TCA solution (P<0.05).

[0075] In summary, by comparing the extraction of free amino acids from chicken meat using different extraction solvents, it can be shown that using distilled water as the extraction solvent results in better extraction and has the highest extraction efficiency.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made in accordance with the claims of the present invention are included within the protection scope of the present invention.

Claims

1. A method for extracting and detecting free amino acids in meat, characterized in that, Includes the following steps: (1) After pretreatment, the meat sample is dried, pulverized, and sieved to obtain meat powder; (2) Mix meat powder and extraction solvent at a ratio of 1g:(10-50)ml, extract by vortexing and constant temperature water bath shaking, and centrifuge to obtain crude extract; (3) Add sulfosalicylic acid to the crude extract to precipitate the protein, and centrifuge to separate the supernatant; (4) Adjust the pH of the supernatant to 1-2 with dilute hydrochloric acid, filter it and then use an amino acid analyzer for detection.

2. The method according to claim 1, characterized in that, Step (1) involves removing tendons and fat from the fresh meat sample, cutting it into small pieces, freeze-drying and pulverizing it, and sieving it to obtain meat powder for later use. The freeze-dried meat sample is a meat sample that has been thoroughly freeze-dried by a vacuum freeze dryer and sieved through a mesh size of 16 to 24.

3. The method according to claim 1, characterized in that, Step (2) is as follows: Weigh the meat powder, add 10-50 ml of extraction solvent per gram of meat powder, vortex mix, and then extract by constant temperature shaking in a water bath. After taking it out, invert it 10-20 times and centrifuge to obtain crude extract. The extraction solvent is one of water, 15% trichloroacetic acid solution, 0.1M hydrochloric acid solution, or 4% sulfosalicylic acid solution.

4. The method according to claim 1, characterized in that: The temperature conditions for water bath oscillation extraction in step (2) are 0-50℃ and the oscillation time is 5-30min.

5. The method according to claim 1, characterized in that: The centrifugation in steps (2) and (3) is controlled at 4000 r / min at 4℃ for 15 min.

6. The method according to claim 3, characterized in that: The extraction solvent mentioned in step (2) is distilled water.

7. The method for extracting and detecting free amino acids in meat according to claim 6, characterized in that: In step (1), the meat powder is pulverized to 18 mesh, and in step (2), the temperature conditions for water bath oscillation extraction are 40°C and the oscillation time is 25 min.

8. The method according to claim 6, characterized in that: The amino acid analyzer uses an ion exchange column with a specification of 4.6×60mm, a packing material of 3μm particle cation exchange resin, and a separation column temperature of 57℃.

9. The method according to claim 6, characterized in that: The amino acid analyzer uses a visible spectrophotometer with detection wavelengths of 570 nm and 440 nm.

10. The method according to claim 1, characterized in that, Includes the following steps: (1) After removing the tendons and fat from the fresh meat sample, cut it into small pieces, freeze-dry the meat sample thoroughly using a vacuum freeze dryer, grind it in a grinder, and pass it through an 18-mesh sieve to obtain meat powder for later use. (2) Weigh 1g of spare meat powder, add 25ml of distilled water, vortex for 10s to mix thoroughly, then shake in a water bath at a constant temperature. After taking it out, invert it 20 times to fully extract the free amino acids in the sample. Centrifuge to obtain crude extract. (3) Add 10% sulfosalicylic acid to the crude extract, mix by inverting, centrifuge, and separate the supernatant. The supernatant is the extract. (4) Add 0.02M dilute hydrochloric acid to the extract to adjust the pH to 1-2, then filter it through a 0.22μm pinhole filter membrane and transfer it to a sample bottle for testing using a fully automated amino acid analyzer. (5) The detection method is set to the physiological liquid analysis method in the L-8900 high-speed amino acid analyzer.

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