Method for determining content of amino acid component in fermented soybean

Abstract

The invention relates to the technical field of detection of amino acid components in fermented soybean, in particular to a method for determining the content of amino acid components in fermented soybean. Comprising the following steps: (1) preparing a fermented soybean sample into a fermented soybean extraction solution; (2) preparing a fermented soybean test solution from the fermented soybean extraction solution; (3) diluting the amino acid solution into different concentration gradients to obtain a mixed amino acid reference substance solution; preparing the mixed amino acid reference substance solution into an amino acid derivatization reference substance solution; (4) preparing a blank derivatization reagent from the hydrochloric acid solution by using the derivatization reagent; and (5) carrying out ultra-high performance liquid chromatography on the fermented soybean test solution, the amino acid derivatization reference substance solution and a blank derivatization reagent. The invention provides an efficient, accurate and reliable method for amino acid analysis of fermented soybean and other fermented food, and the method has important significance for improving the product quality, optimizing the production process and promoting related research.

Description

Technical Field

[0001] The invention relates to the technical field of amino acid component detection in light fermented black beans, in particular to a method for determining the content of amino acid components in light fermented black beans. Background Art

[0002] Soybeans, a high-protein plant, undergo fermentation to produce fermented black beans. Their macromolecular proteins can be degraded into free amino acids. These free amino acids are not only important nutrients but also serve as precursors for bioactive components such as biogenic amines and melanoidins, significantly regulating the color and flavor of the product. Amino acids play crucial roles in regulating immune responses, antioxidant defense, energy metabolism, and signal transduction. Notably, the free amino acid content in light black beans fluctuates significantly under different environmental conditions, and these changes directly impact the quality of the final product. Therefore, systematically studying the dynamic changes in the amino acid composition of light black beans during fermentation is crucial for uncovering the mechanisms underlying their characteristic "strong flavor." However, the fact that most amino acids are colorless crystals and lack UV- or fluorescent-active groups presents significant technical challenges for traditional analytical detection methods. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for determining the content of amino acid components in fermented black beans.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for determining the content of amino acid components in fermented black beans, comprising the following steps:

[0006] (1) preparing a light fermented black bean sample into a light fermented black bean extract solution;

[0007] (2) using a derivatization reagent to prepare a light fermented black bean extract solution into a light fermented black bean test solution;

[0008] (3) diluting the amino acid solution into different concentration gradients to obtain a mixed amino acid reference solution; using a derivatization reagent to prepare an amino acid derivatization reference solution from the mixed amino acid reference solution;

[0009] (4) using a derivatization reagent to prepare a blank derivatization reagent from a hydrochloric acid solution;

[0010] (5) The light fermented black bean test solution, amino acid derivatization reference solution, and blank derivatization reagent were subjected to ultra-high performance liquid chromatography; wherein mobile phase A was sodium acetate buffer and mobile phase B was acetonitrile; and the gradient elution conditions were as shown in Table 1:

[0011] Table 1 Mobile phase gradient elution conditions

[0012]

[0013]

[0014] The norleucine solution is used as the internal standard solution with a concentration of 0.25-0.25 mg / mL; the derivatization reagent is an acetonitrile solution of 0.3-0.5 mol / L phenyl isothiocyanate and an acetonitrile solution of 1.0-1.3 mol / L triethylamine.

[0015] Preferably, the chromatographic column of the ultra-high performance liquid chromatography in step (5) is ACQUITY BEH C18, specifications: 2.1×100 mm, 1.7 μm; injection volume: 2-5 μL; flow rate: 0.3-0.5 mL / min; detection wavelength: 254-280 nm; column temperature: 40-45° C.; the concentration of the sodium acetate buffer: 0.1-0.2 mol / L, pH: 6.5-6.7.

[0016] Preferably, the method for preparing the light fermented black bean sample in step (4) is: mixing black beans, mulberry leaf decoction, Artemisia annua decoction of equal mass, soaking for 10 to 16 hours, and steaming for 1.5 to 2 hours; spreading mulberry leaf residue and Artemisia annua residue on the bottom of the fermentation mold, putting in the steamed material, fermenting for 6 to 8 days, washing, fermenting again for 15 to 20 days, steaming for 0.5 to 1 hour, and drying.

[0017] Preferably, the preparation method of the light fermented black beans extract solution is: mixing the light fermented black beans sample with water and ultrasonicating at 600-700w for 20-25min, cooling, adding hydrochloric acid solution, centrifuging at 14000-15000rpm for 10-12min, and taking the supernatant to obtain the light fermented black beans extract solution.

[0018] Preferably, the preparation method of the light fermented black bean test solution is: the light fermented black bean extract, the internal standard solution, the acetonitrile solution with phenyl isothiocyanate and the acetonitrile solution with triethylamine are mixed and derivatized in a volume ratio of 200:15-25:90-110:90-110, and twice the volume of n-hexane of the light fermented black bean extract is added and shaken, and the mixture is allowed to stand and separate, and the lower layer solution is taken and added with an equal amount of water to mix, and the mixture is centrifuged at 14000-15000 rpm for 10-12 minutes, and the supernatant is taken to obtain the product; and the amino acid derivatization reference solution and blank derivatization reagent are prepared according to the same method.

[0019] Preferably, the amino acid solution is prepared from components with the following concentrations: Asp 133.60 μg / mL, Glu 274.75 μg / mL, Ser 71.90 μg / mL, Gly 52.80 μg / mL, Gln 50.60 μg / mL, His 42.45 μg / mL, Arg 181.80 μg / mL, Thr 544.00 μg / mL, Ala 211.75 μg / mL, Pro 137.86 μg / mL, Tyr 103.25 μg / mL, Val 107.88 μg / mL, Met 27.45 μg / mL, Ile 112.50 μg / mL, Leu 192.25 μg / mL, Phe 126.13 μg / mL, and Lys 192.40 μg / mL.

[0020] Preferably, the dilution into different concentration gradients is: diluting the amino acid solution stepwise by 2, 4, 8, 16, 32, and 64 times with 0.1 mol / L hydrochloric acid solution.

[0021] The invention also provides application of the method in quality control of light fermented black beans.

[0022] The invention also provides application of the method in monitoring fermentation of light fermented black beans.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] To reveal the material basis for the "strong flavor" characteristic of light fermented black beans after fermentation, this paper established a quantitative analysis method for 17 amino acid components in light fermented black beans. The amino acids in light fermented black beans were detected during the fermentation process and the changing trends of each amino acid were analyzed. The results showed that as fermentation progressed, the protein in light fermented black beans gradually converted into amino acids, with significant changes in the pre-fermentation period, especially the rate of change of alanine, which remained stable in the post-fermentation period. Among them, the proportion of umami amino acids gradually increased in the pre-fermentation stage, while the amino acid content did not change much in the late fermentation period, which is presumably due to weakened microbial metabolism.

[0025] The present invention provides an efficient, accurate and reliable method for amino acid analysis of fermented black beans and other fermented foods, which is of great significance for improving product quality, optimizing production processes and promoting related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the ultra-high performance liquid chromatogram of the amino acid mixed reference solution.

[0027] Figure 2 This is the ultra-high performance liquid chromatogram of the light fermented black bean sample solution.

[0028] Figure 3Ultra-high performance liquid chromatogram of the derivatization reagent.

[0029] Figure 4 The changes in amino acid content during the pre-fermentation process of light fermented black beans.

[0030] Figure 5 The changes in amino acid content during the post-fermentation process of light fermented black beans.

[0031] Figure 6 is the change rate of amino acid composition after fermentation of light fermented black beans.

[0032] Figure 7 The percentage of amino acid components in light fermented black beans at different fermentation stages. DETAILED DESCRIPTION

[0033] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] 1.1 Instruments and Materials

[0036] Table 2 Instruments

[0037]

[0038] Table 3 Reagents

[0039]

[0040]

[0041] Reagents: Aspartic acid (H10J10H90034, Asp), Glutamic acid (J30S9R71489, Glu), Serine (H02N9H73849, Ser), Glycine (H04J10Z79020, Gly), Glutamine (J22J9R64037, Gln), Histidine (A31HS192979, His), Arginine (H11M10Y82633, Arg), Threonine (J24N8R48850, Thr), Alanine (J08N8R47577, Ala), Proline (H09N 9T74404, Pro), tyrosine (J30M10R84464, Tyr), valine (H02J10Y91720, Val), methionine (H28M9H62331, Met), isoleucine (H23M9H56575, Ile), leucine (H27F10Y81177, Leu), norleucine (H05JS1, NIe), phenylalanine (H20N8H48638, Phe), and lysine (J27A9Y59981, Lys) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. with a purity of ≥98%.

[0042] Medicinal materials: Black beans, Artemisia annua, and mulberry leaves were provided by Hebei Chunkai Pharmaceutical Co., Ltd.

[0043] 1.2 Experimental methods

[0044] 1.2.1 Preparation of light fermented black beans sample

[0045] Take 90g of mulberry leaves and 100g of Artemisia annua, add 10 and 8 times the amount of the raw herbs in water, and decoct for 1 hour. Filter, combine the filtrates, and concentrate to 1000mL. Place 1000g of black beans in the mixture and soak for 12 hours until fully expanded. After steaming thoroughly in water for 1.5 hours, place the mixture in a fermentation mold, layer the bottom with the decoction of mulberry leaves and Artemisia annua residue, and place the beans on top. Place the mixture in an incubator at a constant temperature and humidity of (30±2)°C and 70% for 6 days of "yellow clothing upper layer" fermentation (pre-fermentation). Remove the residue from the sample after the 6-day pre-fermentation, rinse it clean, and place it in a ceramic jar. Keep it in a constant temperature and humidity of (30±2)°C and 70% for 15 days (post-fermentation). Once fermentation is complete and the aroma is strong, remove the sample, steam it in boiling water for 0.5 hours, and dry it. During the pre-fermentation, samples are taken every two days and are designated as Q0, Q2, Q4, and Q6. During the post-fermentation process, samples were taken every 3 days and recorded as H0, H3, H6, H9, H12 and H15, and refrigerated at -80°C.

[0046] 1.2.2 Sample mass calibration

[0047] The average particle mass (M) of different samples was obtained by weighing, and the correction coefficient of the sample relative to the raw material was calculated according to formula (1-1).

[0048] Correction factor (f) = M sample / M black beans (1-1)

[0049] 1.2.3 Chromatographic conditions

[0050] ACQUITY Column BEH C18 (2.1×100 mm, 1.7 μm); mobile phase: 0.1 mol / L sodium acetate buffer (adjusted to pH 6.5 with acetic acid, A) - acetonitrile (B), gradient elution program see Table 4; injection volume 2 μL; flow rate 0.3 mL / min; detection wavelength 254 nm; column temperature 40°C.

[0051] Table 4 Mobile phase gradient elution conditions

[0052]

[0053] 1.2.4 Preparation of amino acid reference solution

[0054] Take appropriate amounts of 17 reference substances, accurately weigh them, and dissolve them separately in 0.1 mol / L hydrochloric acid solution to prepare reference substance stock solutions with concentrations of Asp 5.34 mg / mL, Glu 5.495 mg / mL, Ser 3.60 mg / mL, Gly 2.64 mg / mL, Gln 2.53 mg / mL, His 4.25 mg / mL, Arg 4.55 mg / mL, Thr 5.44 mg / mL, Ala 4.24 mg / mL, Pro 5.52 mg / mL, Tyr 4.13 mg / mL, Val 4.32 mg / mL, Met 2.75 mg / mL, Ile 4.50 mg / mL, Leu 3.85 mg / mL, Phe 5.05 mg / mL, and Lys 4.81 mg / mL.

[0055] An appropriate amount of each stock solution was taken to prepare a mixed reference solution containing Asp 133.60μg / mL, Glu 274.75μg / mL, Ser 71.90μg / mL, Gly 52.80μg / mL, Gln 50.60μg / mL, His 42.45μg / mL, Arg 181.80μg / mL, Thr 544.00μg / mL, Ala 211.75μg / mL, Pro 137.86μg / mL, Tyr 103.25μg / mL, Val 107.88μg / mL, Met 27.45μg / mL, Ile 112.50μg / mL, Leu 192.25μg / mL, Phe The above mixed reference solution was diluted with 0.1 mol / L hydrochloric acid solution by 2, 4, 8, 16, 32, and 64 times to obtain a series of mixed reference solutions with different concentrations.

[0056] Weigh 5.00 mg of the internal standard Nle into a 5 mL volumetric flask and dissolve it in 0.1 mol / L hydrochloric acid to prepare a 1 mg / mL stock solution. Dilute the internal standard solution to 0.25 mg / mL with 0.1 mol / L hydrochloric acid and store in a refrigerator at 4°C.

[0057] 1.2.5 Preparation of light fermented black bean extract solution

[0058] Take 0.2g of the corrected mass of light fermented black bean powder (passed through No. 2 sieve) of different fermentation times, accurately weigh, place in a 50mL conical flask, add appropriate amount of water, ultrasonically treat (power 600W) for 20min, let cool, add 2mL of 1mol / L hydrochloric acid solution, and adjust the volume to 20mL. Take appropriate amount of 14000rpm centrifugation for 10min, and take the supernatant, which is the light fermented black bean extract. 1.2.6 Preparation of amino acid derivatization reference solution and light fermented black bean test solution

[0059] 1.2.6.1 Preparation of derivatization reagents

[0060] (1) 0.3 mol / L PITC acetonitrile solution: Place 360 ​​μL of PITC in a 10 mL volumetric flask, add acetonitrile to the mark, and shake well.

[0061] (2) 1 mol / L triethylamine acetonitrile solution: Take 1.4 mL of triethylamine and place it in a 10 mL volumetric flask. Add acetonitrile to the mark and shake well.

[0062] 1.2.6.2 Preparation of light fermented black bean sample solution

[0063] Take 200 μL of the light fermented black bean extract under item "1.2.5" and place it in a centrifuge tube, add 20 μL of the internal standard solution, add 100 μL of 1 mol / L triethylamine acetonitrile solution and 100 μL of 0.3 mol / L PITC acetonitrile solution respectively, mix well, and place at 40℃ for 60 minutes for derivatization. Then add 400 μL of n-hexane, shake, and let it stand to separate the layers. Take 100 μL of the lower layer solution, dilute it with 100 μL of water, mix well, centrifuge at 14000 rpm for 10 minutes, and take the supernatant.

[0064] 1.2.6.3 Preparation of Amino Acid Derivatization Reference Solution

[0065] Take 200 μL of each series of mixed reference solution under “1.2.4” and place them in a centrifuge tube. The subsequent derivatization steps are the same as “1.2.6.2”.

[0066] 1.2.6.4 Preparation of blank derivatization reagent

[0067] Take 200 μL of 0.1 mol / L hydrochloric acid solution and place it in a centrifuge tube. The subsequent derivatization steps are the same as those in “1.2.6.2”.

[0068] 1.3 Experimental Results

[0069] 1.3.1 Calibration of sample quality

[0070] As fermentation time increases, mass loss gradually increases. For the same amount of black beans, the post-fermentation mass is lower than the raw material. In quantitative testing, directly calculating by weight will result in an overestimation of the measured substance content and fail to truly reflect the changes in soy isoflavones during the fermentation process. Therefore, it is necessary to use the formula (1-2) to correct the sample mass based on the raw material mass. Table 5 shows the M values ​​for samples at different fermentation stages.

[0071] Sample calibration mass = sample true mass / f(1-2)

[0072] Table 5 Grain weight and correction coefficient of light fermented black bean samples at different processing stages using black beans as fermentation raw materials

[0073]

[0074] 1.3.2 Optimization of amino acid extraction conditions for fermented black beans

[0075] 1.3.2.1 Optimization of extraction solvent

[0076] Take about 0.2 g of the light fermented black bean powder after 15 days of fermentation, place it in a 50 mL conical flask, and accurately add 0.05 mol / L, 0.1 mol / L, 0.2 mol / L hydrochloric acid solution and 5 mL of water respectively. Ultrasonic treatment (power 600 W) for 30 min, cool, take an appropriate amount, centrifuge at 14000 rpm for 10 min, and filter. To ensure consistent acid-base conditions for amino acid derivatization, take 200 μL of the supernatant from the water-extracted sample and add 200 μL of 0.2 mol / L hydrochloric acid solution. Take 200 μL of the supernatant from the sample extracted with 0.05 mol / L hydrochloric acid solution and add 200 μL of 0.15 mol / L hydrochloric acid solution. Take 200 μL of the supernatant from the sample extracted with 0.1 mol / L hydrochloric acid solution and add 200 μL of 0.1 mol / L hydrochloric acid solution. Take 200 μL of the supernatant from the sample extracted with 0.2 mol / L hydrochloric acid solution and add 200 μL of the aqueous solution. Prepare the test samples according to "1.2.6.2." Each condition was processed in triplicate. The peak area of ​​each compound was determined using "1.2.3." Relative peak areas were calculated using the internal standard. The results are shown in Table 6.

[0077] Table 6 Optimal results of extraction solvents (n=3)

[0078]

[0079] It can be seen from Table 6 that when water is used as the extraction solvent, the measured values ​​of amino acids in light fermented black beans are relatively high, so water is selected as the solvent for extraction.

[0080] 1.3.2.2 Optimization of material-liquid ratio

[0081] Place approximately 0.2 g of light fermented black bean powder (15 days post-fermentation) in a 50 mL conical flask. Add appropriate amounts of water, then add 0.5 mL, 1 mL, 2 mL, and 3 mL of 1 mol / L hydrochloric acid solution, respectively. Dose to 5, 10, 20, and 30 mL with water, respectively, and shake well. Follow the same steps as in "1.2.6.2" to obtain the test sample. Each condition was processed in triplicate. Determine the peak area of ​​each compound using "1.2.3" and calculate the relative peak area using the internal standard. The results are shown in Table 7.

[0082] Table 7 Optimization results of material-liquid ratio (n=3)

[0083]

[0084]

[0085] The results are shown in Table 7. When the material-liquid ratio is 1:100, the measured values ​​of most amino acids in light fermented black beans are the highest. Therefore, the optimal material-liquid ratio is determined to be 1:100.

[0086] 1.3.2.3 Optimization of extraction time

[0087] Take approximately 0.2 g of post-fermentation light fermented black bean powder (calibrated mass) and place it in a 50 mL conical flask. Add an appropriate amount of water and sonicate (600 W) for 10, 20, and 30 min, respectively. Allow to cool, then add 2 mL of 1 mol / L hydrochloric acid solution and adjust the volume to 20 mL. Follow the same steps as in "1.2.6.2" to obtain the test solution. Each condition was processed in triplicate. Determine the peak area of ​​each compound using "1.2.3" and calculate the relative peak area using the internal standard. The results are shown in Table 8.

[0088] Table 8 Optimization results of extraction time (n=3)

[0089]

[0090] As can be seen from Table 8, ultrasonic treatment for 20 min can obtain a higher content than that for 10 min and 30 min. Based on this, 20 min was determined to be the optimal extraction time.

[0091] 1.3.3 Optimization of amino acid derivatization conditions for fermented black beans

[0092] 1.3.3.1 Optimal Derivatization Reagent Concentration

[0093] Take 200 μL of the light fermented black bean extract from "1.2.5," add 20 μL of the internal standard, and add 100 μL of 0.1, 0.2, 0.3, and 0.4 mol / L PITC acetonitrile solutions, respectively. Add 100 μL of a 1 mol / L triethylamine acetonitrile solution, mix thoroughly, and perform subsequent derivatization procedures as in "1.2.6.2" to obtain the test sample. Each condition was processed in triplicate. Determine the peak area of ​​each compound using "1.2.3." Calculate the relative peak area using norleucine as the internal standard. The results are shown in Table 9.

[0094] Table 9 Optimal results of PITC solution concentration (n=3)

[0095]

[0096] As shown in Table 9, the contents of 17 amino acids increased with the increase of the derivatization reagent concentration, and good results were obtained at 0.3 mol / L and 0.4 mol / L. After comprehensive consideration, the optimal concentration was determined to be 0.3 mol / L. 1.3.3.2 Optimization of derivatization time

[0097] Take 200 μL of the light fermented black bean extract from "1.2.5," add 20 μL of norleucine as the internal standard, and then add 100 μL each of 0.3 M / L PITC in acetonitrile and 1 M / L triethylamine in acetonitrile. Mix thoroughly and incubate at 40°C for 20, 40, 60, and 80 min, respectively. Follow the same steps as in "1.2.6.2" to obtain the test sample. Each condition is processed in triplicate. Determine the peak area of ​​each compound using "1.2.3." Calculate the relative peak area using the internal standard. The results are shown in Table 10.

[0098] Table 10 Preferred results of derivatization time (n=3)

[0099]

[0100]

[0101] As shown in Table 10, the amino acid content increased with increasing derivatization time, reaching a peak at 60 minutes. At 80 minutes, the content of some amino acids decreased, suggesting that prolonged derivatization time may lead to side reactions. Therefore, a derivatization time of 60 minutes was selected.

[0102] 1.3.3.3 Optimal Derivatization Temperature

[0103] Take 200 μL of the fermented black bean extract from "1.2.5," add 20 μL of the internal standard, norleucine, and then add 100 μL each of 0.3 mol / L PITC in acetonitrile and 1 mol / L triethylamine in acetonitrile. Mix thoroughly and incubate at 20°C, 30°C, 40°C, and 50°C for 60 min. Follow the same steps as in "1.2.6.2" to obtain the test sample. Each condition is processed in triplicate. Determine the peak area of ​​each compound using "1.2.3." Calculate the relative peak area using the internal standard. The results are shown in Table 11.

[0104] Table 11 Preferred results of derivatization temperature (n=3)

[0105]

[0106]

[0107] As shown in Table 11, at 40°C, the measured values ​​of the amino acid components basically reached their peak values, while too high a temperature may cause degradation of the components. Therefore, 40°C was selected as the optimal derivatization temperature.

[0108] In summary, the optimal extraction conditions for amino acids from fermented black beans are as follows: Accurately weigh 0.2 g of fermented black bean powder in a 50 mL stoppered conical flask and add an appropriate amount of aqueous solution. Ultrasonicate at room temperature (600 W) for 20 minutes. Cool, add 2 mL of 1 mol / L hydrochloric acid solution, and adjust the volume to 20 mL. Shake well, and centrifuge an appropriate amount of the supernatant at 14,000 rpm for 10 minutes before filtering. The derivatization method of light fermented black bean amino acids is as follows: 200 μL of the extract is placed in a centrifuge tube, 20 μL of the internal standard solution is added, 100 μL of 1 mol / L triethylamine acetonitrile solution and 100 μL of 0.3 mol / L PITC acetonitrile solution are added respectively, mixed, and placed at 40°C for 60 minutes for derivatization, followed by adding 400 μL of n-hexane, shaking, standing to separate layers, taking 100 μL of the lower layer solution, adding 100 μL of water to dilute, mixing, centrifuging at 14000 rpm for 10 minutes, and taking the supernatant.

[0109] 1.3.4 Methodological Study on the Determination of Amino Acid Content in Fermented Soya Beans

[0110] 1.3.4.1 Study on the linear relationship and detection limit of 17 amino acids in fermented black beans

[0111] Prepare serially diluted mixed reference solution from "1.2.4" and derivatize according to "1.2.6.3" to obtain derivatized reference solutions of varying concentrations. Determine the peak area of ​​each amino acid using "1.2.3" and calculate the relative peak area using the internal standard. Perform two parallel injections, plotting a regression equation with the reference concentration (x) as the x-axis (μg / mL) and the relative peak area as the y-axis. Using a signal-to-noise ratio (S / N) of 3 as the limit of detection (LOD) and an S / N of 10 as the limit of quantification (LOQ), the limits of detection and quantification (LOQs) for 17 amino acids were determined. The results are shown in Table 12.

[0112] Table 1 Linear regression equation, linear range, correlation coefficient, detection limit and quantification limit of 1217 amino acids

[0113]

[0114]

[0115] As shown in Table 12, each component has a good linear relationship within its own concentration range (r 2 ≥0.999), the detection limit was 0.0416-0.3340 μg / mL, and the quantification limit was 0.1248-1.002 μg / mL, indicating that the method for determining the content of amino acid components in light fermented black beans has high sensitivity.

[0116] 1.3.4.2 Precision test

[0117] (1) Intraday precision test

[0118] Prepare the test sample according to the methods in "1.2.5" and "1.2.6.2". Accurately aspirate the same aliquot of the derivatized test sample solution and repeat the injection 6 times. Use "1.2.3" to determine the peak area of ​​each amino acid and calculate the RSD value based on the relative peak area. The results are shown in Table 13.

[0119] Table 13 Intra-day precision test results of 17 amino acids in fermented black beans (n=6)

[0120]

[0121]

[0122] As shown in Table 13, the intra-day precision RSD of the 17 amino acid components was ≤2.0%, which was a good result.

[0123] (2) Daytime precision test

[0124] Prepare the test sample according to the methods in "1.2.5" and "1.2.6.2". For 3 consecutive days, accurately aspirate the same aliquot of the derivatized test sample solution and repeat the injection 6 times. Determine the peak area of ​​each amino acid according to "1.2.3". Calculate the RSD values ​​based on the relative peak areas of the 17 amino acids. The results are shown in Table 14.

[0125] Table 14 Results of inter-day precision test of 17 amino acids in fermented black beans (n=3)

[0126]

[0127] As shown in Table 14, the inter-day precision RSD of the amino acid components in fermented black beans was ≤2.9%, which was a good result.

[0128] 1.3.4.3 Stability test

[0129] Prepare the test solution according to the methods in "1.2.5" and "1.2.6.2" and store it at room temperature. Take samples every 2 hours. Determine the stability of the test solution within 12 hours according to "1.2.3". Calculate the RSD values ​​based on the relative peak areas of the 17 amino acids. The results are shown in Table 15.

[0130] Table 15 Stability test results of 17 amino acids in fermented black beans

[0131]

[0132]

[0133] As shown in Table 15, the RSD of the stability test of 17 amino acids in light fermented black beans was ≤ 2.7%, indicating that the test solution was good at room temperature within 12 h.

[0134] 1.3.4.4 Repeatability test

[0135] Prepare the test sample according to the methods in "1.2.5" and "1.2.6.2" in parallel. Measure the peak areas of 17 amino acids according to "1.2.3" and calculate the contents and RSD values ​​of 17 amino acids using the standard curve method. The results are shown in Table 16.

[0136] Table 16 Repeatability test results of the determination of amino acid content in light fermented black beans (n=6, mg / g)

[0137]

[0138]

[0139] As shown in Table 16, the repeatability results of the 17 amino acid assays showed that the contents of Asp, Glu, Ser, Gly, Gln, His, Arg, Thr, Ala, Pro, Tyr, Val, Met, Ile, Leu, Phe, and Lys were 7.202 mg / g, 14.490 mg / g, 5.298 mg / g, 2.698 mg / g, 4.227 mg / g, 2.665 mg / g, 13.167 mg / g, 28.737 mg / g, 7.264 mg / g, 7.466 mg / g, 6.303 mg / g, 5.856 mg / g, 0.689 mg / g, 5.824 mg / g, 13.016 mg / g, 8.569 mg / g, and 13.179 mg / g, respectively. The RSD of the repeatability test was ≤2.7%, indicating that the method had good repeatability.

[0140] 1.3.4.5 Sample recovery

[0141] 0.2 g of light fermented black bean powder was taken and accurately weighed, and 3 mL of mixed reference solution (containing Asp 17.90 μg / mL, Glu 36.36 μg / mL, Ser 9.152 μg / mL, Gly 7.741 μg / mL, Gln 1.870 μg / mL, His 7.108 μg / mL, Arg 25.71 μg / mL, Thr 76.13 μg / mL, Ala 13.76 μg / mL, Pro 18.15 μg / mL, Tyr 15.07 μg / mL, Val 14.21 μg / mL, Met 2.276 μg / mL, Ile 13.78 μg / mL, Leu 33.67 μg / mL, Phe 2. 22.37 μg / mL, Lys 33.13 μg / mL). Prepare six extraction solutions according to "1.2.5" and perform derivatization according to the steps under "1.2.6.2" to obtain the light fermented black bean curd test solution. Determine the test solution using "1.2.3" in duplicate. Calculate the recoveries and RSDs of the 17 amino acids using recovery formulas 1-3 to assess the accuracy of this method.

[0142] The recovery formula of the sample addition is as follows:

[0143]

[0144] Table 17: Results of sample recovery test for determination of amino acid content in fermented black beans (n=6)

[0145]

[0146]

[0147]

[0148]

[0149] As shown in Table 17, the average recovery rate of the method for determining the content of amino acids in light fermented black beans is between 85.0% and 105.5%, with RSD ≤ 6.0%, indicating that the recovery rate of each compound basically meets the requirements and the method is accurate and feasible.

[0150] In summary, the established method for determining the content of amino acids in fermented black beans is reasonable and feasible.

[0151] 1.3.5 Study on the Changes of Amino Acids in Fermented Soybean

[0152] Based on the method established above, the content of amino acids in fermented fermented soybeans with different fermentation times was determined. The retention time of the chromatographic peaks of 17 amino acids in the test solution was consistent with that of the mixed reference solution, and they were well separated within 28 minutes. The results are as follows: Figure 1 Figure 2 The blank derivatization solvent and internal standard compound have no interference with amino acid analysis, see Figure 3 The results show that the method has good specificity. The changes in the content of amino acid components during the fermentation of light fermented black beans are shown in Table 18.

[0153] Table 18 Changes in the content of amino acid components during the fermentation process of light fermented black beans (n=6, mg / g)

[0154]

[0155]

[0156] Note: “–” means below the detection limit

[0157] Fermentation, a key technology for improving the nutritional, texture, and flavor of soybeans, is one of the oldest food processing methods. Douchi (fermented black beans) can be divided into three categories based on the dominant fermenting microorganisms: bacterial, Aspergillus, and Mucor. Douchi fermented with Aspergillus and Mucor is particularly favored for the unique umami characteristics of its metabolites. Research has shown that umami-producing substances primarily include umami amino acids (such as Glu and Asp), nucleotides, organic acids, organic bases, and umami peptides. During the post-fermentation stage of douchi, soy protein is hydrolyzed by proteases secreted by Mucor and Aspergillus into amino acids, organic acids, and small peptides, significantly enhancing the flavor profile. Because soy protein is rich in Glu and Asp, its fermentation products (such as douchi, soy sauce, and soybean paste) typically exhibit excellent umami properties. Many Glu-containing oligopeptides have been identified as core umami components of fermented soy products, and several protease peptides with umami properties have also been identified. The primary free amino acids contributing to umami are glycine, alanine, serine, glutamic acid, aspartic acid, and glutamine. During the fermentation process of black beans into light fermented black beans, the protein is degraded to produce a variety of amino acids, forming the sensory characteristics of "strong smell and fragrance", which is the material basis of the unique and delicious flavor.

[0158] As shown in Table 18, Figure 4 、 Figure 5As shown, the amino acid content of light fermented black beans changes significantly during the pre-fermentation process. Umami amino acids such as glycine increase from below the detection limit to 4.46±0.197 mg / g, alanine increase from below the detection limit to 46.19±0.757 mg / g, serine increase from below the detection limit to 12.32±0.150 mg / g, glutamate increase from 1.77±0.057 mg / g to 26.47±0.261 mg / g, aspartic acid increase from 0.68±0.023 to 13.85±0.186 mg / g, and glutamine increase from below the detection limit to 19.52±0.324 mg / g. Other amino acids also show a similar upward trend during the pre-fermentation stage. Proteases hydrolyze soybean proteins into small molecules such as peptides and free amino acids, enhancing their bioactivity and bioavailability. During the pre-fermentation process, as protease activity continues to increase, the amino acid content also gradually increases. During the post-fermentation process, the amino acid content fluctuated slightly but remained generally stable. This is presumably because the microorganisms enter a stable phase during post-fermentation, their metabolic activity weakens, and the amino acids produced by the pre-fermentation undergo a series of fermentation metabolism and biochemical reactions (such as the Maillard reaction) with other flavor precursors (such as peptides, fatty acids, and sugars), forming complex flavor compounds.

[0159] The change rate of amino acids after fermentation was calculated using formula (1-4), where VR is the change rate, is the content of the amino acid to be tested in the fermented light fermented black bean curd sample, n is the number of fermentation days, m represents different amino acids, The amino acid content of fermented light fermented soybeans on day 0 was compared with the change rate before and after fermentation. Figure 6 .

[0160]

[0161] Depend on Figure 6 The results showed that the contents of 17 amino acids increased after fermentation of light fermented black beans. Among them, the change rates of lysine and alanine were above 10, the change rates of proline, valine, isoleucine, threonine, tyrosine, serine, glutamine and glutamic acid were between 5 and 10, and the change rates of methionine, arginine, glycine and histidine were less than 5. Figure 7 As can be seen, the proportion of umami amino acids gradually increases with the duration of pre-fermentation, reaching its peak on the sixth day of pre-fermentation. Umami amino acids remain relatively stable during the post-fermentation period. During the fermentation process, in addition to these distinct umami amino acids, several other amino acids also exhibit distinct flavors. Sweet amino acids interact synergistically with umami, enhancing umami flavor. This study, by exploring the changes in umami amino acids during the fermentation of light fermented black beans, illustrates the characteristic characteristic of light fermented black beans that they develop a "rich" flavor.

Claims

1. A method for determining the content of amino acid components in light fermented black beans, characterized in that: The steps include: (1) preparing a light fermented black bean sample into a light fermented black bean extract solution; (2) using a derivatization reagent to prepare a light fermented black bean extract solution into a light fermented black bean test solution; (3) diluting the amino acid solution into different concentration gradients to obtain a mixed amino acid reference solution; Using a derivatization reagent, preparing an amino acid derivatization reference solution from a mixed amino acid reference solution; (4) using a derivatization reagent to prepare a blank derivatization reagent from a hydrochloric acid solution; (5) The light fermented black bean test solution, amino acid derivatization reference solution, and blank derivatization reagent were subjected to ultra-high performance liquid chromatography; wherein mobile phase A was sodium acetate buffer and mobile phase B was acetonitrile; and the gradient elution conditions were as shown in Table 1: Table 1 Mobile phase gradient elution conditions The norleucine solution is used as the internal standard solution with a concentration of 0.25-0.25 mg / mL; the derivatization reagent is an acetonitrile solution of 0.3-0.5 mol / L phenyl isothiocyanate and an acetonitrile solution of 1.0-1.3 mol / L triethylamine.

2. The method according to claim 1, characterized in that The chromatographic column of the ultra-high performance liquid chromatography in step (5) is ACQUITY BEH C18, specifications: 2.1×100 mm, 1.7 μm; injection volume: 2-5 μL; flow rate: 0.3-0.5 mL / min; detection wavelength: 254-280 nm; column temperature: 40-45° C.; the concentration of the sodium acetate buffer: 0.1-0.2 mol / L, pH: 6.5-6.

7.

3. The method according to claim 1, characterized in that The method for preparing the light fermented black bean sample in step (4) is as follows: black beans, mulberry leaf decoction, and Artemisia annua decoction of equal mass are mixed and soaked for 10 to 16 hours, and then steamed for 1.5 to 2 hours; mulberry leaf residue and Artemisia annua residue are spread on the bottom of a fermentation mold, the steamed materials are placed in, fermented for 6 to 8 days, washed, fermented again for 15 to 20 days, steamed for 0.5 to 1 hour, and dried.

4. The method according to claim 1, wherein The preparation method of the light fermented black beans extract solution comprises the following steps: mixing a light fermented black beans sample with water, ultrasonicating at 600-700W for 20-25 minutes, cooling, adding hydrochloric acid solution, centrifuging at 14000-15000rpm for 10-12 minutes, and collecting the supernatant to obtain the light fermented black beans extract solution.

5. The method according to claim 1, wherein The preparation method of the light fermented black bean test solution is as follows: a light fermented black bean extract, an internal standard solution, an acetonitrile solution with phenyl isothiocyanate added, and an acetonitrile solution with triethylamine are mixed and derivatized in a volume ratio of 200:15-25:90-110:90-110, and n-hexane twice the volume of the light fermented black bean extract is added and shaken, and the mixture is allowed to stand and separate, and the lower layer solution is taken and added with an equal amount of water to mix evenly, and the mixture is centrifuged at 14000-15000 rpm for 10-12 minutes, and the supernatant is taken to obtain the solution; and an amino acid derivatization reference solution and a blank derivatization reagent are prepared according to the same method.

6. The method according to claim 1, wherein The amino acid solution was prepared from components with the following concentrations: Asp 133.60 μg / mL, Glu 274.75 μg / mL, Ser 71.90 μg / mL, Gly 52.80 μg / mL, Gln 50.60 μg / mL, His 42.45 μg / mL, Arg 181.80 μg / mL, Thr 544.00 μg / mL, Ala 211.75 μg / mL, Pro 137.86 μg / mL, Tyr 103.25 μg / mL, Val 107.88 μg / mL, Met 27.45 μg / mL, Ile 112.50 μg / mL, Leu 192.25 μg / mL, Phe 126.13 μg / mL, and Lys 192.40 μg / mL.

7. The method according to claim 1, characterized in that The dilution into different concentration gradients is as follows: the amino acid solution is diluted stepwise by 2, 4, 8, 16, 32, and 64 times with 0.1 mol / L hydrochloric acid solution.

8. Use of the method according to any one of claims 1 to 7 in the quality control of light fermented black beans.

9. Use of the method according to any one of claims 1 to 7 in monitoring fermentation of light fermented black beans.

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