A method for rapid detection of amino acid content

Abstract

The application discloses a method for rapidly detecting amino acid content, which comprises the following steps: firstly, subjecting a substrate such as earthworm protein powder to microbial fermentation; and then rapidly detecting the free amino acid content in the fermentation liquor by means of an optimized indantrione color developing method (boiling water bath for 2-3 minutes, determination at 570 nm). The rapid detection technology is deeply combined with a strain screening process, and the amino acid production capacity of bacillus, lactic acid bacteria and mixed strains in a ratio of F14:MLB36:LAB2 (2:3:3) is utilized, so that high-quality strains capable of significantly improving the amino acid yield are directly screened. The application overcomes the problems of low efficiency and high cost of a traditional detection method, and provides a solution for efficient biological manufacturing and rapid detection of amino acids.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection, in particular to a method for rapidly detecting the content of amino acids. BACKGROUND

[0002] As a class of biological molecules with important physiological activity, the content and composition of free amino acids have core guiding significance in key fields such as food, medicine, and agriculture, in product quality control, functional research, and production practice. In the food industry, free amino acids are key substances that give food characteristic flavors such as umami and sweetness, and are also important indicators for measuring the nutritional value of food protein, directly determining the sensory quality and market competitiveness of food; in the medical field, many free amino acids can be used as core raw materials for drug synthesis, supporting the production of antibiotics, vitamins, and other drugs, and the abnormal content of specific amino acids can be used as an auxiliary diagnostic basis for liver diseases, metabolic syndrome, and other conditions, providing data support for clinical diagnosis and treatment; in the agricultural field, the content of free amino acids in soil is a key parameter for assessing soil fertility level, and the accumulation of free amino acids in plants is closely related to crop stress resistance (such as drought resistance, disease and pest resistance) and growth and development status, providing scientific reference for precision fertilization and crop variety improvement; therefore, building an accurate, efficient, and widely applicable free amino acid content detection method is an important foundation for promoting technology upgrading and high-quality development in related fields.

[0003] Currently, the mainstream amino acid content detection technologies in the industry mainly include high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and amino acid analyzer method; among them, high performance liquid chromatography requires sample derivatization pretreatment (such as pre-column derivatization, post-column derivatization), the operation process is complicated and the derivatization reagent is easy to introduce interference, which leads to prolonged detection period and increased error risk; gas chromatography-mass spectrometry has high sensitivity, but requires a special thermal cracking sampling device, which has high equipment purchase and maintenance costs, and requires high professional skills of the operator; the amino acid analyzer method has low detection efficiency (more than 30 minutes for single sample analysis) and high price of instrument consumables, which is difficult to adapt to large-scale sample batch detection scenarios. SUMMARY

[0004] In order to solve the problems of long detection time and low efficiency of amino acids, the purpose of the present application is to provide a method for rapidly detecting the content of amino acids, which improves the content of free amino acids and rapidly detects the content of free amino acids by screening strains and ninhydrin colorimetric method.

[0005] The present application provides a method for rapidly detecting the content of amino acids, comprising the following steps:

[0006] (1) sample pretreatment: take 5g earthworm protein powder in a 150ml flask, add water to 50ml, sterilize at 121℃, 101kpa for 30min, then add 0.005g alkaline protease and bacterial culture solution to the fermentation substrate at a ratio of 1%, stir evenly, cultivate at 30℃, 200r / min, measure pH every 12h during cultivation, when pH<5, add 0.005g acid protease, cultivate for 36h to obtain microbial fermentation product, then determine the content of free amino acid by ninhydrin colorimetry; wherein the bacterial culture solution is any one of bacillus or lactic acid bacteria, take the microbial fermentation product, centrifuge, take the supernatant and dilute to obtain the extract;

[0007] (2) color development: take the extract and add to 2ml with ammonia-free water, add ninhydrin color developing solution and vitamin C solution, mix well, then heat in boiling water bath for 2-3min, cool to room temperature, add ethanol solution and mix well to obtain the sample to be tested;

[0008] (3) take the amino acid standard solution and the sample to be tested to read wavelength, take the content of amino acid standard as abscissa and absorbance as ordinate to draw standard curve, then substitute the sample absorbance into the standard curve to obtain the content of free amino acid in the sample, the formula is:

[0009]

[0010] Wherein, ρ is the mass concentration of free amino acid in microbial fermentation product, μg / ml; A is the absorbance value of the sample; a is the intercept of the standard curve; b is the slope of the standard curve; f is the dilution factor.

[0011] Preferably, the color developing method of the amino acid standard solution in step (3) is the same as step (2), take 5μg / ml of leucine standard solution 0, 0.2, 0.4, 0.6, 0.8, 1.0ml in 6 25ml colorimetric tubes, add to 2ml with ammonia-free water; then add 3.0ml ninhydrin color developing solution, 0.1ml 1g / ml vitamin C solution, mix well, cover the test tube opening; heat in boiling water bath for 3min, cool to room temperature with tap water, add 15ml 60% ethanol for dilution, mix well, obtain the leucine standard solution to be tested.

[0012] Preferably, the centrifugation in step (1) is 4℃, 10000r / min for 5min.

[0013] Preferably, the dilution to constant volume in step (1) is dilution to constant volume with pure water to 250ml.

[0014] Preferably, the concentration of ninhydrin color developing solution in step (2) is 2%(m / v).

[0015] Preferably, the concentration of the vitamin C solution in step (2) is 1 g / mL, and the added amount is 0.1 mL.

[0016] Preferably, the ethanol solution in step (2) is 60% ethanol.

[0017] Preferably, the amino acid standard solution in step (3) is a leucine standard solution, and the concentration is 5 μg / mL.

[0018] Preferably, the wavelength in step (3) is 570 nm.

[0019] The embodiment of the present application has the following beneficial effects:

[0020] Through microbial strain screening and fermentation process optimization, it is determined that the spore bacillus F14, MLB36 and lactic acid bacteria LAB2 (ratio 2:3:3) are the dominant mixed strains, and the enzyme hydrolysis process of "alkaline protease pretreatment + adding acid protease at pH <5" can efficiently release the free amino acids in the earthworm protein powder, providing stable and sufficient target substances for subsequent detection, and reducing the detection error caused by the low or fluctuating content of free amino acids in the sample.

[0021] The traditional indophenol colorimetric method is optimized, and the color reaction time is shortened from the conventional ten minutes to 2-3 minutes, realizing the ultra-fast determination of amino acid content. At the same time, through the operation specification of "standard solution and sample under the same condition coloration", the system error in the coloration process is further eliminated. The experimental data shows that the standard error of the same sample detected by the spore bacillus F14 strain is only 3.51575, and the result stability is excellent.

[0022] Compared with the traditional high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and amino acid analyzer method (more than 30 minutes for single analysis), the method realizes the rapid connection of "sample pretreatment-coloration-absorbance determination" through the optimization of color reaction and detection process. The coloration step only needs to be heated in a boiling water bath for 2-3 minutes, and the whole single detection time can be controlled within 10 minutes, which greatly shortens the detection period and solves the problems of low efficiency and long time of traditional methods.

[0023] The indophenol coloration detection is combined with the microbial fermentation process to improve the content of free amino acids in the product. The content of free amino acids in the fermentation product of mixed strains is as high as 140.636 μg / mL, which is much higher than that of single strain. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The leucine standard solution coloration time and absorbance value curve in Example 4 of the present application;

[0025] Figure 2 The color development time experiment graph of the leucine standard solution in Example 4 of the present application;

[0026] Figure 3 The concentration and absorbance value curve graph of the leucine standard solution in Example 4 of the present application;

[0027] Figure 4 The leucine standard curve graph in the single strain screening in Example 5 of the present application;

[0028] Figure 5 The result statistical difference graph of the Bacillus sp. in Example 5 of the present application;

[0029] Figure 6 The result statistical difference graph of the Trichoderma sp. in Example 5 of the present application;

[0030] Figure 7 The result statistical difference graph of the Lactic acid bacteria in Example 5 of the present application;

[0031] Figure 8 The experiment graph of the MLB36 strain and the LAB2 strain in Example 6 of the present application;

[0032] Figure 9 The experiment graph of the F14 strain and the LAB2 strain in Example 6 of the present application;

[0033] Figure 10 The experiment graph of the F14 strain and the MLB36 strain in Example 6 of the present application;

[0034] Figure 11 The result statistical difference graph of the strain ratio in Example 6 of the present application. DETAILED DESCRIPTION

[0035] The technical solutions of the present application will be described in detail below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0036] In the following preparation examples and embodiments, the strains involved are all from the laboratory, and the remaining reagents are all commercially available reagents.

[0037] Preparation Example

[0038] Preparation of reagents:

[0039] (1) 2% ninhydrin solution: weigh 0.2 g of ninhydrin, dissolve in anhydrous ethanol and dilute to 10 mL (freshly prepared for use);

[0040] (2) 5 μg / mL leucine standard solution: 0.0234 g of leucine dried at 80°C to constant weight was dissolved in 10% isopropyl alcohol and diluted to 50 mL, 5 mL of the solution was diluted to 50 mL with distilled water, and then transferred into a reagent bottle to obtain 5 mg / mL amino acid standard solution;

[0041] (3) 60% ethanol solution: 60 mL of anhydrous ethanol was diluted to 100 mL with pure water;

[0042] (4) 1 g / mL vitamin C solution: 1.00 g of vitamin C was diluted to 10 mL with pure water.

[0043] Example 1

[0044] Bacillus strain

[0045] (1) Experimental method: 5 g of earthworm protein powder was taken in each of 150 mL of a flask, a total of 11, and the bacillus strain number was added, distilled water was added to 50 mL, sterilized at 121°C, 101 kPa for 30 min, 0.005 g of alkaline protease and bacillus strain culture solution were added to the fermentation substrate at a ratio of 1%, stirred uniformly, and cultured at 30°C, 200 r / min. During the culture, the pH was measured every 12 h using a pH meter, and when the pH was <5, 0.005 g of acidic protease was added. After 36 h of culture, 20 mL of bacillus fermentation product was taken and the free amino acid content was determined by the ninhydrin colorimetric method;

[0046] (2) The bacillus strain numbers were: XP271, MX27, XTB1, MFL102, MX66, XP248, XP181, F14, MX38, MFL379, MLB36.

[0047] Example 2

[0048] Trichoderma strain

[0049] (1) Experimental method: 5 g of earthworm protein powder was taken in each of 150 mL of a flask, a total of 8, and the trichoderma strain number was added, distilled water was added to 50 mL, sterilized at 121°C, 101 kPa for 30 min, 0.005 g of alkaline protease and trichoderma strain culture solution were added to the fermentation substrate at a ratio of 1%, stirred uniformly, and cultured at 30°C, 200 r / min. During the culture, the pH was measured every 12 h using a pH meter, and when the pH was <5, 0.005 g of acidic protease was added. After 36 h of culture, 20 mL of trichoderma fermentation product was taken and the free amino acid content was determined by the ninhydrin colorimetric method;

[0050] (2) The trichoderma strain numbers were: M1, M2, M3, M4, M5, M6, M7, M8.

[0051] Example 3

[0052] Lactic acid bacteria strain

[0053] (1) Experimental method: 5 g of earthworm protein powder was taken in a 150 mL flask, a total of 6, and the lactic acid bacteria strain number was added. Distilled water was added to 50 mL, sterilized at 121 ℃, 101 kPa for 30 min, then 0.005 g of alkaline protease and lactic acid bacteria strain culture solution was added to the fermentation substrate at a ratio of 1%, stirred uniformly, and cultured at 30 ℃, 200 r / min. During the culture period, the pH was measured every 12 h using a pH meter, and when the pH was <5, 0.005 g of acid protease was added. After 36 h of culture, 20 mL of lactic acid bacteria fermentation product was taken and the free amino acid content was determined by ninhydrin colorimetry;

[0054] (2) The lactic acid bacteria strain number is: lab4.4, LAB3.2, LAB2, Y2-8, Y1-3, Y1-2.1.

[0055] Example 4

[0056] Leucine standard curve parameter determination

[0057] (1) Determination of coloration time:

[0058] 1) Take 5 μg / mL of leucine standard solution 1.0 mL in 6 25 mL colorimetric tubes, respectively, and add distilled water to 2 mL; then add 3.0 mL of ninhydrin color developing solution, 0.1 mL of 1 g / mL vitamin C solution, mix thoroughly, and cover the test tube opening; heat in a boiling water bath for 0 min, 1 min, 2 min, 3 min, 4 min, and 5 min, respectively; cool to room temperature with tap water, dilute with 15 mL of 60% ethanol, mix thoroughly, and measure the absorbance at 570 nm with a spectrophotometer. The results are shown in Table 1. The time-absorbance curve is shown in Figure 1 , where the abscissa is time (min) and the ordinate is ABS absorbance. The experimental coloration graph is shown in Figure 2 .

[0059] Table 1 Reaction time and absorbance data

[0060]

[0061] 2) Coloration time results: According to Table 1, Figure 1 and Figure 2 , it is found that the coloration effect is best when the boiling water bath is at 2-3 min, and 2 min is better than 3 min.

[0062] (2) Determination of standard curve range:

[0063] 1) Take 0, 1, 2, 3, 4, 5, 6, 7 μg / mL of leucine standard solution 1.0 mL in 8 25 mL cuvettes, respectively, and add up to 2 mL with ammonia-free water; then add 3.0 mL of ninhydrin color developing solution, 0.1 mL of 1 g / mL vitamin C solution, mix thoroughly, and cover the test tube opening; heat in a boiling water bath for 2 min; cool to room temperature with tap water, dilute with 15 mL of 60% ethanol, mix thoroughly, and measure the 570 nm absorbance value with a spectrophotometer. The results are shown in Table 2. The concentration vs. absorbance curve is shown in FIG. 1, where the abscissa is the concentration (μg / mL) and the ordinate is the ABS absorbance value. Figure 3

[0064] Table 2 Standard amino acid concentration vs. absorbance data

[0065]

[0066] 3) Leucine standard solution concentration range results: from Table 2 Figure 3 It is obtained that the absorbance value data of the standard amino acid concentration of 5 μg / mL is the best.

[0067] Example 5

[0068] Single strain screening

[0069] (1) Leucine standard curve preparation: take 0, 0.2, 0.4, 0.6, 0.8, 1.0 mL of 5 μg / mL leucine standard solution in 6 25 mL cuvettes, respectively, and add up to 2 mL with ammonia-free water; then add 3.0 mL of ninhydrin color developing solution, 0.1 mL of 1 g / mL vitamin C solution, mix thoroughly, cover the test tube opening, and heat in a boiling water bath for 3 min; cool to room temperature with tap water, dilute with 15 mL of 60% ethanol, mix thoroughly, and measure the 570 nm absorbance value. The data are shown in Table 3, and the standard curve is shown in FIG. 2, where the ordinate is the absorbance value and the abscissa is the amino acid content. Figure 4

[0070] Table 3 Leucine standard solution absorbance

[0071]

[0072] (2) Fermentation samples: take 20 mL of each of 11 Bacillus fermentation products of Example 1, 8 Trichoderma fermentation products of Example 2, and 6 lactic acid bacteria fermentation products of Example 3 in 150 mL conical flasks;

[0073] ​​(3) Sample pretreatment: Place the conical flask containing the fermentation sample in a shaker at 150 r / min and extract at 20℃ for 30 min. After extraction, transfer all the material in the conical flask into a 50 mL centrifuge tube. Rinse the conical flask with distilled water and transfer the rinsing liquid into the centrifuge tube. Centrifuge at 10000 r / min and 4℃ for 5 min. After centrifugation, transfer the supernatant into a 250 mL volumetric flask. Transfer the residue into the conical flask, add 30 mL of distilled water and continue extraction. Repeat the extraction 3 times. Make up the volume of the volumetric flask to 250 mL. Accurately transfer 1.00 mL of sample into a 50 mL colorimetric tube and make up the volume with pure water to obtain the sample to be tested.

[0074] (4) Determination of free amino acid content in samples: Take 1.0 mL of each sample to be tested into a 25 mL colorimetric tube, and make up to 2 mL with ammonia-free water; then add 3.0 mL of ninhydrin colorimetric solution and 0.1 mL of 1 g / mL vitamin C solution, mix thoroughly, and cover the mouth of the test tube; heat in a boiling water bath for 3 min, cool to room temperature with tap water, add 15 mL of 60% ethanol for dilution, shake thoroughly, and measure the absorbance at 570 nm using a spectrophotometer to calculate the free amino acid content in the sample: Wherein, ρ - mass concentration of free amino acids in microbial fermentation products, μg / mL; A - absorbance value of the sample; a - intercept of the standard curve; b - slope of the standard curve; f - dilution factor;

[0075] (5) Test results: The results for Bacillus are shown in Table 4, and the statistical graph of the results is shown in the figure below. Figure 5 As shown in Table 5; the results for Trichoderma are shown in the statistical graph below. Figure 6 As shown in Table 6; the lactic acid bacteria are shown in Table 7, and the statistical results are shown in the following graph. Figure 7 As shown;

[0076] Table 4 Results of Bacillus subtilis (n=3)

[0077]

[0078] Table 5 Results of Trichoderma (n=3)

[0079]

[0080] Table 6 Results of lactic acid bacteria (n=3)

[0081]

[0082] (6) Results Analysis: Through Table 4 and Figure 5 Bacillus strains F14 and MLB36 were found to have the highest free amino acid content and the best effect; as shown in Table 5 and... Figure 6Trichoderma strain free amino acid content results lowest, poor effect; by table 6 and Figure 7 Get LAB2 lactic acid bacteria strain number free amino acid content highest, the best effect.

[0083] Example 6

[0084] Mixed strain screening

[0085] (1) leucine standard curve drawing: same as example 5;

[0086] (2) affinity determination between strains: cross each other with bacillus F14 and MLB36 strain, lactic acid bacteria LAB2 strain, 24h after observing whether there is antagonism between strains, MLB36 strain and LAB2 strain experimental diagram as shown in Figure 8 ; F14 strain and LAB2 strain experimental diagram as shown in Figure 9 ; F14 strain and MLB36 strain experimental diagram as shown in Figure 10 ;

[0087] (3) strain ratio screening: set different inoculation amount (1%, 2%, 3%, ) to mix fermentation of each strain, as shown in table 1, set L9(3 3 ) orthogonal test, after activation of each strain in LB plate, inoculate in liquid LB medium (liquid volume: 50mL / 150 mL flask), 30℃, 180 r / min shaking table culture 36-48h, after each strain culture concentration reaches 108 CFU / mL, according to the treatment ratio in table 7, 5g / 50mL earthworm protein culture medium is added again, when pH<5, 0.005 g acid protease is added and cultured for 36h, then the free amino acid content is determined by ninhydrin colorimetry, the results are shown in table 7, and the results statistical diagram is shown in Figure 11 ;

[0088] Table 7 best ratio of each strain experiment

[0089]

[0090] (4) results: from Figure 8 , Figure 9 and Figure 10 , F14, MLB36 and LAB2 strain have no antagonism, can be mixed in proportion; from table 7 and Figure 11 , T5 group has the highest free amino acid content, the ratio of F14:MLB36:LAB2 is 2:3:3.

[0091] In summary, the present application establishes a high-efficiency and rapid free amino acid content detection method by optimizing microbial fermentation process and ninhydrin colorimetric detection process; in the fermentation process aspect, the present application screens out Bacillus F14, MLB36 and lactic acid bacteria LAB2 as dominant strains, and the three strains have no antagonism, and when mixed fermentation according to the inoculation ratio of 2:3:3, the release amount of free amino acids in earthworm protein powder can be significantly improved; in the detection process aspect, the present application determines the optimal coloration condition as follows: boiling water bath heating for 2-3 min, dilution with 60% ethanol after cooling, and determination of the absorbance value at 570 nm wavelength; and the standard curve is drawn by using 5 μg / mL leucine standard solution, the detection period is short, the operation is simple and no complex and expensive equipment is needed, compared with the traditional HPLC, GC-MS and other methods, the detection efficiency is greatly improved, and the detection cost is reduced, at the same time, the strain screening and process optimization ensure the accuracy and stability of the detection results, and the method is suitable for batch detection of large-scale samples in the fields of food, medicine, agriculture and the like, and has wide application prospect and practical value.

[0092] Although the embodiments of the present application have been shown and described, it is to be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present application, and any equivalent changes and improvements made within the scope of the present application shall still belong to the patent coverage scope of the present application.

Claims

1. A method for rapid detection of amino acid content, characterized by, The method comprises the following steps: (1) sample pretreatment: taking earthworm protein powder as a substrate, adding alkaline protease and strain culture solution for fermentation, adding acid protease when pH<5 and culturing for 36 hours to obtain a microbial fermentation product, wherein the strain culture solution is any one of bacillus or lactic acid bacteria, taking the microbial fermentation product, extracting, centrifuging, taking supernatant and constant volume to obtain an extract; (2) color development: taking the extract, adding 2 mL of ammonia-free water, adding indantrione color developing solution and vitamin C solution, mixing, boiling water bath heating for 2-3 minutes, cooling to room temperature, adding ethanol solution and mixing to obtain a sample to be tested; (3) taking amino acid standard solution and the sample to be tested to read wavelength, taking amino acid standard content as abscissa and absorbance as ordinate to draw a standard curve, and substituting sample absorbance into the standard curve to obtain the content of free amino acid in the sample.

2. The method for rapid detection of amino acid content according to claim 1, wherein, The color developing method of the amino acid standard solution in step (3) is the same as step (2).

3. The method for rapid detection of amino acid content according to claim 1, wherein, The centrifugation in step (1) is 10000 r / min at 4℃ for 5 minutes.

4. The method for rapid detection of amino acid content according to claim 1, wherein, The constant volume in step (1) is constant volume to 250 mL with pure water.

5. The method for rapid detection of amino acid content according to claim 1, wherein, The concentration of the indantrione color developing solution in step (2) is 2% (m / v).

6. The method for rapid detection of amino acid content according to claim 1, wherein, The concentration of the vitamin C solution in step (2) is 1 g / mL, and the addition amount is 0.1 mL.

7. The method for rapid detection of amino acid content according to claim 1, wherein, The ethanol solution in step (2) is 60% ethanol.

8. The method for rapid detection of amino acid content according to claim 1, wherein, The amino acid standard solution in step (3) is leucine standard solution, and the concentration is 5 μg / mL.

9. The method for rapid detection of amino acid content according to claim 1, wherein, The wavelength in step (3) is 570 nm.

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