Quantitative detection method for content of soybean protein component

Abstract

The invention relates to the technical field of protein detection, in particular to a quantitative detection method for the content of soybean protein components. According to the quantitative detection method for the component content of the soybean protein, the capillary electrophoresis method is adopted, the detection method is optimized, efficient separation of all components of the soybean protein can be achieved, quantitative calculation can be achieved, the analysis time is short, the scheme is reliable, and reproducibility is good.

Description

Technical Field

[0001] The invention relates to the technical field of protein detection, and in particular to a method for quantitatively detecting the content of soybean protein components. Background Art

[0002] Mature soybean seeds contain 35-46% protein, and soy protein is considered "high-quality protein." In 1999, the U.S. Food and Drug Administration (FDA) issued a statement: consuming 25 grams of soy protein per day can reduce the risk of cardiovascular and cerebrovascular diseases.

[0003] There are many types of soy protein subcomponents, and the common components are: trypsin inhibitor, cytochrome C, hemagglutinin, lipoxygenase, β-amylase, β-conglycinin, and glycinin. The composition of each component of soy protein is directly related to its nutritional value and functional properties. The specific protein component ratios are different between different soybean varieties. For soy protein products, such as soy protein powder, soy milk, soy-based drinks, etc., it is an important task to determine their protein composition and ratio.

[0004] At present, gel electrophoresis optical density scanning, liquid chromatography and immunoadsorption are mainly used in the detection of soybean protein components. The main problem of gel electrophoresis is that the resolution is low, and the separation degree of each protein component in the limited electrophoresis channel is not high. Moreover, the quantitative accuracy of protein by gel electrophoresis optical density scanning is not high, and the quantitative results of different software vary greatly, which can only be used as a rough estimate. Liquid chromatography has low separation efficiency and low separation degree. For some components with extremely subtle differences, liquid chromatography cannot be separated, and multi-step and multi-process operations are required. The sample processing of liquid chromatography is more complicated, the sample amount required is large, usually at the μl level, and the separation speed is slow, and many mobile phases need to use organic mobile phases, which are less environmentally friendly. The problem with immunoadsorption is that the process is cumbersome and requires the preparation of high-purity antigens and antibodies.

[0005] Therefore, it is urgent to develop a quantitative detection method that can detect the content of each component of soybean protein with good separation effect and high resolution. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. To this end, one object of the present invention is to provide a quantitative detection method for the content of soybean protein components. The quantitative detection method for the content of soybean protein components provided by the present invention adopts capillary electrophoresis, and by optimizing the detection method, it can achieve efficient separation of various components of soybean protein, quantitative calculation, short analysis time, reliable scheme, and good reproducibility.

[0007] To this end, the first aspect of the present invention provides a quantitative detection method for the content of soybean protein components. According to an embodiment of the present invention, the quantitative detection method comprises:

[0008] S1: Pre-treating the soybean protein sample to be tested so as to dissociate the protein components and obtain a dissociation solution;

[0009] S2: performing capillary electrophoresis separation on the dissociation solution to obtain a capillary electrophoresis pattern of the soybean protein sample to be tested;

[0010] S3: Obtain capillary electrophoresis profile of standard products based on proteins of known molecular weight;

[0011] S4: Based on the capillary electrophoresis spectrum of the standard, determining the molecular weight of each protein to be tested and the peak position of each component in the capillary electrophoresis spectrum of the soybean protein sample to be tested;

[0012] S5: Based on the molecular weight of each protein to be tested and the peak position of each component, the content of each component is determined by normalization and integration.

[0013] The electrophoresis buffer used in capillary electrophoresis separation includes urea, Tris, SDS and methyl cellulose.

[0014] Capillary electrophoresis can achieve efficient separation of some components with extremely subtle differences, and the separation speed is fast. Moreover, capillary electrophoresis is suitable for the analysis of components with very small sample amounts, and can realize the injection analysis of nl samples. Capillary electrophoresis has a variety of separation methods and is suitable for different separation needs. More importantly, capillary electrophoresis has excellent quantitative analysis functions and can achieve quantitative chromatogram integration of each component, which cannot be achieved by gel electrophoresis and optical density scanning. The consumables and reagents used in capillary electrophoresis analysis are low in cost and low in expense.

[0015] The inventors of the present application have obtained a quantitative detection method for soybean protein components by using capillary electrophoresis to detect the content of soybean protein components, and further optimized the composition of the electrophoresis buffer used in capillary electrophoresis separation, thereby obtaining a quantitative detection method for soybean protein components that can be accurately quantified, has good repeatability, and is highly reliable. Through this method, the content of soybean protein components can be more accurately quantified by an integral method, and the contents of multiple components in soybean protein can be detected simultaneously.

[0016] According to an embodiment of the present invention, the electrophoresis buffer is DTT and / or mercaptoethanol.

[0017] According to an embodiment of the present invention, the concentration of urea in the electrophoresis buffer is -4-8M.

[0018] According to an embodiment of the present invention, the concentration of Tris in the electrophoresis buffer is 50-200 mM.

[0019] According to an embodiment of the present invention, the concentration of DTT in the electrophoresis buffer is 0.05-0.3 wt %.

[0020] According to an embodiment of the present invention, the concentration of SDS in the electrophoresis buffer is 0.1-1.5 wt %.

[0021] According to an embodiment of the present invention, the concentration of methylcellulose in the electrophoresis buffer is 0.05-5 wt %.

[0022] According to an embodiment of the present invention, the pH of the electrophoresis buffer is 7.0-9.2.

[0023] According to an embodiment of the present invention, uncoated capillaries are used when performing capillary electrophoresis separation.

[0024] According to an embodiment of the present invention, the length of the uncoated capillary is 30-80 cm.

[0025] According to an embodiment of the present invention, the inner diameter of the uncoated capillary is 25-70 μm.

[0026] By utilizing the specifications of the uncoated capillary explored by the present invention, the desired separation effect can be obtained, and multiple components in soybean protein can be separated.

[0027] According to an embodiment of the present invention, the separation voltage used in capillary electrophoresis separation is 10-30 kV, and the separation time is 40-180 min.

[0028] According to an embodiment of the present invention, in step S1, the soybean protein sample to be tested is pre-treated with a sample buffer.

[0029] Wherein, the sample buffer comprises urea, Tris, DTT and SDS.

[0030] Urea and SDS in the sample buffer are conducive to the full dissolution of proteins, and DTT can dissociate disulfide bonds. According to an embodiment of the present invention, the concentration of urea in the sample buffer is 4-8M, preferably 6-8M.

[0031] According to an embodiment of the present invention, the concentration of Tris in the sample buffer is 50-200 mM.

[0032] According to an embodiment of the present invention, the concentration of DTT in the sample buffer is 0.05-0.3 wt %, preferably 0.1-0.3 wt %.

[0033] According to an embodiment of the present invention, the concentration of SDS in the sample buffer is 0.1-0.5 wt %.

[0034] According to an embodiment of the present invention, the pH of the sample buffer is 7.0-9.2.

[0035] According to an embodiment of the present invention, the protein of known molecular weight is the following a or b:

[0036] a. Protein with the same molecular weight as the protein component to be measured contained in soybean protein;

[0037] b. Standard samples of the protein components to be determined in soy protein.

[0038] According to an embodiment of the present invention, step S5 further comprises:

[0039] The peak area normalization method was used to quantitatively analyze each component.

[0040] According to an embodiment of the present invention, each component is quantitatively analyzed by the following method:

[0041] The content X of a certain protein component in the soybean protein sample to be tested is calculated by the following formula:

[0042]

[0043] In the formula, A m is the area of ​​peak m corresponding to a certain protein component;

[0044] A T is the sum of the total areas of n peaks;

[0045] n is the total number of peaks integrated in the electrophoresis spectrum of the soybean protein sample to be tested. The method of the present invention can achieve relatively accurate quantitative determination of the content of soybean protein components, and can simultaneously detect the content of multiple components in soybean protein, with good separation effect, high separation degree and high separation efficiency. In particular, each component can be quantitatively calculated, and the computability is good. It is economical, and the reagents used are non-toxic and harmless, and have good reproducibility.

[0046] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0048] Figure 1 A flow chart showing a method for quantitatively detecting the content of soybean protein components according to one embodiment of the present invention is shown;

[0049] Figure 2 The figure shows the separation result obtained by capillary electrophoresis in Example 1;

[0050] Figure 3 The figure shows the separation result obtained by capillary electrophoresis in Example 2;

[0051] Figure 4 The figure shows the separation result obtained by capillary electrophoresis in Example 3;

[0052] Figure 5 The figure shows the separation result obtained by capillary electrophoresis in Example 4;

[0053] Figure 6 The figure shows the separation result obtained by capillary electrophoresis in Example 5;

[0054] Figure 7 The figure shows the separation result obtained by capillary electrophoresis in Comparative Example 1;

[0055] Figure 8 The figure shows the separation result obtained by capillary electrophoresis in Comparative Example 2;

[0056] Fig. 9 The gel image of the molecular weight standard and soybean protein detected by SDS-PAGE method in Comparative Example 3 is shown. DETAILED DESCRIPTION

[0057] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0058] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0059] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0060] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0061] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0062] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0063] The purity of some protein subunits in soybean protein was determined by SDS-PAGE gel electrophoresis. However, the separation of bands by gel electrophoresis is limited, and the bands overlap with each other. In addition, the definition of bands by various analysis software is different, so the analysis results often have large differences and inaccuracies. Gel chromatography is to separate soybean protein into various components through chromatographic classification, and then detect and integrate them. However, the problem is that for protein separation, many additives such as mercaptoethanol and SDS need to be added. These reagents often damage the chromatographic filler and remain in the filler and cannot be removed, causing irreversible damage to the filler and reducing the separation efficiency. The problem with immunoadsorption is that the process is cumbersome and requires the preparation of high-purity antigen antibodies. For qualitative analysis, immunoadsorption has certain advantages and convenience, but for quantitative analysis, the accuracy of the results is not very high.

[0064] Based on this, the inventors used high-pressure capillary electrophoresis to separate and detect soybean protein, and the test results can be used to determine the content of each protein component using the peak area normalization method.

[0065] According to a specific embodiment of the present invention, the present invention provides a method for quantitatively detecting the content of soybean protein components, such as Figure 1 As shown, including:

[0066] S1: Pre-treating the soybean protein sample to be tested so as to dissociate the protein components and obtain a dissociation solution;

[0067] S2: performing capillary electrophoresis separation on the dissociation solution to obtain a capillary electrophoresis pattern of the soybean protein sample to be tested;

[0068] S3: Obtain capillary electrophoresis profile of standard products based on proteins of known molecular weight;

[0069] S4: Based on the capillary electrophoresis spectrum of the standard, determining the molecular weight of each protein to be tested and the peak position of each component in the capillary electrophoresis spectrum of the soybean protein sample to be tested;

[0070] S5: Based on the molecular weight of each protein to be tested and the peak position of each component, the content of each component is determined by normalization and integration.

[0071] The electrophoresis buffer used in capillary electrophoresis separation includes urea, Tris, SDS and methyl cellulose.

[0072] According to a specific embodiment of the present invention, the electrophoresis buffer is DTT and / or mercaptoethanol.

[0073] According to a specific embodiment of the present invention, the concentration of urea in the electrophoresis buffer is 4-8 M. Preferably, the concentration of urea in the electrophoresis buffer is 6-8 M, more preferably 6 M.

[0074] According to a specific embodiment of the present invention, the concentration of Tris in the electrophoresis buffer is 50-200 mM.

[0075] According to a specific embodiment of the present invention, the concentration of DTT in the electrophoresis buffer is 0.1-0.3 wt %. Preferably, the concentration of DTT in the electrophoresis buffer is 0.1 wt %.

[0076] According to a specific embodiment of the present invention, the concentration of SDS in the electrophoresis buffer is 0.1-0.5 wt %.

[0077] According to a specific embodiment of the present invention, the concentration of methylcellulose in the electrophoresis buffer is 0.05-5wt%.

[0078] According to a specific embodiment of the present invention, the pH of the electrophoresis buffer is 7.0-9.2. It should be noted that the electrophoresis buffer provided by the present invention can be adjusted to a pH of 7.0-9.2 using HCl.

[0079] According to a specific embodiment of the present invention, the electrophoresis buffer used for capillary electrophoresis separation comprises:

[0080] 4-8 M urea, 50-200 mM Tris, 0.1-0.3 wt % DTT, 0.1-0.5 wt % SDS and 0.05-5 wt % methylcellulose, pH adjusted to 7.0-9.2 with HCl.

[0081] According to a specific embodiment of the present invention, the protein of known molecular weight is the following a or b:

[0082] a. Protein with the same molecular weight as the protein component to be measured contained in soybean protein;

[0083] b. Standard samples of the protein components to be determined in soy protein.

[0084] It should be noted that the protein of known molecular weight may be a pure protein of one molecular weight, or a mixture of proteins of multiple different molecular weights.

[0085] According to a specific embodiment of the present invention, uncoated capillaries are used for capillary electrophoresis separation.

[0086] According to a specific embodiment of the present invention, the length of the uncoated capillary is 30-80 cm.

[0087] According to a specific embodiment of the present invention, the inner diameter of the uncoated capillary is 25-70 μm.

[0088] According to a preferred embodiment of the present invention, the uncoated capillary has a length of 60 cm and an inner diameter of 50 μm.

[0089] According to a specific embodiment of the present invention, the separation voltage used in capillary electrophoresis separation is 10-30 kV, and the separation time is 40-180 min.

[0090] According to a preferred embodiment of the present invention, the separation voltage used in capillary electrophoresis separation is 20 kV and the separation time is 120 min.

[0091] According to a specific embodiment of the present invention, in step S1, the soybean protein sample to be tested is pre-treated with a sample buffer, wherein the sample buffer comprises urea, Tris, DTT, and SDS.

[0092] According to a specific embodiment of the present invention, the concentration of urea in the sample buffer is 4-8M.

[0093] According to a specific embodiment of the present invention, the concentration of Tris in the sample buffer is 50-200 mM.

[0094] According to a specific embodiment of the present invention, the concentration of DTT in the sample buffer is 0.05-0.3 wt %.

[0095] According to a specific embodiment of the present invention, the concentration of SDS in the sample buffer is 0.1-1.5 wt %.

[0096] According to a specific embodiment of the present invention, the pH of the sample buffer is 7.0-9.2.

[0097] It should be noted that the pH of the sample buffer provided by the present invention can be adjusted to 7.0-9.2 using HCl.

[0098] According to a specific embodiment of the present invention, the sample buffer comprises:

[0099] 4-8 M urea, 50-200 mM Tris, 0.05-0.3 wt % DTT, 0.1-0.5 wt % SDS, pH adjusted to 7.0-9.2 with HCl.

[0100] According to a specific embodiment of the present invention, step S5 further comprises:

[0101] Using the peak area normalization method, the components were quantitatively analyzed by the following method:

[0102] The content X of a certain protein component in the soybean protein sample to be tested is calculated by the following formula:

[0103]

[0104] In the formula, A m is the area of ​​peak m corresponding to a certain protein component;

[0105] A T is the sum of the total areas of n peaks;

[0106] n is the total number of integrated peaks in the electrophoresis spectrum of the soybean protein sample to be tested.

[0107] According to a specific embodiment of the present invention, the quantitative detection method of the soybean protein component content provided by the present invention comprises:

[0108] ①Use pretreatment methods to completely dissociate the components of soybean protein, and then separate the components by capillary electrophoresis according to their molecular weight;

[0109] ② Use standard molecular weight substances to make a standard curve and calibrate the molecular weight of each substance in the protein separation spectrum;

[0110] ③ Combined with the molecular weight of each band in SDS-PAGE, the names of substances corresponding to each peak in capillary electrophoresis are verified;

[0111] ④ Normalize and integrate each peak in the capillary electrophoresis spectrum to determine the content of each component.

[0112] The quantitative detection method of the soybean protein component content provided by the present invention does not use dangerous toxic and harmful chemical reagents, is safe and environmentally friendly, and each protein component can be efficiently separated, and the content of each protein component can be quantitatively calculated. At the same time, the method provided by the present invention has a short analysis time (the sample analysis time of SDS-PAGE usually takes 1 day, and the steps are cumbersome. The method of the present invention usually only takes about 3 hours), the scheme is reliable, the reproducibility is good, and the analysis efficiency is high.

[0113] According to a specific embodiment of the present invention, in the quantitative detection method of soybean protein component content provided by the present invention, the sample pretreatment includes:

[0114] Accurately weigh 5.0g of powder (converted after nitrogen determination of liquid sample), add 200ml of water and shake well, stir in a water bath at 25-30℃ for 2 hours, take out the mixed liquid, transfer it to a 250ml volumetric flask, and make up to 250ml with water. Take 10ml of the mixed liquid in a centrifuge tube, centrifuge (3000r / min, 10min), and keep the supernatant.

[0115] Take the supernatant and mix it with the sample buffer to make the final protein concentration between 1-2 mg / ml. Filter it with a 0.45 μm water system membrane and keep it for later use.

[0116] According to a specific embodiment of the present invention, in the quantitative detection method of soybean protein component content provided by the present invention, the sample pretreatment reagent is prepared as follows:

[0117] 50-200 Mm Tris, 6 M urea, 0.1% DTT, 0.1-1.5% SDS, adjust pH to 7.0-9.2 with HCl solution, and make up to volume. Filter the prepared solution with a 0.45 μm water filter membrane, sonicate, and set aside.

[0118] According to a specific embodiment of the present invention, in the quantitative detection method of soybean protein component content provided by the present invention, the electrophoresis buffer is prepared as follows:

[0119] 50-200 Mm Tris, 6 M urea, 0.1% DTT, 0.1-1.5% SDS, 0.05-5% methylcellulose, adjust pH to 7.0-9.2 with HCl solution, and make up to volume. Filter the prepared solution with a 0.45 μm water filter membrane, sonicate, and set aside.

[0120] According to a specific embodiment of the present invention, in the quantitative detection method of soybean protein component content provided by the present invention, an uncoated capillary is used, preferably with a length of 30-80 cm, an inner diameter of 25-70 μm, a separation voltage of 10-30 kV, and a separation time of 40-180 min.

[0121] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0122] Example 1

[0123] Sample buffer preparation:

[0124] 150mM Tris, 6M urea, 0.1% DTT, 0.5% SDS, adjust pH to 9.0 with HCl solution, and make up to volume. Filter the prepared solution with a 0.45μm water filter membrane, sonicate, and set aside.

[0125] Preparation of electrophoresis buffer:

[0126] 150mM Tris, 6M urea, 0.1% DTT, 0.2% SDS, 2% methylcellulose, adjust pH to 9.2 with HCl solution, and make up to volume. Filter the prepared solution with a 0.45μm water filter membrane, sonicate, and set aside.

[0127] (1) Accurately weigh 5.0g of soy protein isolate, add 200ml of water and shake well, stir in a water bath at 25-30℃ for 2 hours, take out the mixed liquid, transfer it to a 250ml volumetric flask, and make up to 250ml with water. Take 10ml of the mixed liquid in a centrifuge tube, centrifuge (3000r / min, 10min), and keep the supernatant;

[0128] (2) Take 1 ml of the supernatant from the previous step, add 4 ml of sample buffer, shake well, and boil in a boiling water bath for 10 min. Take 200 μl for capillary electrophoresis test;

[0129] (3) Use an uncoated capillary with a length of 60 cm and an inner diameter of 50 μm; the separation voltage is 20 kV and the separation time is 120 min.

[0130] Molecular weight standards were tested under the same conditions.

[0131] The results are as follows Figure 2 As shown, it is shown that the separation degree of each component of soybean protein is higher by using the method of the present invention.

[0132] Example 2

[0133] Sample buffer preparation:

[0134] 100 mM Tris, 4 M urea, 0.1% DTT, 1% SDS, adjust pH to 8.0 with HCl solution, and make up to volume. Filter the prepared solution with a 0.45 μm water filter membrane, sonicate, and set aside.

[0135] Preparation of electrophoresis buffer:

[0136] 100 mM Tris, 4 M urea, 0.1% DTT, 0.5% SDS, 2% methylcellulose, adjust pH to 9.0 with HCl solution, and make up to volume. Filter the prepared solution with a 0.45 μm water filter membrane, sonicate, and set aside.

[0137] (1) Accurately weigh 5.0g of soy protein isolate, add 200ml of water and shake well, stir in a water bath at 25-30℃ for 2 hours, take out the mixed liquid, transfer it to a 250ml volumetric flask, and make up to 250ml with water. Take 10ml of the mixed liquid in a centrifuge tube, centrifuge (3000r / min, 10min), and keep the supernatant;

[0138] (2) Take 1 ml of the supernatant from the previous step, add 4 ml of sample buffer, shake well, and boil in a boiling water bath for 10 min. Take 200 μl for capillary electrophoresis test;

[0139] (3) Use an uncoated capillary with a length of 60 cm and an inner diameter of 50 μm; the separation voltage is 15 kV and the separation time is 120 min.

[0140] Molecular weight standards were tested under the same conditions.

[0141] The results are as follows Figure 3 As shown, it is shown that the separation degree of each component of soybean protein is higher by using the method of the present invention.

[0142] Example 3

[0143] Sample buffer preparation:

[0144] 50mM Tris, 8M urea, 0.1% DTT, 1.5% SDS, adjust pH to 7.0 with HCl solution, and make up to volume. Filter the prepared solution with a 0.45μm water filter membrane, sonicate, and set aside.

[0145] Preparation of electrophoresis buffer:

[0146] 150mM Tris, 8M urea, 0.1% DTT, 0.5% SDS, 5% methylcellulose, adjust pH to 7.0 with HCl solution, and make up to volume. Filter the prepared solution with a 0.45μm water filter membrane, sonicate, and set aside.

[0147] (1) Accurately weigh 5.0g of soy protein isolate, add 200ml of water and shake well, stir in a water bath at 25-30℃ for 2 hours, take out the mixed liquid, transfer it to a 250ml volumetric flask, and make up to 250ml with water. Take 10ml of the mixed liquid in a centrifuge tube, centrifuge (3000r / min, 10min), and keep the supernatant;

[0148] (2) Take 1 ml of the supernatant from the previous step, add 4 ml of sample buffer, shake well, and boil in a boiling water bath for 10 min. Take 200 μl for capillary electrophoresis test;

[0149] (3) Use an uncoated capillary with a length of 60 cm and an inner diameter of 50 μm; the separation voltage is 20 kV and the separation time is 120 min.

[0150] Molecular weight standards were tested under the same conditions.

[0151] The results are as follows Figure 4 As shown, it is shown that the separation degree of each component of soybean protein is higher by using the method of the present invention.

[0152] Example 4

[0153] Sample buffer preparation:

[0154] 50mM Tris, 4M urea, 0.1% DTT, 1.5% SDS, adjust pH to 7.5 with HCl solution, and make up to volume. Filter the prepared solution with a 0.45μm water filter membrane, sonicate, and set aside.

[0155] Preparation of electrophoresis buffer:

[0156] 150mM Tris, 6M urea, 0.1% DTT, 0.5% SDS, 2.5% methylcellulose, adjust pH to 8.5 with HCl solution, and make up to volume. Filter the prepared solution with a 0.45μm water filter membrane, sonicate, and set aside.

[0157] (1) Accurately weigh 5.0g of soy protein isolate, add 200ml of water and shake well, stir in a water bath at 25-30℃ for 2 hours, take out the mixed liquid, transfer it to a 250ml volumetric flask, and make up to 250ml with water. Take 10ml of the mixed liquid in a centrifuge tube, centrifuge (3000r / min, 10min), and keep the supernatant;

[0158] (2) Take 1 ml of the supernatant from the previous step, add 4 ml of sample buffer, shake well, and boil in a boiling water bath for 10 min. Take 200 μl for capillary electrophoresis test;

[0159] (3) Use an uncoated capillary with a length of 60 cm and an inner diameter of 50 μm; the separation voltage is 30 kV and the separation time is 60 min.

[0160] Molecular weight standards were tested under the same conditions.

[0161] The results are as follows Figure 5 As shown, it is shown that the separation degree of each component of soybean protein is higher by using the method of the present invention.

[0162] Example 5

[0163] Sample buffer preparation:

[0164] 50mM Tris, 4M urea, 0.1% DTT, 1.5% SDS, adjust pH to 7.5 with HCl solution, and make up to volume. Filter the prepared solution with a 0.45μm water filter membrane, sonicate, and set aside.

[0165] Preparation of electrophoresis buffer:

[0166] 150mM Tris, 6M urea, 0.1% DTT, 0.5% SDS, 2.5% methylcellulose, adjust pH to 8.5 with HCl solution, and make up to volume. Filter the prepared solution with a 0.45μm water filter membrane, sonicate, and set aside.

[0167] (1) Accurately weigh 5.0g of soy protein isolate, add 200ml of water and shake well, stir in a water bath at 25-30℃ for 2 hours, take out the mixed liquid, transfer it to a 250ml volumetric flask, and make up to 250ml with water. Take 10ml of the mixed liquid in a centrifuge tube, centrifuge (3000r / min, 10min), and keep the supernatant;

[0168] (2) Take 1 ml of the supernatant from the previous step, add 4 ml of sample buffer, shake well, and boil in a boiling water bath for 10 min. Take 200 μl for capillary electrophoresis test;

[0169] (3) An uncoated capillary with a length of 60 cm and an inner diameter of 50 μm was used; the separation voltage was 30 kV and the separation time was 180 min.

[0170] Molecular weight standards were tested under the same conditions.

[0171] The results are as follows Figure 6 As shown, it is shown that the separation degree of each component of soybean protein is higher by using the method of the present invention.

[0172] Based on the result graphs obtained by the methods in Examples 1-5, the peak area normalization method can be used to perform quantitative analysis on each protein component to be measured.

[0173] The content of a certain protein component X is calculated by the following formula:

[0174]

[0175] In the formula, A m is the area of ​​peak m corresponding to a certain protein component;

[0176] A T is the sum of the total areas of n peaks;

[0177] n is the total number of integrated peaks in the electrophoresis spectrum of the soybean protein sample to be tested.

[0178] Comparative Example 1

[0179] Sample buffer preparation:

[0180] 40mmol trisodium citrate, 6M urea, 0.1% DTT, adjust pH to 8.0, and make up to volume. Filter the prepared solution with a 0.45μm water filter membrane, sonicate, and set aside.

[0181] Preparation of electrophoresis buffer:

[0182] 32mmol citric acid, 20mmol trisodium citrate, 0.05% hydroxypropyl methylcellulose, 6M urea, 0.1% DTT, adjust pH to 3.0, and make up to volume. Filter the prepared solution with a 0.45μm water filter membrane, sonicate, and set aside.

[0183] (1) Accurately weigh 5.0g of soy protein isolate, add 200ml of water and shake well, stir in a water bath at 25-30℃ for 2 hours, take out the mixed liquid, transfer it to a 250ml volumetric flask, and make up to 250ml with water. Take 10ml of the mixed liquid in a centrifuge tube, centrifuge (3000r / min, 10min), and keep the supernatant;

[0184] (2) Take 1 ml of the supernatant from the previous step, add 4 ml of sample buffer, shake well, and boil in a boiling water bath for 10 min. Take 200 μl for capillary electrophoresis test;

[0185] (3) Use an uncoated capillary with a length of 60 cm and an inner diameter of 50 μm; the separation voltage is 20 kV and the separation time is 120 min.

[0186] The results are as follows Figure 7 As shown, it is shown that the separation degree of each component of soybean protein is not high when using methods other than the present invention.

[0187] Comparative Example 2

[0188] Sample buffer preparation:

[0189] 10mmol sodium dihydrogen phosphate, 6M urea, 0.1% DTT, adjust pH to 8.0, and make up to volume. Filter the prepared solution with a 0.45μm water filter membrane, sonicate, and set aside.

[0190] Preparation of electrophoresis buffer:

[0191] 20mmol sodium dihydrogen phosphate, 20mmol disodium hydrogen phosphate, 0.05% hydroxypropyl methylcellulose, 6M urea, 0.1% DTT, adjust pH to 8.0, and make up to volume. Filter the prepared solution with a 0.45μm water filter membrane, sonicate, and set aside.

[0192] (1) Accurately weigh 5.0g of soy protein isolate, add 200ml of water and shake well, stir in a water bath at 25-30℃ for 2 hours, take out the mixed liquid, transfer it to a 250ml volumetric flask, add water to make up to 250ml. Take 10ml of the mixed liquid in a centrifuge tube, centrifuge (3000r / min, 10min), and keep the supernatant;

[0193] (2) Take 1 ml of the supernatant from the previous step, add 4 ml of sample buffer, shake well, and boil in a boiling water bath for 10 min. Take 200 μl for capillary electrophoresis test;

[0194] (3) Use an uncoated capillary tube with a length of 60 cm and an inner diameter of 50 μm; separation voltage 20 kV, separation time 120 min. The results are as follows Figure 8 As shown, it is shown that the separation degree of each component of soybean protein is not high when using methods other than the present invention.

[0195] Comparative Example 3

[0196] The protein distribution of soybean protein standard and soybean protein isolate was detected by conventional SDS-PAGE method. Fig. 9 As shown, it is difficult to clearly separate the various soybean protein components using the SDS-PAGE method, and some bands cannot be distinguished, resulting in difficulty in quantifying some protein components and inaccurate test results.

[0197] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0198] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for quantitatively detecting the content of soybean protein components, characterized in that: include: S1: Pre-treating the soybean protein sample to be tested so as to dissociate the protein components and obtain a dissociation solution; S2: performing capillary electrophoresis separation on the dissociation solution to obtain a capillary electrophoresis pattern of the soybean protein sample to be tested; S3: Obtain capillary electrophoresis profile of standard products based on proteins of known molecular weight; S4: Based on the capillary electrophoresis spectrum of the standard, determining the molecular weight of each protein to be tested and the peak position of each component in the capillary electrophoresis spectrum of the soybean protein sample to be tested; S5: Based on the molecular weight of each protein to be tested and the peak position of each component, the content of each component is determined by normalization and integration. The electrophoresis buffer used in capillary electrophoresis separation includes urea, Tris, SDS and methyl cellulose.

2. The quantitative detection method according to claim 1, characterized in that: The electrophoresis buffer DTT and / or mercaptoethanol; Optionally, the concentration of urea in the electrophoresis buffer is 4-8 M; Optionally, the concentration of Tris in the electrophoresis buffer is 50-200 mM; Optionally, the concentration of DTT in the electrophoresis buffer is 0.05-0.3 wt %; Optionally, the concentration of SDS in the electrophoresis buffer is 0.1-1.5wt%; Optionally, the concentration of methylcellulose in the electrophoresis buffer is 0.05-5wt%; Optionally, the pH of the electrophoresis buffer is 7.0-9.

2.

3. The quantitative detection method according to claim 1, characterized in that: Uncoated capillaries were used for capillary electrophoresis separation; Optionally, the length of the uncoated capillary is 30-80 cm; Optionally, the inner diameter of the uncoated capillary is 25-70 μm.

4. The quantitative detection method according to claim 1, characterized in that: The separation voltage used in capillary electrophoresis separation is 10-30 kV and the separation time is 40-180 min.

5. The quantitative detection method according to claim 1, characterized in that: In step S1, the soybean protein sample to be tested is pre-treated with a sample buffer. Wherein, the sample buffer comprises urea, Tris, DTT, and SDS; Optionally, the concentration of urea in the sample buffer is 4-8 M; Optionally, the concentration of Tris in the sample buffer is 50-200 mM; Optionally, the concentration of DTT in the sample buffer is 0.05–0.3 wt %; Optionally, the concentration of SDS in the sample buffer is 0.1-1.5wt%; Optionally, the pH of the sample buffer is 7.0-9.

2.

6. The quantitative detection method according to claim 1, characterized in that: The protein of known molecular weight is a or b below: a. Protein with the same molecular weight as the protein component to be measured contained in soybean protein; b. Standard samples of the protein components to be determined in soy protein.

7. The quantitative detection method according to claim 1, characterized in that: Step S5 further comprises: The peak area normalization method was used to quantitatively analyze each component.

8. The quantitative detection method according to claim 7, characterized in that: The components were quantitatively analyzed by the following methods: The content X of a certain protein component in the soybean protein sample to be tested is calculated by the following formula: In the formula, A m is the area of ​​peak m corresponding to a certain protein component; A T is the sum of the total areas of n peaks; n is the total number of integrated peaks in the electrophoresis spectrum of the soybean protein sample to be tested.

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