Method for measuring protein content of corn kernels

Abstract

The invention belongs to the technical field of protein content determination, and particularly relates to a method for determining the protein content of corn kernels. The method comprises the following steps: extraction of corn kernel proteins: extracting different types of corn kernel proteins respectively; determining the protein content of the corn kernels: drawing a standard curve by using a bovine serum standard protein solution; calculating to obtain different types of corn kernel proteins; the sum of the contents of the proteins of the different types of corn kernels is the total protein content of the corn kernel sample. According to the method, the protein components in the corn kernel sample are extracted by adopting a sequential extraction method, each protein component can be completely extracted, the use amount of the sample is small, the operation process is simple, the cost is relatively low, and a basis is provided for accurately measuring the content of the protein components in the corn kernels. According to the method, toxic reagents such as concentrated sulfuric acid, lauryl sodium sulfate, phenolic substances and mercaptoethanol are not used, so that the health of operators is not damaged, and the safety in the experiment process is ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of protein content determination, and particularly relates to a method for determining the protein content of corn kernels. Background Art

[0002] Corn is one of the three major crops in the world. It is not only an important food and feed crop, but also an important industrial raw material. Protein is one of the most important nutrients in corn kernels, and its extract plays an important role in anti-oxidation, lowering blood pressure, lowering blood sugar, and anti-tumor.

[0003] According to different solubility, the protein in seeds can be divided into four categories: albumin, globulin, alcohol-soluble protein and gluten. Different protein components have different food functional properties. Albumin and globulin have good solubility, water retention, emulsification and foaming properties, and can be used as food additives to improve the nutritional value and quality characteristics of foods such as bread, hamburgers, and ice cream. Alcohol-soluble protein has good film-forming, gelling, emulsification and waterproofing properties, and can be used as a raw material for the production of edible packaging films. It has a certain effect on alleviating the decline in moisture and quality of fresh vegetables and fruits during storage. Gluten hydrolyzate has good antioxidant capacity and can be used as an antioxidant to effectively inhibit the spoilage of sausages and increase their storage time. In summary, corn kernel protein and component content have good application prospects in food processing and medical industries.

[0004] Currently, methods for determining protein include the Kjeldahl method, the biuret method and the bispyribacic acid method, but the above methods can only detect the total protein content in the sample, and there is no systematic detection for the determination of protein components. Summary of the invention

[0005] In order to solve the problem that the prior art can only detect the total protein content in a sample, but there is no systematic detection for the determination of protein components, the present invention provides a method for determining the protein content of corn kernels. The method can quickly and accurately determine the protein content of a variety of different types of corn kernels, and has simple operation, less time and low cost.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for determining the protein content of corn kernels, comprising the following steps:

[0008] Extraction of corn kernel protein: extract corn kernel protein from corn kernel samples using a sequential extraction method to obtain different types of corn kernel protein; the different types of corn kernel protein include any two or more of albumin extract, globulin extract, alcohol-soluble protein extract and gluten extract.

[0009] Determination of corn kernel protein content: Dilute different types of corn kernel protein by 1.5 to 2.5 times, add Coomassie Brilliant Blue solution to each of them, mix well, and measure the absorbance at a wavelength of 595 nanometers.

[0010] The standard curve was drawn using bovine serum standard protein solution.

[0011] The measured absorbance value is substituted into the standard curve for calculation to obtain the protein content of different types of corn kernels; the sum of the protein content of different types of corn kernels is the total protein content of the corn kernel sample.

[0012] The method for determining the protein content of corn kernels provided by the present invention can not only detect the total protein content in a sample, but also can determine the protein content of various types of corn kernels in a sample, and has a systematic detection of the determination of protein components.

[0013] Preferably, the sequential extraction method comprises the following steps:

[0014] A portion of the corn kernel sample is first mixed with distilled water, extracted and centrifuged, and the supernatant is collected to obtain the albumin extract; the lower layer of precipitation is further mixed with the first extract, extracted and centrifuged again to obtain the globulin extract.

[0015] Another part of corn kernel samples is first mixed with the second extract, extracted and centrifuged, and the supernatant is collected to obtain an alcohol-soluble protein extract; the lower precipitate is further mixed with the third extract, extracted and centrifuged again to obtain a gluten extract.

[0016] Among them, the first extracting solution is a 10% by mass sodium chloride solution, and the solvent is water; the second extracting solution is a 50% to 60% by mass n-propanol solution, and the solvent is water; the third extracting solution is a mixed solution containing 0.3% to 0.5% by mass potassium sodium tartrate, 0.20% to 0.25% by mass copper sulfate, 1.50% to 1.70% by mass potassium hydroxide and 45% to 55% by mass n-propanol, and the solvent is water.

[0017] Preferably, the conditions for the extraction of the distilled water and the first extracting solution are: extraction is carried out at room temperature, and shaking is required for 15 minutes to 20 minutes during the extraction process.

[0018] Preferably, the centrifugal conditions are: centrifugation at 10,000 to 12,000 rpm at room temperature for 10 to 15 minutes.

[0019] Preferably, the second extracting solution and the third extracting solution are both extracted under the following conditions: extraction is performed under heating conditions, and shaking is required for 8 minutes to 16 minutes during the extraction process.

[0020] The heating temperature is 45°C to 60°C.

[0021] Preferably, the second extracting solution is extracted at 50°C to 60°C for 10 to 15 minutes; and the third extracting solution is extracted at 50°C to 55°C for 10 to 15 minutes.

[0022] Preferably, the standard curve is a standard regression equation obtained by plotting the absorbance values ​​of bovine serum albumin solutions of four different concentrations measured at 595 nanometers.

[0023] The concentrations of the four different concentrations of bovine serum albumin solutions are 0.1 mg / mL, 0.1 mg / mL, 0.9 mg / mL and 0.5 mg / mL respectively.

[0024] Preferably, the corn kernel sample is corn kernel powder obtained by drying and removing the bran from corn kernels, crushing the kernels, and passing through an 80-mesh sieve.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a method for determining the protein content of corn kernels. The method for determining the protein content of corn kernels provided by the present invention can not only detect the total protein content in a sample, but also determine the protein content of various types of corn kernels in a sample, and has a systematic detection of the determination of protein components.

[0027] The method for determining the protein content of corn kernels provided by the present invention has the following advantages:

[0028] (1) The present invention requires less sample volume, has a simple pretreatment process, and is easy to operate.

[0029] (2) The present invention does not use strong acids, such as concentrated sulfuric acid, phenolic substances, mercaptoethanol and other toxic reagents, in the process of treating the samples to be tested, which does not harm the health of the operators and ensures the safety of the experimental process.

[0030] (3) The present invention adopts a sequential extraction method to extract the protein components in the sample, and can measure multiple protein-related indicators at one time. The processing cycle is short, and corn samples can be measured in batches.

[0031] (4) The entire extraction and determination process of the present invention only takes about 2 hours, which greatly improves the current defect of long determination cycle of corn kernel protein.

[0032] (5) The extraction and determination reagents used in the present invention are relatively low in price, thus saving costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A standard curve of albumin content was drawn for different concentrations of bovine serum albumin in the present invention.

[0034] Figure 2 A standard curve of globulin content was drawn for different concentrations of bovine serum albumin in the present invention.

[0035] Figure 3 A standard curve of the content of prolamin was drawn for different concentrations of bovine serum albumin in the present invention.

[0036] Figure 4 A standard curve of gluten content was drawn for different concentrations of bovine serum albumin in the present invention.

[0037] Figure 5 The protein components and total protein content of the two corn varieties in the present invention. DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0039] Experimental materials and instruments

[0040] 1. Reagents

[0041] Commercially available chemical reagents were selected: sodium chloride, n-propanol, sodium potassium tartrate, copper sulfate, potassium hydroxide, bovine serum albumin and distilled water.

[0042] The reagent grade of the above chemical reagents is AR.

[0043] 2. Materials

[0044] Common corn varieties include CIMBL30 and CmL468, which were provided by the State Key Laboratory of Crop Genetic Improvement of Huazhong Agricultural University.

[0045] The corn kernels were crushed by a YB-4500A high-speed crusher and passed through an 80-mesh sieve to obtain corn kernel powder for the extraction of corn kernel protein.

[0046] 3. Instruments

[0047] BS-1E full-temperature shaking incubator: Jintan Jingda Instrument Manufacturing Co., Ltd.; 5424R high-speed refrigerated centrifuge: Aibende; FA224 electronic balance: Shanghai Sunny Hengping Scientific Instrument Co., Ltd.; UV-1200 UV-visible spectrophotometer: Shanghai Aoyi Instrument Co., Ltd.; DK-S24 electric constant temperature water bath: Shanghai Jinghong Experimental Equipment Co., Ltd.; KQ-500DE CNC ultrasonic cleaner: Kunshan Ultrasonic Instrument Co., Ltd.; YB-4500A high-speed crusher: Yongkang Sufeng Industry and Trade Co., Ltd.

[0048] Example 1

[0049] A method for determining the protein content of corn kernels comprises the following steps:

[0050] (1) Extraction of corn kernel protein

[0051] The corn kernel protein of the variety CIMBL30 was extracted by sequential extraction method. Among them, corn kernel protein includes albumin, globulin, alcohol-soluble protein and glutenin. The specific extraction method is as follows:

[0052] Separation of albumin: Weigh 0.05 g of corn kernel powder and place it in a 2 ml centrifuge tube, add 2 ml of distilled water and shake for 10 minutes, then centrifuge at 12000 rpm at room temperature for 10 minutes, transfer the supernatant to a 10 ml glass test tube, repeatedly add 2 ml of distilled water and shake for 10 minutes, then centrifuge for 10 minutes as above, and transfer the supernatant to a 10 ml glass test tube, combine the two supernatants to obtain the albumin extract.

[0053] The corn kernel powder is obtained by crushing corn kernels of the CIMBL30 variety with a YB-4500A high-speed crusher and passing through an 80-mesh sieve.

[0054] Separation of globulin: Add 2 ml of the first extract to the residue in the centrifuge tube after collecting the supernatant, shake and mix for 10 minutes, then centrifuge at 12000 rpm at room temperature for 10 minutes, transfer the supernatant to a 10 ml glass test tube, repeatedly add 2 ml of the first extract and shake and mix for 10 minutes. After centrifugation as above, transfer the supernatant to a 10 ml glass test tube, and combine the two supernatants to obtain the globulin extract.

[0055] The first extract is a sodium chloride solution containing 10% by mass, and the solvent is water. The specific preparation method is: add 10 grams of sodium chloride solid powder into 100 milliliters of distilled water and fully dissolve it.

[0056] Separation of alcohol-soluble protein: Weigh 0.05 g of corn kernel powder again and place it in a 2 ml centrifuge tube, add 2 ml of the second extract, heat the mixture in a 55°C water bath for 10 minutes, shake it every 2 minutes, take it out and oscillate and mix it for 10 minutes, then centrifuge it at 12,000 rpm at room temperature for 10 minutes, transfer the supernatant to a 10 ml glass test tube, repeat adding 2 ml of the second extract, heat the mixture in a 55°C water bath for 10 minutes, shake it every 2 minutes, take it out and oscillate and mix it for 10 minutes, centrifuge it for 10 minutes as above, and transfer the supernatant to a 10 ml glass test tube, combine the two supernatants to obtain the alcohol-soluble protein extract.

[0057] The second extract is a 55% by mass n-propanol solution, and the solvent is water. The specific preparation method is: dilute 55 ml of n-propanol solution to 100 ml with distilled water and mix well to obtain the extract.

[0058] The corn kernel powder is obtained by crushing corn kernels of the variety CIMBL30 by a YB-4500A high-speed crusher and passing through an 80-mesh sieve.

[0059] Separation of gluten: add 2 ml of the third extract to the residue in the centrifuge tube after collecting the supernatant, heat the mixture in a 50°C water bath for 10 minutes, shake it every 2 minutes, take it out and oscillate for 10 minutes, then centrifuge it as above, transfer the supernatant to a 10 ml glass test tube, repeat the addition of 2 ml of the third extract, heat the mixture in a 50°C water bath for 10 minutes, shake it every 2 minutes, take it out and oscillate for 10 minutes, then centrifuge it as above for 10 minutes, transfer the supernatant to a 10 ml glass test tube, and combine the two supernatants to obtain the gluten extract.

[0060] The third extract is a mixed solution composed of equal volumes of 0.5% by mass of sodium potassium tartrate, 0.24% by mass of copper sulfate, 1.68% by mass of potassium hydroxide and 50% by mass of n-propanol, and the solvent is water.

[0061] Among them, the preparation method of 0.5% potassium sodium tartrate by mass percentage is: add 0.5 grams of potassium sodium tartrate solid powder into 100 milliliters of distilled water and fully dissolve it. The preparation method of 0.24% copper sulfate by mass percentage is: add 0.24 grams of potassium sodium tartrate solid powder into 100 milliliters of distilled water and fully dissolve it. The preparation method of 1.68% potassium hydroxide by mass percentage is: add 1.68 grams of potassium hydroxide solid powder into 100 milliliters of distilled water and fully dissolve it. The preparation method of 50% n-propanol by mass percentage is: dilute 50 milliliters of n-propanol solution to 100 milliliters with distilled water and mix well.

[0062] (2) Determination of protein content in corn kernels

[0063] The albumin extract and the gluten extract were respectively diluted to 10 ml and mixed to obtain albumin dilution and gluten dilution. The globulin extract and the alcohol-soluble protein extract were respectively diluted to 6 ml and mixed to obtain globulin dilution and alcohol-soluble protein dilution.

[0064] 1 ml of the albumin dilution, glutenin dilution, globulin dilution and alcohol-soluble protein dilution were taken respectively, and 5 ml of Coomassie brilliant blue solution was added to each of them, and the absorbance was measured at 595 nm. The results of the measurement are shown in Table 1.

[0065] Table 1 Absorbance values ​​of CIMBL30 corn kernel protein

[0066]

[0067] Note: The numbers in the table represent the mean of 3 replicates.

[0068] (3) Standard curve drawing

[0069] Prepare 0.1 mg / mL, 0.1 mg / mL, 0.9 mg / mL and 0.5 mg / mL bovine serum standard protein solutions, add 5 ml of Coomassie brilliant blue solution, measure the absorbance at 595 nm, draw a standard curve based on the bovine serum standard protein concentration and absorbance, and obtain a regression equation. Take 6 10 ml glass test tubes, numbered af, prepare the bovine serum standard protein solutions and the corresponding corn kernel protein solution to be tested according to the formulas in Tables 2 to 5, and the measured regression equation is as follows: Figure 1-Figure 4 The standard curve was drawn with the protein concentration (mg / mL) as the horizontal axis and the absorbance value as the vertical axis to obtain the regression equation of each protein.

[0070] The test solutions of corn kernel protein to be tested include corn kernel albumin test solution, corn kernel globulin test solution, corn kernel alcohol-soluble protein test solution and corn kernel glutenin test solution.

[0071] The corn grain albumin test solution is prepared by using albumin diluent and diluent; the corn grain globulin test solution is prepared by using globulin diluent and diluent; the corn grain alcohol-soluble protein test solution is prepared by using alcohol-soluble protein diluent and diluent; the corn grain gluten test solution is prepared by using gluten diluent and diluent.

[0072] Table 2 Standard curve drawing of corn kernel albumin content

[0073]

[0074] Note: “Extracting solution” refers to “distilled water”.

[0075] Table 3 Drawing of standard curve of corn kernel globulin content

[0076]

[0077] Note: “Extract solution” refers to “10% by mass sodium chloride solution”.

[0078] Table 4 Standard curve drawing of corn kernel alcohol-soluble protein content

[0079] serial number a b c d e f 0.9mg / mL standard protein 0.1 0.2 0.4 0.6 0.8 1 Extraction solution / mL 0.9 0.8 0.6 0.4 0.2 0

[0080] Note: “Extract solution” refers to “55% by mass n-propanol solution”.

[0081] Table 5 Drawing of standard curve for glutenin content in corn kernels

[0082] serial number a b c d e f 0.5mg / mL standard protein 0.1 0.2 0.4 0.6 0.8 1 Extraction solution / mL 0.9 0.8 0.6 0.4 0.2 0

[0083] Note: “Extract solution” refers to “a mixed solution consisting of equal volumes of 0.5% sodium potassium tartrate, 0.24% copper sulfate, 1.68% potassium hydroxide and 50% n-propanol”.

[0084] The standard curve equation of albumin is: Y=7.8293X+0.0147, wherein Y represents the absorbance value and X represents the albumin concentration in mg / mL.

[0085] The standard curve equation of globulin is: Y = 7.7216X-0.0279, where Y represents the absorbance value and X represents the globulin concentration in mg / mL.

[0086] The standard curve equation of prolamin is: Y=1.4982X+0.0758, where Y represents the absorbance value and X represents the concentration of prolamin in mg / mL.

[0087] The standard curve equation of gluten is: Y=2.0591X+0.3711, where Y represents the absorbance value and X represents the gluten concentration in mg / mL.

[0088] (4) Calculation of corn kernel protein content

[0089] According to the method of step (2), the absorbance values ​​of the test solution of the corn kernel protein to be tested are respectively substituted into the corresponding standard curve to calculate the content of each protein. The specific calculation method is as follows:

[0090] Content of each protein and component (%) = X*V*N / M / 1000*100%

[0091] Total protein content (%) = albumin (%) + globulin (%) + prolamin (%) + glutenin (%)

[0092] Among them, X represents the horizontal axis value in the standard curve, in mg / mL; V represents the total volume of the extract, in mL; N represents the dilution factor; M is the sample mass, in g.

[0093] The results of the measured protein content of CIMBL30 corn kernels are shown in Figure 5 .

[0094] Depend on Figure 5 It can be seen that the albumin, globulin, alcohol-soluble protein, glutenin and total protein contents of CIMBL30 corn kernels are 0.872%, 0.304%, 7.842%, 2.572% and 11.59% respectively.

[0095] Example 2

[0096] A method for determining the protein content of corn kernels comprises the following steps:

[0097] (1) Extraction of corn kernel protein

[0098] The corn kernel protein of the variety CML468 was extracted by sequential extraction method. Among them, corn kernel protein includes albumin, globulin, alcohol-soluble protein and glutenin. The specific extraction method is as follows:

[0099] Separation of albumin: Weigh 0.05 g of corn kernel powder and place it in a 2 ml centrifuge tube, add 2 ml of distilled water and shake for 10 minutes, then centrifuge at 12000 rpm at room temperature for 10 minutes, transfer the supernatant to a 10 ml glass test tube, repeatedly add 2 ml of distilled water and shake for 10 minutes, then centrifuge for 10 minutes as above, and transfer the supernatant to a 10 ml glass test tube, combine the two supernatants to obtain the albumin extract.

[0100] The corn kernel powder is obtained by crushing corn kernels of the variety CML468 by a YB-4500A high-speed crusher and passing through an 80-mesh sieve.

[0101] Separation of globulin: Add 2 ml of the first extract to the residue in the centrifuge tube after collecting the supernatant, shake and mix for 10 minutes, then centrifuge at 12000 rpm at room temperature for 10 minutes, transfer the supernatant to a 10 ml glass test tube, repeatedly add 2 ml of the first extract and shake and mix for 10 minutes. After centrifugation as above, transfer the supernatant to a 10 ml glass test tube, and combine the two supernatants to obtain the globulin extract.

[0102] The first extract is a sodium chloride solution containing 10% by mass, and the solvent is water. The specific preparation method is: add 10 grams of sodium chloride solid powder into 100 milliliters of distilled water and fully dissolve it.

[0103] Separation of alcohol-soluble protein: Weigh 0.05 g of corn kernel powder again and place it in a 2 ml centrifuge tube, add 2 ml of the second extract, heat the mixture in a 55°C water bath for 10 minutes, shake it every 2 minutes, take it out and oscillate and mix it for 10 minutes, then centrifuge it at 12,000 rpm at room temperature for 10 minutes, transfer the supernatant to a 10 ml glass test tube, repeat adding 2 ml of the second extract, heat the mixture in a 55°C water bath for 10 minutes, shake it every 2 minutes, take it out and oscillate and mix it for 10 minutes, centrifuge it for 10 minutes as above, and transfer the supernatant to a 10 ml glass test tube, combine the two supernatants to obtain the alcohol-soluble protein extract.

[0104] The second extract is a 55% by mass n-propanol solution, and the solvent is water. The specific preparation method is: dilute 55 ml of n-propanol solution to 100 ml with distilled water and mix well to obtain the extract.

[0105] The corn kernel powder is obtained by crushing corn kernels of the variety CML468 with a YB-4500A high-speed crusher and passing through an 80-mesh sieve.

[0106] Separation of gluten: add 2 ml of the third extract to the residue in the centrifuge tube after collecting the supernatant, heat the mixture in a 50°C water bath for 10 minutes, shake it every 2 minutes, take it out and oscillate for 10 minutes, then centrifuge it as above, transfer the supernatant to a 10 ml glass test tube, repeat the addition of 2 ml of the third extract, heat the mixture in a 50°C water bath for 10 minutes, shake it every 2 minutes, take it out and oscillate for 10 minutes, then centrifuge it as above for 10 minutes, transfer the supernatant to a 10 ml glass test tube, and combine the two supernatants to obtain the gluten extract.

[0107] The third extract is a mixed solution composed of equal volumes of 0.5% by mass of sodium potassium tartrate, 0.24% by mass of copper sulfate, 1.68% by mass of potassium hydroxide and 50% by mass of n-propanol, and the solvent is water.

[0108] Among them, the preparation method of 0.5% potassium sodium tartrate by mass percentage is: add 0.5 grams of potassium sodium tartrate solid powder into 100 milliliters of distilled water and fully dissolve it. The preparation method of 0.24% copper sulfate by mass percentage is: add 0.24 grams of potassium sodium tartrate solid powder into 100 milliliters of distilled water and fully dissolve it. The preparation method of 1.68% potassium hydroxide by mass percentage is: add 1.68 grams of potassium hydroxide solid powder into 100 milliliters of distilled water and fully dissolve it. The preparation method of 50% n-propanol by mass percentage is: dilute 50 milliliters of n-propanol solution to 100 milliliters with distilled water and mix well. (2) Determination of protein content in corn kernels

[0109] The albumin extract and the gluten extract were respectively diluted to 10 ml and mixed to obtain albumin dilution and gluten dilution. The globulin extract and the alcohol-soluble protein extract were respectively diluted to 6 ml and mixed to obtain globulin dilution and alcohol-soluble protein dilution.

[0110] 1 ml of the albumin dilution, glutenin dilution, globulin dilution and alcohol-soluble protein dilution were taken respectively, and 5 ml of Coomassie brilliant blue solution was added to each of them, and the absorbance was measured at 595 nm. The results of the measurement are shown in Table 6.

[0111] Table 6 Absorbance values ​​of CML 468 corn kernel protein

[0112]

[0113] Note: The numbers in the table represent the mean of 3 replicates.

[0114] (3) Standard curve drawing

[0115] Prepare 0.1 mg / mL, 0.1 mg / mL, 0.9 mg / mL and 0.5 mg / mL bovine serum standard protein solutions, add 5 ml of Coomassie brilliant blue solution, measure the absorbance at 595 nm, draw a standard curve based on the bovine serum standard protein concentration and absorbance, and obtain a regression equation. Take 6 10 ml glass test tubes, numbered af, prepare the bovine serum standard protein solutions and the corresponding corn kernel protein solution to be tested according to the formulas in Tables 2 to 5, and the measured regression equation is as follows: Figure 1-Figure 4The standard curve was drawn with the protein concentration (mg / mL) as the horizontal axis and the absorbance value as the vertical axis to obtain the regression equation of each protein.

[0116] The test solutions of corn kernel protein to be tested include corn kernel albumin test solution, corn kernel globulin test solution, corn kernel alcohol-soluble protein test solution and corn kernel glutenin test solution.

[0117] The corn grain albumin test solution is prepared by using albumin diluent and diluent; the corn grain globulin test solution is prepared by using globulin diluent and diluent; the corn grain alcohol-soluble protein test solution is prepared by using alcohol-soluble protein diluent and diluent; the corn grain gluten test solution is prepared by using gluten diluent and diluent.

[0118] The standard curve equation of albumin is: Y=7.8293X+0.0147, wherein Y represents the absorbance value and X represents the albumin concentration in mg / mL.

[0119] The standard curve equation of globulin is: Y = 7.7216X-0.0279, where Y represents the absorbance value and X represents the globulin concentration in mg / mL.

[0120] The standard curve equation of prolamin is: Y=1.4982X+0.0758, where Y represents the absorbance value and X represents the concentration of prolamin in mg / mL.

[0121] The standard curve equation of gluten is: Y=2.0591X+0.3711, where Y represents the absorbance value and X represents the gluten concentration in mg / mL.

[0122] (4) Calculation of corn kernel protein content

[0123] According to the method of step (2), the absorbance values ​​of the test solution of the corn kernel protein to be tested are respectively substituted into the corresponding standard curve to calculate the content of each protein. The specific calculation method is as follows:

[0124] Content of each protein and component (%) = X*V*N / M / 1000*100%

[0125] Total protein content (%) = albumin (%) + globulin (%) + prolamin (%) + glutenin (%)

[0126] Among them, X represents the horizontal axis value in the standard curve, in mg / mL; V represents the total volume of the extract, in mL; N represents the dilution factor; M is the sample mass, in g.

[0127] The results of the measured protein content of CIMBL30 corn kernels are shown in Figure 5.

[0128] Depend on Figure 5 It can be seen that the albumin, globulin, alcohol-soluble protein, glutenin and total protein contents of CML468 corn kernels are 1.298%, 0.36%, 3.912%, 5.166% and 10.736%, respectively.

[0129] In summary, the present invention provides a method for determining the protein content of corn kernels. This method can simultaneously detect the content of multiple proteins in corn kernels, and takes a short time. The sample and chemical reagents required for the test are small, and the operation is simple. It is suitable for the detection of large quantities of corn kernel samples in laboratories and scientific research institutions, and is convenient and fast. At the same time, this method has repeatedly detected the protein content in the above two corn kernels, and the obtained test data has a small fluctuation, indicating that this method is reliable.

[0130] The method for determining the protein content of corn kernels provided by the present invention can not only detect the total protein content in a sample, but also can determine the protein content of various types of corn kernels in a sample, and has a systematic detection of the determination of protein components.

[0131] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0132] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A method for determining the protein content of corn kernels, characterized in that: The steps include: Extraction of corn kernel protein: extracting corn kernel protein from corn kernel samples by sequential extraction method to obtain different types of corn kernel protein; the different types of corn kernel protein include any two or more of albumin extract, globulin extract, alcohol-soluble protein extract and gluten extract; Determination of corn kernel protein content: dilute different types of corn kernel protein by 1.5 to 2.5 times, add Coomassie Brilliant Blue solution to each, mix well, and measure the absorbance at 595 nm. A standard curve was drawn using bovine serum standard protein solution; The measured absorbance value is substituted into the standard curve for calculation to obtain the protein content of different types of corn kernels; the sum of the protein content of different types of corn kernels is the total protein content of the corn kernel sample.

2. The method for determining the protein content of rice grains according to claim 1, characterized in that: The sequential extraction method includes the following steps: A portion of the corn kernel sample is first mixed with distilled water, extracted, centrifuged, and the supernatant is collected to obtain the albumin extract; the lower precipitate is further mixed with the first extract, extracted, and centrifuged to obtain the globulin extract; Another portion of corn kernel samples is first mixed with the second extract, extracted, centrifuged, and the supernatant is collected to obtain an alcohol-soluble protein extract; The lower precipitate is further mixed with the third extract, and then extracted and centrifuged to obtain a gluten extract; Among them, the first extracting solution is a 10% by mass sodium chloride solution, and the solvent is water; the second extracting solution is a 50% to 60% by mass n-propanol solution, and the solvent is water; the third extracting solution is a mixed solution containing 0.3% to 0.5% by mass potassium sodium tartrate, 0.20% to 0.25% by mass copper sulfate, 1.50% to 1.70% by mass potassium hydroxide and 45% to 55% by mass n-propanol, and the solvent is water.

3. The method for determining the protein content of rice grains according to claim 2, characterized in that: The conditions for the extraction of the distilled water and the first extracting solution are: extraction is carried out at room temperature, and shaking is required for 15 minutes to 20 minutes during the extraction process.

4. The method for determining the protein content of rice grains according to claim 2, characterized in that: The centrifugal conditions are: centrifugation at 10000 rpm to 12000 rpm at room temperature for 10 minutes to 15 minutes.

5. The method for determining the protein content of rice grains according to claim 2, characterized in that: The conditions of the second extracting solution and the third extracting solution during extraction are: extraction under heating conditions, and shaking for 8 minutes to 16 minutes during the extraction process; The heating temperature is 45°C to 60°C.

6. The method for determining the protein content of rice grains according to claim 5, characterized in that: The second extracting solution is extracted at 50° C. to 60° C. for 10 to 15 minutes; the third extracting solution is extracted at 50° C. to 55° C. for 10 to 15 minutes.

7. The method for determining the protein content of rice grains according to claim 1, characterized in that: The standard curve is a standard regression equation obtained by plotting the absorbance values ​​of bovine serum albumin solutions of four different concentrations measured at 595 nanometers; The concentrations of the four different concentrations of bovine serum albumin solutions are 0.1 mg / mL, 0.1 mg / mL, 0.9 mg / mL and 0.5 mg / mL respectively.

8. The method for determining the protein content of rice grains according to claim 1, characterized in that: The corn kernel sample is corn kernel powder obtained by drying corn kernels, removing the bran, crushing the corn kernels, and passing through an 80-mesh sieve.

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