Method for preparing glutenin monomer by using saturated urea dissolution and coacervation method

Abstract

The method comprises the following steps: mixing vital wheat gluten and 65-70% ethanol according to a mass ratio of 1: 20-1: 30, stirring and extracting in a water bath kettle at the temperature of 30-40 DEG C for 2-3 hours at the stirring speed of 1.5-6 m / s; and centrifuging the extracted solution for 5-10 minutes by using a centrifugal machine under the condition of 3500r / min, and taking the lower layer precipitate, namely glutenin. The preparation method comprises the following steps: dissolving glutenin in a saturated urea solution, stirring and dissolving for 10-15 hours at normal temperature, centrifuging for 5-10 minutes under the condition of 3500 r / min to remove precipitate to obtain glutenin-urea compound liquid, and standing at room temperature to separate out urea crystals; and dialyzing the residual protein liquid by using an MD77 dialysis bag to remove small molecular proteins and ions, freeze-drying at-60 DEG C for 2-6 days, crushing, and sieving by using a 100-mesh sieve to obtain powder which is a glutenin monomer, and the preparation rate is 20-50%.

Description

Technical Field

[0001] The invention relates to a method for preparing glutenin monomer by utilizing a saturated urea dissolution and coagulation method. Background Art

[0002] Gluten is one of the main components of gluten flour (gluten protein). Its main function in the processing of flour products is to promote the formation of dough viscosity and enhance the dough extensibility. Gluten can quickly absorb water and swell to form a network structure, which is the main component of gluten skeleton formation.

[0003] High molecular weight glutenin constitutes the skeleton of the gluten network, and low molecular weight glutenin is connected to high molecular weight glutenin to form a chain structure with fine branches. The gluten network is a complex three-dimensional structure. When low molecular weight glutenin is lacking, the gluten network is more like a sheet structure; the role of alcohol-soluble protein in the entire gluten network structure is mainly a clustered protein that fills the gaps. Therefore, the composition and proportion of glutenin directly affect the final functional properties of gluten. The composition and content of glutenin in different wheat flours are different. In most cases, its composition and content are not conducive to the formation of the best functional gluten network, and additional glutenin needs to be added. In addition, in order to better study the formation mechanism of the gluten network, especially the formation mechanism of disulfide bonds therein, it is necessary to prepare a large number of glutenin monomers with a relatively narrow molecular weight distribution range. Accordingly, this patent uses a saturated urea dissolution and coagulation method to prepare glutenin monomers, dissolves urea-soluble glutenin from gluten powder alcohol extraction residue with saturated urea, and then uses a room temperature urea coagulation crystallization method to effectively separate protein and urea to prepare urea-soluble glutenin monomers. This preparation method provides a new technology and method for adding glutenin components to flour products to improve their quality. With the continuous deepening of research, the application field of glutenin monomers will continue to expand, providing green processing technology guarantee for the production of high-quality flour products. Summary of the invention

[0004] The present invention aims to overcome the deficiencies in the prior art and provides a method for preparing glutenin monomers by using a saturated urea dissolution and coagulation method. The preparation method is simple, low in cost and high in safety.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for preparing glutenin monomers by saturated urea dissolution and coagulation method comprises the following steps:

[0007] The gluten powder and 65-70% ethanol are mixed in a mass ratio of 1:20-1:30, and stirred and extracted in a water bath at 30-40°C for 2-3 hours at a stirring speed of 1.5-6 m / s. The extracted solution is centrifuged at 3500 r / min for 5-10 minutes, and the lower layer of precipitation is taken out to be glutenin. Glutenin is added to a saturated urea solution (solid-liquid ratio 1:25) and stirred and dissolved for 12 hours at a stirring speed of 1.5-6 m / s, and the undissolved glutenin is removed by centrifugation to obtain a glutenin-urea solution complex, and the complex is placed at room temperature for 72 hours, and urea is crystallized from the glutenin-urea solution complex, which is in the upper layer of the complex, and the protein is in the lower layer of the complex, and the protein is taken out to obtain non-aggregated glutenin after urea treatment, and the non-aggregated glutenin is dialyzed to remove ionic impurities, and finally the glutenin monomer is obtained.

[0008] The present invention has the following technical effects:

[0009] The method of the present invention does not use an organic solvent, but only uses a saturated urea solution to open the disulfide bonds in the glutenin aggregates, so that the glutenin monomers are released from the aggregates into the solution. The small molecule protein with a molecular weight less than 10000 g / mol released at the same time is removed by dialysis. This type of protein has a narrow molecular weight distribution range (all components are protein molecules with a molecular weight of 38000 g / mol, the number average molecular weight and the weight average molecular weight are basically equal, and the dispersity index is 1.0), and is very easy to crystallize in a solution of a certain concentration, providing conditions for the rapid, environmentally friendly and low-cost preparation of this type of protein.

[0010] The glutenin monomers prepared by the method of the present invention are arranged in a spherical and linear manner under a microscope (see Figure 1 ), the scanning electron micrograph showed that the protein monomer had a regular tetrahedral morphology (see Figure 2 ), the X-ray diffraction pattern showed that the protein had a diffraction shape similar to that of metal salt crystals, with strong diffraction angles of 2θ at 22° (strong), 24° (medium), 29° (medium), 31° (weak), 35° (medium), 37° (weak), 40° (weak), 41° (weak), 45° (weak), 49° (weak) and 54° (weak) (see Figure 3 ).

[0011] Instruction Manual

[0012] Figure 1 is a micrograph of glutenin monomer (×400);

[0013] Figure 2 is a scanning electron micrograph of glutenin monomer (×3000);

[0014] Figure 3 This is the X-ray diffraction pattern of glutenin monomer. DETAILED DESCRIPTION

[0015] The present invention is described in detail below with reference to specific embodiments.

[0016] Example 1

[0017] Mix 200g gluten powder and 4000mL 65% ethanol, stir and extract in a 30℃ water bath for 2h, stirring at a speed of 1.5m / s. After extraction, the solution was centrifuged at 3500r / min for 5min, precipitated as glutenin, dried at 45℃ to constant weight, corresponding to a dry weight of 152.0g. Dissolve glutenin in 3800ml saturated urea solution, stir and dissolve at room temperature 10-30℃ for 12h, stirring at a speed of 1.5-6m / s, centrifuge the solution at 3500r / min for 10min, remove the undissolved impurity precipitate, dry the impurity precipitate at 45℃ to constant weight, corresponding to a dry weight of 28.0g, and obtain a solution of glutenin-urea solution complex, place it at 25℃ for 80h, urea crystallizes, and the lower layer is taken out as non-condensed protein. The non-condensed protein was placed in an MD77 dialysis bag and dialyzed 7 times to remove low molecular weight proteins and ions to obtain glutenin monomers, which were freeze-dried at -60°C for 2 days and then crushed through a 100-mesh sieve to obtain a powder of glutenin monomers. The corresponding dry weight was 96.7 g, and the preparation rate was 48.35%.

[0018] Example 2

[0019] Mix 1000g gluten powder with 30000mL 75% ethanol, stir and extract in a 40℃ water bath for 3h, stirring at a speed of 6m / s. After extraction, the solution was centrifuged at 3500r / min for 10min, precipitated as glutenin, dried at 60℃ to constant weight, corresponding to a dry weight of 753.3g. Dissolve glutenin in 18835ml saturated urea solution, stir and dissolve at room temperature 10-30℃ for 12h, stirring at a speed of 1.5-6m / s, centrifuge the solution at 3500r / min for 10min, remove the undissolved impurity precipitate, dry the impurity precipitate at 45℃ to constant weight, corresponding to a dry weight of 156.4g, and obtain a solution of glutenin-urea solution complex, place it at 4℃ for 80h, urea crystallizes, and the lower layer is taken out as non-condensed protein. The non-condensed protein was placed in an MD77 dialysis bag and dialyzed seven times to remove low molecular weight proteins and ions to obtain glutenin monomers. After freeze-drying at -60°C for 6 days, the monomers were crushed and passed through a 100-mesh sieve. The resulting powder was glutenin monomers with a corresponding dry weight of 287.3 g and a preparation rate of 28.7%.

Claims

1. A method for preparing glutenin monomers using saturated urea dissolution and coagulation method, characterized in that: The steps include: (1) Mix gluten powder and 65-70% ethanol in a mass ratio of 1:20-1:30, stir and extract in a water bath at 30-40° C. for 2-3 hours to dissolve wheat alcohol-soluble protein in the gluten powder in the ethanol; centrifuge the extracted solution at 3500 r / min for 5-10 minutes, and dry the precipitate to obtain glutenin; (2) adding glutenin to a saturated urea solution (solid-liquid ratio of 1:20 to 1:30) and stirring and dissolving for 10 to 15 hours at a stirring speed of 1.5 to 6 m / s, centrifuging at 3500 r / min for 5 to 10 minutes to remove undissolved impurities and precipitate, thereby obtaining a glutenin-urea solution complex; (3) The complex is placed at room temperature for 60 to 80 hours. Urea is crystallized from the glutenin-urea solution complex and is located in the upper layer of the complex. The protein is located in the lower layer of the complex. The protein is taken out to obtain non-aggregated glutenin treated with urea. The small molecular proteins, ions, etc. in the non-aggregated glutenin are removed by dialysis, and the glutenin monomer is obtained by freeze-drying.

2. The method for preparing glutenin monomers by saturated urea dissolution and coagulation method according to claim 1, characterized in that: In step 1 (1), the stirring speed of the alcohol extraction process of wheat alcohol-soluble protein in gluten powder is 1.5-6 m / s, the room temperature dissolution temperature is 10-30°C, and the precipitation drying temperature is 45°C.

3. The method for preparing glutenin monomers by saturated urea dissolution and coagulation method according to claim 1, characterized in that: In step 1(2), saturated urea solution can be used to dissolve glutenin, and the stirring speed is 3 to 6 m / s.

4. The method for preparing glutenin monomers by saturated urea dissolution and coagulation method according to claim 1, characterized in that: In step 1 (3), the glutenin-urea solution complex can be allowed to stand at room temperature to precipitate crystals to precipitate urea. The temperature range of room temperature condensation is 15-30°C.

5. The method for preparing glutenin monomers by saturated urea dissolution and coagulation method according to claim 1, characterized in that: After the urea crystals are taken out from the glutenin-urea solution complex, the remaining solution is dialyzed 7 times with an MD77 dialysis bag to remove small molecular proteins, ions, etc., and then freeze-dried at -60°C for 2-6 days and crushed through a 100-mesh sieve to obtain a powder that is a glutenin monomer.

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