Preparation method of soybean 11S protein

Abstract

The present invention provides a soybean 11S protein preparation method, which comprises: (1) alcohol washing: dissolving a soybean protein raw material in an oligosaccharide-containing ethanol solution, uniformly mixing, and filtering to obtain an alcohol washed protein; (2) performing enzymolysis, namely performing enzymolysis on the alcohol-washed protein in the step (1) by using protease to obtain a protein solution; according to the method disclosed by the invention, 7S protein in soybean protein is denatured while 11S protein is not denatured by utilizing the ethanol solution containing oligosaccharide, so that protease can specifically hydrolyze the 7S protein, and the yield of the 11S protein is improved; meanwhile, in the preparation method disclosed by the invention, the 7S protein can be thoroughly subjected to enzymolysis, and the 11S protein is almost not lost, so that the 11S protein with high purity and high yield is obtained.

Description

Technical Field

[0001] The present invention relates to a method for preparing soy protein, and particularly to a method for preparing soy 11S protein with high purity and high yield. Background Art

[0002] Soy proteins mainly include 7S protein and 11S protein, and the two proteins have their respective advantageous functional properties. In the production process of traditional soy protein products, 7S protein and 11S protein are generally extracted together and used as raw materials, such as products like soy protein isolate and soy protein concentrate. Subsequent research found that obtaining a single protein component by certain means to fully exhibit its unique physical properties or physiological functions will greatly expand the application of soy protein in the food field.

[0003] 11S protein has significantly better heat gel properties than 7S protein, and at the same time its allergenicity is lower than that of 7S protein; therefore, obtaining high-purity 11S protein is of great significance for the food industry or other fields. Currently, the methods for preparing 11S protein mainly include the "isoelectric point method" and the "enzymatic hydrolysis method". The "isoelectric point method" utilizes the isoelectric point difference between 7S and 11S to precipitate 11S protein at a relatively high pH (usually pH 6.4), but this process requires standing overnight at 4 °C and is difficult to industrialize. The "enzymatic hydrolysis method" usually first denatures 7S in soy protein using a temperature of 70 - 75 °C, then uses a protease (usually papain) to hydrolyze it, and then combines acid precipitation to obtain 11S protein. However, after the preheating treatment, the structure of 11S protein will also become loose and is prone to partial hydrolysis during enzymatic hydrolysis, ultimately resulting in a reduced yield. In addition, high-temperature enzyme inactivation treatment will also cause a significant decrease in the solubility of 11S protein. Therefore, developing an easy-to-operate method for preparing soy 11S protein, while ensuring protein purity and improving protein yield and solubility, is of great significance. Summary of the Invention

[0004] In the first aspect of the present invention, there is provided a method for preparing soy 11S protein, the method comprising:

[0005] (1) Alcohol washing: Dissolve the soy protein raw material in an ethanol solution containing oligosaccharides, mix evenly and then filter to obtain alcohol-washed protein;

[0006] (2) Enzymatic hydrolysis: Use protease to perform enzymatic hydrolysis on the alcohol-washed protein in step (1) to obtain a protein solution.

[0007] In one or more embodiments, the soy protein raw material is low-denatured soy protein isolate.

[0008] In one or more embodiments, the concentration of the ethanol solution is 20 - 50% (v / v), preferably 30 - 40% (v / v).

[0009] In one or more embodiments, based on the volume of the ethanol solution, the addition amount of the oligosaccharide is 8 - 35% (w / v), preferably 10 - 30% (w / v).

[0010] In one or more embodiments, the mass ratio of the soybean protein raw material to the ethanol solution is 1:5 - 10 (w / w).

[0011] In one or more embodiments, the oligosaccharide is selected from one or more of sucrose, maltose, lactose, trehalose, maltotriose, isomaltooligosaccharide, xylooligosaccharide, and soybean oligosaccharide.

[0012] In one or more embodiments, based on the dry matter mass of the alcohol-washed protein, the addition amount of the protease is 0.02 - 2%, preferably 0.05 - 0.2%.

[0013] In one or more embodiments, the protease is selected from one or more of neutral protease (such as Novozymes Neutrase 0.8L, Xiasheng FDG-2230, Donghenghuadao 1-11, Pangbo Biology PBD-01-3), papain (such as Xiasheng FDG-2203, Donghenghuadao 2-10, Pangbo Biology PBD-01-1), and bromelain (such as Xiasheng FDG-2201, Donghenghuadao 2-55, Pangbo Biology PBD-01-2).

[0014] In one or more embodiments, the enzymatic hydrolysis temperature is 30 - 60 °C, preferably 40 - 50 °C.

[0015] In one or more embodiments, the enzymatic hydrolysis time is 10 - 100 min, preferably 20 - 60 min.

[0016] In one or more embodiments, the method further comprises one or more of the following steps:

[0017] (3) Acid precipitation and neutralization: Adjust the protein solution in step (2) to acidic, collect the protein precipitate, then redissolve it with water and adjust it to neutral to obtain a 11S protein solution;

[0018] (4) Drying: Dry the 11S protein solution; preferably spray drying.

[0019] In a second aspect of the present invention, there is provided a soybean 11S protein prepared by the method described in any one of the present invention.

[0020] In one or more embodiments, the purity of the soybean 11S protein ≥ 90%; and / or, the solubility of the soybean 11S protein ≥ 90%.

[0021] In a third aspect of the present invention, there is provided a food product, which comprises the soy 11S protein prepared by the method according to any one of the present invention, or is prepared from the soy 11S protein according to the present invention.

[0022] Preferably, the food product is a hot pot product, a dairy product, a baked product, a soy product, a sauce product, a beverage product, a chocolate product or a meat product, and more preferably a meat product.

[0023] In a fourth aspect of the present invention, there is provided a method for preparing a food product, which comprises: obtaining the soy 11S protein prepared by the method according to any one of the present invention, and using the soy 11S protein to prepare the food product.

[0024] In a fifth aspect of the present invention, there is provided the use of an ethanol solution containing oligosaccharides in increasing the yield of soy 11S protein, wherein the soy protein raw material is washed with the ethanol solution containing oligosaccharides, and then enzymatically hydrolyzed with a protease.

[0025] In one or more embodiments, the concentration of the ethanol solution is 20-50% (v / v), preferably 30-40% (v / v); based on the volume of the ethanol solution, the addition amount of the oligosaccharides is 8-35% (w / v), preferably 10-30% (w / v).

[0026] In one or more embodiments, the oligosaccharides are selected from one or more of sucrose, maltose, lactose, trehalose, maltotriose, isomaltooligosaccharide, xylooligosaccharide and soy oligosaccharide.

[0027] Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : The position of the 11S protein band on the electrophoresis pattern. DETAILED DESCRIPTION

[0029] To enable those skilled in the art to understand the features and effects of the present invention, the following provides a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein shall have the ordinary meaning understood by those skilled in the art for the present invention, and in case of conflict, the definition in this specification shall prevail.

[0030] The theories or mechanisms described and disclosed herein, whether correct or incorrect, shall not in any way limit the scope of the present invention, that is, the content of the present invention can be implemented without being limited by any specific theory or mechanism.

[0031] In this text, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are only for the sake of brevity and convenience. Accordingly, the description of a numerical range or percentage range should be regarded as having covered and specifically disclosed all possible sub-ranges and individual numerical values within the range (including integers and fractions). In this text, unless otherwise specified, percentages refer to mass percentages and ratios refer to mass ratios.

[0032] In this text, for the sake of concise description, all possible combinations of all technical features in each embodiment or example are not described. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each embodiment or example can be combined arbitrarily, and all possible combinations should be considered as the scope described in this specification.

[0033] In this text, unless otherwise specified, the raw materials mentioned can adopt the conventional raw materials in the art, and the processes mentioned can adopt the conventional operation methods in the art, and those skilled in the art can reasonably determine them according to the content disclosed in this text and the prior art.

[0034] The present invention discovers that in the preparation process of soybean 11S protein, first, the protein raw material is washed with an ethanol solution containing oligosaccharides, and then enzymatically hydrolyzed with a protease, so as to obtain soybean 11S protein with high purity and high solubility, and at the same time, the yield of soybean 11S protein can be improved. Thus, the present invention is completed.

[0035] The present invention provides a method for preparing soybean 11S protein, comprising the following steps:

[0036] (1) Alcohol washing: Dissolve the soybean protein raw material in an ethanol solution containing oligosaccharides, mix evenly and then filter to obtain alcohol-washed protein;

[0037] (2) Enzymatic hydrolysis: Enzymatically hydrolyze the alcohol-washed protein in step (1) with a protease to obtain a protein solution.

[0038] In the present invention, the soybean 11S protein is a well-known soybean protein component in the technical field, referring to soy globulin composed of acidic subunits (A subunits) and basic subunits (B subunits), with a molecular mass of about 300 - 380 kDa.

[0039] In the present invention, the soy protein raw material refers to low-denatured soy protein isolate, which can be commercially available or prepared by oneself through alkali dissolution and acid precipitation. Specifically, low-temperature defatted soybean meal is mixed with water at a mass ratio of 1:(8 - 12), the alkalinity is adjusted with sodium hydroxide, stirred and extracted at room temperature, and the supernatant is collected by filtration; the supernatant is adjusted to be acidic with hydrochloric acid, and the precipitate is collected; preferably, the precipitate can be redissolved in water, adjusted to be neutral with sodium hydroxide, and then spray-dried (controlling the outlet temperature at 60 - 65 °C) or freeze-dried to obtain low-denatured soy protein isolate powder. The low denaturation means that the soy protein isolate has not been treated under physical or chemical conditions such as heat, pressure, radiation, heavy metal salts, strong acids or bases, and the protein molecules basically retain the original tertiary structure.

[0040] In the present invention, the ethanol solution is used to alcohol-wash the soy protein raw material, and the denaturation degree of 7S and 11S proteins in soybeans can be controlled by the concentration of the ethanol solution. The ethanol concentration used in the present invention is 20 - 50% (v / v), preferably 30 - 40% (v / v). Too high ethanol concentration will cause no denaturation of both 7S and 11S proteins, and too low ethanol concentration will cause a significant decrease in the denaturation degree of 7S. The present invention reasonably controls the denaturation degree of 7S and 11S proteins by controlling the ethanol solution.

[0041] In the present invention, adding an appropriate amount of oligosaccharide to the ethanol solution can protect 11S protein from denaturation during alcohol-washing of soy protein. Based on the volume of the ethanol solution, the addition amount of the oligosaccharide is 8 - 35% (w / v), preferably 10 - 30% (w / v). Too much addition amount of oligosaccharide will cause a decrease in the denaturation degree of 7S and the sample is prone to absorb moisture and agglomerate, and too little addition amount of oligosaccharide will cause partial denaturation of 11S protein, which will be enzymatically hydrolyzed in the subsequent enzymatic hydrolysis treatment, reducing the yield of 11S protein.

[0042] In the present invention, the oligosaccharide refers to a compound polymerized by 2 - 10 glycosidic bonds. The most common oligosaccharides are disaccharides, trisaccharides and tetrasaccharides, which can be extracted from plants or commercially available. The oligosaccharide can be selected from one or more of sucrose, maltose, lactose, trehalose, maltotriose, isomaltooligosaccharide, xylooligosaccharide, soy oligosaccharide.

[0043] In the alcohol-washing step of the present invention, the mass ratio of the soy protein raw material to the ethanol solution is 1:5 - 10 (w / w). The alcohol-washing is a conventional washing method in the art. For example, the soy protein raw material is mixed with the ethanol solution containing oligosaccharide, stirred at room temperature for 20 - 50 min, and then the ethanol solution is removed by filtration or centrifugation. The obtained protein precipitate is the alcohol-washed protein. In some embodiments, the protein precipitate obtained by alcohol-washing is first dried at 40 - 60 °C and then ground to less than 60 mesh to obtain the alcohol-washed protein.

[0044] In this text, the enzyme preparation used in enzymatic hydrolysis is protease, and the purpose of enzymatic hydrolysis is to hydrolyze 7S protein. The protease of the present invention is obtained by microbial fermentation or extraction from plant raw materials, and can be selected from neutral protease (such as Novozymes Neutrase 0.8L, Xiasheng FDG-2230, Donghenghuadao 1-11, Pangbo Biology PBD-01-3), papain (such as Xiasheng FDG-2203, Donghenghuadao 2-10, Pangbo Biology PBD-01-1), bromelain (such as Xiasheng FDG-2201, Donghenghuadao 2-55, Pangbo Biology PBD-01-2), or one or more of them. Based on the dry matter mass of alcohol-washed protein, the addition amount of the protease is 0.02-2%, preferably 0.05-0.2%. To ensure a relatively high reaction rate, preferably, the enzyme activity of the protease is 0.5 AU / g or more than 50,000 u / g, for example, 0.8 AU / g or 100,000 u / g.

[0045] In some embodiments, during enzymatic hydrolysis, the pH of the alcohol-washed protein solution is adjusted to the applicable range of the protease, for example, the pH is adjusted to 5-8, preferably 6-7, then the protease is added, and the reaction is stirred at an appropriate temperature for a period of time, for example, stirred at 40-50 °C for 20-60 min, and then the protein precipitate in the enzymatic hydrolysate is separated to obtain the enzymatically hydrolyzed protein. Centrifugation can be used for separation, such as centrifuging at 5000 rpm for 10 min.

[0046] The method for preparing soybean 11S protein of the present invention further includes one or more steps of conventional acid precipitation, neutralization, and drying.

[0047] Exemplary acid precipitation includes adjusting the pH of the supernatant to 4.5-5.0 with acid, and separating the precipitate after standing for a period of time. The acid commonly used in the acid precipitation step can be used for acid precipitation treatment, such as hydrochloric acid. The standing time can be selected according to factors such as the material, and the precipitate can also be collected by centrifugation.

[0048] Exemplary neutralization includes redissolving the protein precipitate in water and adjusting it to neutral, such as adjusting the pH to 7.0-7.5 with sodium hydroxide and stirring to dissolve the precipitate.

[0049] The drying step of the present invention can be a drying method well-known in the art, such as spray drying or freeze drying.

[0050] In some embodiments, the present invention provides a method for preparing soybean 11S protein. More specifically, the method includes:

[0051] (1) Alcohol washing: Dissolve the soybean protein raw material in an ethanol solution containing oligosaccharides, mix evenly and filter to obtain alcohol-washed protein;

[0052] (2) Enzymatic hydrolysis: Use protease to enzymatically hydrolyze the alcohol-washed protein in step (1) to obtain an enzymatically hydrolyzed protein solution;

[0053] (3) Acid precipitation and neutralization: Adjust the enzymatically hydrolyzed protein solution in step (2) to acidic, collect the protein precipitate, redissolve it in water, and then adjust it to neutral to obtain a soy 11S protein solution.

[0054] (4) Optional drying: Dry the soy 11S protein solution to obtain soy 11S protein.

[0055] The soy 11S protein obtained by the method of the present invention can be used as part of food. Therefore, the present invention provides a food containing the soy 11S protein described in any embodiment herein, or prepared from the soy 11S protein described in any embodiment herein.

[0056] As a food, the soy 11S protein of the present invention can be incorporated into and used in various foods. The foods include but are not limited to hot pot products (such as surimi products, hot pot balls), dairy products, baked products, soy products (such as thousand-layer tofu), sauces (such as mayonnaise), chocolate products, and meat products (such as ham, bacon).

[0057] In this article, the preparation method of the food includes the step of preparing the food using the soy 11S protein described in any embodiment herein. For example, the preparation of hot pot balls: Mix the soy 11S protein of the present invention with starch paste, minced meat, and other ingredients and stir evenly, then chop, shape, steam, and cool to obtain hot pot balls.

[0058] Advantages or positive effects of the present invention compared with the prior art.

[0059] (1) In the present invention, by using an ethanol solution containing oligosaccharides to denature the 7S protein in soy protein while keeping the 11S protein unchanged, protease can specifically hydrolyze the 7S protein, improving the yield of 11S protein; at the same time, in the preparation method of the present invention, the 7S protein can be completely enzymatically hydrolyzed with almost no loss of 11S protein, thereby obtaining 11S protein with high purity and high yield.

[0060] (2) The structure of the 11S protein prepared by the method of the present invention is well-preserved and has anti-enzymatic hydrolysis properties, making the enzymatic hydrolysis process easy to control, without the need for high-temperature enzyme inactivation treatment, reducing costs, and maintaining the solubility of the protein.

[0061] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources without special instructions.

[0062] Experimental methods

[0063] 1. Determination of solubility

[0064] Prepare a protein solution with a concentration of 1% (w / v) using deionized water, and stir and dissolve it at room temperature for 2 h. Centrifuge the protein solution at 1500 rpm for 10 min, and take the supernatant to measure the protein content (mg / mL).

[0065] Solubility (%) = (supernatant protein content / protein solution concentration) × 100%.

[0066] 2. Determination of the yield of 11S protein:

[0067] Yield (%) = mass of 11S protein powder / mass of protein in the original soybean meal × 100%.

[0068] Among them, the mass of the 11S protein powder refers to the mass (g) of the 11S protein powder sample prepared by the present invention, and the mass (g) of the protein in the original soybean meal is calculated according to the following formula:

[0069] Mass of protein in the original soybean meal (g) = mass of the original soybean meal (g) × protein content (%) of the original soybean meal

[0070] Among them, the protein content of the original soybean meal is measured by the Kjeldahl method.

[0071] 3. Determination of the purity of 11S protein:

[0072] Dissolve the 11S protein powder in deionized water to prepare a solution with a concentration of 5 mg / mL (w / v), and perform reduced SDS-PAGE electrophoresis. Use Scion Image to perform optical density scanning on two electrophoresis maps to obtain the gray values of each protein band.

[0073] Purity (%) = gray value of 11S protein band / total gray value of bands * 100%

[0074] Example 1

[0075] Preparation of protein raw material: Mix the low-temperature defatted soybean meal (commercially available) obtained after soybean oil extraction with water at a mass ratio of 1:10, adjust the pH to 7.5 with sodium hydroxide, and stir and extract at room temperature for 60 min. Filter the extract with an 80-mesh filter cloth, centrifuge the obtained filtrate at 5000 rpm for 10 min, and collect the supernatant. Adjust the pH of the supernatant to 4.5 with hydrochloric acid, and centrifuge at 5000 rpm for 10 min to collect the precipitate. Add a certain amount of water to the precipitate, adjust the pH to 7.0 with sodium hydroxide, and stir and dissolve to obtain a protein complex solution. Perform spray drying on the obtained protein complex solution, control the outlet temperature at 65 °C, and obtain low-denatured soy protein isolate 1.

[0076] Alcohol washing: Add 10% (w / v) sucrose to ethanol with a concentration of 30% (v / v), stir and dissolve to obtain an ethanol solution containing sucrose. Mix low-denatured soy protein isolate 1 with the ethanol solution at a ratio of 1:10 (w / w), and stir at room temperature for 30 min. Remove the ethanol solution by filtration or centrifugation, dry the obtained precipitate at 50 °C, and crush it to less than 60 mesh to obtain alcohol-washed protein.

[0077] Enzymatic hydrolysis: Mix the alcohol-washed protein with water at a ratio of 1:10 (w / w), adjust the pH to neutral, add neutral protease (Novozymes Neutrase 0.8L, 0.8 AU / g) at 0.2% of the protein mass, and react at 40 °C for 20 min to obtain a protein hydrolysate.

[0078] Acid precipitation and neutralization: Adjust the pH of the protein hydrolysate to 5, let it stand for 30 min, then centrifuge at 5000 rpm for 10 min to collect the protein precipitate. Add a certain amount of water to the protein precipitate, adjust the pH to 7.5 with sodium hydroxide, stir to dissolve the precipitate, and obtain a protein re-solution. Spray-dry the protein re-solution to obtain soy 11S protein sample A.

[0079] Example 2

[0080] Alcohol washing: Add 20% (w / v) sucrose to ethanol with a concentration of 35% (v / v), stir and dissolve to obtain an ethanol solution containing sucrose. Mix the low-denatured soy protein isolate 1 in Example 1 with the ethanol solution at a ratio of 1:8 (w / w), and stir at room temperature for 30 min. Remove the ethanol solution by filtration or centrifugation, dry the obtained precipitate at 50 °C, and crush it to less than 60 mesh to obtain alcohol-washed protein.

[0081] Enzymatic hydrolysis: Mix the alcohol-washed protein with water at a ratio of 1:10 (w / w), adjust the pH to neutral, add neutral protease (Novozymes Neutrase 0.8L, 0.8 AU / g) at 0.1% of the protein mass, and react at 45 °C for 40 min to obtain a protein hydrolysate.

[0082] Acid precipitation and neutralization: Adjust the pH of the protein hydrolysate to 5, let it stand for 30 min, then centrifuge at 5000 rpm for 10 min to collect the protein precipitate. Add a certain amount of water to the protein precipitate, adjust the pH to 7.5 with sodium hydroxide, stir to dissolve the precipitate, and obtain a protein re-solution. Spray-dry the protein re-solution to obtain soy 11S protein sample B.

[0083] Example 3

[0084] Alcohol washing: Add 30% (w / v) sucrose to ethanol with a concentration of 40% (v / v), and stir to dissolve to obtain an ethanol solution containing sucrose. Mix the low-denatured soy protein isolate 1 in Example 1 with the ethanol solution at a ratio of 1:5 (w / w), and stir at room temperature for 30 min. Remove the ethanol solution by filtration or centrifugation, and dry the obtained precipitate at 50 °C and crush it to less than 60 mesh to obtain alcohol-washed protein.

[0085] Enzymatic hydrolysis: Mix the alcohol-washed protein with water at a ratio of 1:10 (w / w), adjust the pH to neutral, add neutral protease (Novozymes Neutrase 0.8L, 0.8 AU / g) at 0.05% of the protein mass, and react at 50 °C for 60 min to obtain a protein hydrolysate.

[0086] Acid precipitation and neutralization: Adjust the pH of the protein hydrolysate to 5, let it stand for 30 min, then centrifuge at 5000 rpm for 10 min to collect the protein precipitate. Add a certain amount of water to the protein precipitate, adjust the pH to 7.5 with sodium hydroxide, and stir to dissolve the precipitate to obtain a protein re-solution. Spray-dry the protein re-solution to obtain soy 11S protein sample C.

[0087] Example 4

[0088] Protein raw material preparation: Mix the low-temperature defatted soybean meal (commercially available) obtained after soybean oil extraction with water at a mass ratio of 1:12, adjust the pH to 8.0 with sodium hydroxide, and stir and extract at room temperature for 40 min. Filter the extraction solution with an 80-mesh filter cloth, centrifuge the obtained filtrate at 5000 rpm for 10 min, and collect the supernatant. Adjust the pH of the supernatant to 4.5 with hydrochloric acid, and centrifuge at 5000 rpm for 10 min to collect the precipitate. Add a certain amount of water to the precipitate, adjust the pH to 7.5 with sodium hydroxide, and stir to dissolve to obtain a protein re-solution. Freeze-dry the obtained protein re-solution to obtain low-denatured soy protein isolate 2.

[0089] Alcohol washing: Add 10% (w / v) lactose to ethanol with a concentration of 30% (v / v), and stir to dissolve to obtain an ethanol solution containing lactose. Mix the low-denatured soy protein isolate 2 with the ethanol solution at a ratio of 1:10 (w / w), and stir at room temperature for 30 min. Remove the ethanol solution by filtration or centrifugation, and dry the obtained precipitate at 50 °C and crush it to less than 60 mesh to obtain alcohol-washed protein.

[0090] Enzymatic hydrolysis: Mix the alcohol-washed protein with water at a ratio of 1:10 (w / w), adjust the pH to neutral, add neutral protease (Novozymes Neutrase 0.8L, 0.8 AU / g) at 0.2% of the protein mass, and react at 40 °C for 20 min to obtain a protein hydrolysate.

[0091] Acid precipitation and neutralization: Adjust the pH of the protein hydrolysate to 5, let it stand for 30 min, then centrifuge at 5000 rpm for 10 min to collect the protein precipitate. Add a certain amount of water to the protein precipitate, adjust the pH to 7.5 with sodium hydroxide, stir to dissolve the precipitate, and obtain a protein re-solution. Spray-dry the protein re-solution to obtain the soybean 11S protein sample D.

[0092] Example 5

[0093] Alcohol washing: Add 10% (w / v) sucrose to ethanol with a concentration of 30% (v / v), stir to dissolve to obtain an ethanol solution containing sucrose. Mix the low-denatured soy protein isolate 1 in Example 1 with the ethanol solution at a ratio of 1:10 (w / w), and stir at room temperature for 30 min. Remove the ethanol solution by filtration or centrifugation, dry the obtained precipitate at 50 °C, and crush it to less than 60 mesh to obtain the alcohol-washed protein.

[0094] Enzymatic hydrolysis: Mix the alcohol-washed protein with water at a ratio of 1:10 (w / w), adjust the pH to neutral, add 0.2% of papain (Xiasheng FDG-2203, 100,000 u / g) based on the protein mass, and react at 40 °C for 20 min to obtain a protein hydrolysate.

[0095] Acid precipitation and neutralization: Adjust the pH of the protein hydrolysate to 5, let it stand for 30 min, then centrifuge at 5000 rpm for 10 min to collect the protein precipitate. Add a certain amount of water to the protein precipitate, adjust the pH to 7.5 with sodium hydroxide, stir to dissolve the precipitate, and obtain a protein re-solution. Spray-dry the protein re-solution to obtain the soybean 11S protein sample E.

[0096] Example 6

[0097] Alcohol washing: Add 10% (w / v) maltotriose to ethanol with a concentration of 30% (v / v), stir to dissolve to obtain an ethanol solution containing maltotriose. Mix the low-denatured soy protein isolate 2 in Example 4 with the ethanol solution at a ratio of 1:10 (w / w), and stir at room temperature for 30 min. Remove the ethanol solution by filtration or centrifugation, dry the obtained precipitate at 50 °C, and crush it to less than 60 mesh to obtain the alcohol-washed protein.

[0098] Enzymatic hydrolysis: Mix the alcohol-washed protein with water at a ratio of 1:10 (w / w), adjust the pH to neutral, add 0.2% of neutral protease (Novozymes Neutrase 0.8L, 0.8 AU / g) based on the protein mass, and react at 40 °C for 20 min to obtain a protein hydrolysate.

[0099] Acid precipitation and neutralization: Adjust the pH of the protein hydrolysate to 5, let it stand for 30 min, then centrifuge at 5000 rpm for 10 min to collect the protein precipitate. Add a certain amount of water to the protein precipitate, adjust the pH to 7.5 with sodium hydroxide, and stir to dissolve the precipitate to obtain a protein re-solution. Spray-dry the protein re-solution to obtain the soybean 11S protein sample F.

[0100] Comparative Example 1

[0101] Prepare soybean 11S protein according to the method of Example 1, wherein the concentration of ethanol in the alcohol washing step is replaced with 10% (v / v), and other conditions remain unchanged, to obtain the soybean 11S protein sample 1.

[0102] Comparative Example 2

[0103] Prepare soybean 11S protein according to the method of Example 1, wherein the concentration of ethanol in the alcohol washing step is replaced with 60% (v / v), and other conditions remain unchanged, to obtain the soybean 11S protein sample 2.

[0104] Comparative Example 3

[0105] Prepare soybean 11S protein according to the method of Example 1, wherein 5% (w / v) of sucrose is added in the alcohol washing step, and other conditions remain unchanged, to obtain the soybean 11S protein sample 3.

[0106] Comparative Example 4

[0107] Prepare soybean 11S protein according to the method of Example 1, wherein 40% (w / v) of sucrose is added in the alcohol washing step, and other conditions remain unchanged, to obtain the soybean 11S protein sample 4.

[0108] Comparative Example 5

[0109] Prepare soybean 11S protein according to the method of Example 1, wherein 0.01% of neutral protease (Novozymes Neutrase 0.8L, 0.8 AU / g) is added in the enzymatic hydrolysis step, and other conditions remain unchanged, to obtain the soybean 11S protein sample 5.

[0110] Comparative Example 6

[0111] Mix low-temperature defatted soybean meal and water at a mass ratio of 1:10, adjust the pH to 7.5 with sodium hydroxide, and stir and extract at room temperature for 60 min. Filter the extract with an 80-mesh filter cloth, centrifuge the obtained filtrate at 5000 rpm for 10 min, and collect the supernatant. Add sodium bisulfite (0.98 g / L) to the supernatant, adjust the pH of the supernatant to 6.4 with hydrochloric acid, let it stand overnight at 4 °C, and centrifuge at 5000 rpm for 10 min to collect the precipitate. Add a certain amount of water to the precipitate, adjust the pH to 7.0 with sodium hydroxide, and stir to dissolve the precipitate to obtain a protein re-solution. Spray-dry the obtained protein re-solution to obtain soybean 11S protein sample 6.

[0112] Comparative Example 7

[0113] Mix low-temperature defatted soybean meal and water at a mass ratio of 1:10, adjust the pH to 7.5 with sodium hydroxide, and stir and extract at room temperature for 60 min. Filter the extract with an 80-mesh filter cloth, centrifuge the obtained filtrate at 5000 rpm for 10 min, and collect the supernatant. Adjust the pH of the supernatant to 4.5 with hydrochloric acid, centrifuge at 5000 rpm for 10 min to collect the precipitate. Add a certain amount of water to the precipitate, adjust the pH to 7.0 with sodium hydroxide, and stir to dissolve to obtain a protein re-solution. Heat the obtained protein re-solution at 75 °C for 10 min, add neutral protease (0.8 AU / g) at 0.2% of the protein mass, react at 40 °C for 20 min, and inactivate the enzyme in a boiling water bath for 5 min. Adjust the pH of the protein hydrolysate to 5, let it stand for 30 min, then centrifuge at 5000 rpm for 10 min, and collect the protein precipitate. Add a certain amount of water to the protein precipitate, adjust the pH to 7.5 with sodium hydroxide, and stir to dissolve the precipitate to obtain a protein re-solution. Spray-dry the protein re-solution to obtain soybean 11S protein sample 7.

[0114] Determine the purity, yield, and solubility of the soybean 11S protein samples in Examples 1-6 and Comparative Examples 1-7 above, and the results are shown in Table 1.

[0115] Table 1: Purity, Yield, and Solubility of Soybean 11S Protein Samples

[0116] Serial Number Protein Sample Protein Purity % Protein Yield % Solubility % Example 1 11S Protein Sample A 92 32 90 Example 2 11S Protein Sample B 93 30 92 Example 3 11S Protein Sample C 92 30 91 Example 4 11S Protein Sample D 92 30 92 Example 5 11S Protein Sample E 93 31 90 Example 6 11S Protein Sample F 91 31 91 Comparative Example 1 11S Protein Sample 1 75 40 91 Comparative Example 2 11S Protein Sample 2 60 15 70 Comparative Example 3 11S Protein Sample 3 67 12 85 Comparative Example 4 11S Protein Sample 4 79 39 90 Comparative Example 5 11S Protein Sample 5 80 38 90 Comparative Example 6 11S Protein Sample 6 92 22 92 Comparative Example 7 11S Protein Sample 7 91 25 73

[0117] Table 1 results show that the soy 11S protein samples prepared in Examples 1-6 have high purity and yield, and can maintain high solubility at the same time. Although the soy 11S protein samples prepared in Comparative Example 1 and Comparative Example 4 have high yields, their purities are low. This is because when the ethanol concentration is very low or the sucrose concentration is high during the alcohol washing process, the denaturation degree of soy 7S protein is low, and the hydrolysis degree is low during the subsequent enzymatic hydrolysis process, resulting in a decrease in the purity of 11S protein; in Comparative Example 5, the addition amount of protease in the enzymatic hydrolysis step is too small, and the hydrolysis degree of soy 7S protein is low, which will also lead to a decrease in the purity of 11S protein; the soy 11S protein sample prepared in Comparative Example 6 has high purity and good solubility, but the yield is low. The soy 11S protein prepared in Comparative Example 7 has high purity, but both the yield and solubility are low.

[0118] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims. At the same time, all the documents mentioned in the present invention are cited herein as references, just as if each document was cited separately as a reference.

Claims

1. A preparation method of soybean 11S protein, characterized in that, The method includes the following: (1) Alcohol washing: Dissolve the soy protein raw material in an ethanol solution containing oligosaccharides, mix evenly and then filter to obtain alcohol-washed protein; (2) Enzymatic hydrolysis: Use protease to perform enzymatic hydrolysis on the alcohol-washed protein in step (1) to obtain a protein solution.

2. The method according to claim 1, wherein The alcohol washing step has one or more of the following characteristics: The soy protein raw material is low-denatured soy protein isolate; The concentration of the ethanol solution is 20-50% (v / v), preferably 30-40% (v / v); Based on the volume of the ethanol solution, the addition amount of the oligosaccharides is 8-35% (w / v), preferably 10-30% (w / v); The mass ratio of the soy protein raw material to the ethanol solution is 1:5-10 (w / w); The oligosaccharides are selected from one or more of sucrose, maltose, lactose, trehalose, maltotriose, isomaltooligosaccharide, xylooligosaccharide and soy oligosaccharides.

3. The method according to claim 1, wherein The enzymatic hydrolysis step has one or more of the following characteristics: Based on the dry matter mass of the alcohol-washed protein, the addition amount of the protease is 0.02-2%, preferably 0.05-0.2%; The protease is selected from one or more of neutral protease, papain and bromelain; The enzymatic hydrolysis temperature is 30-60°C, preferably 40-50°C; The enzymatic hydrolysis time is 10-100 min, preferably 20-60 min.

4. The method according to claim 1, the method further includes one or more of the following steps: (3) Acid precipitation and neutralization: Adjust the protein solution in step (2) to acidic, collect the protein precipitate, redissolve it with water, and adjust it to neutral to obtain an 11S protein solution; (4) Drying: Dry the 11S protein solution; preferably spray drying.

5. A soy 11S protein, which is prepared by the method according to any one of claims 1-4.

6. The 11S protein of soybean according to claim 5, wherein The purity of the soy 11S protein ≥ 90%; and / or, the solubility of the soy 11S protein ≥ 90%.

7. A food, characterized in that, The food contains the soy 11S protein prepared by the method according to any one of claims 1-4, or is prepared from the soy 11S protein described in claim 5. Preferably, the food is a hot pot product, dairy product, baked product, soy product, sauce product, beverage product, chocolate product or meat product; more preferably a meat product.

8. A method for preparing a food, characterized in that, The method includes: obtaining the soy 11S protein prepared by the method according to any one of claims 1-4, and using the soy 11S protein to prepare the food.

9. Use of an ethanol solution containing oligosaccharide in increasing the yield of soybean 11S protein, characterized in that, Wash the soy protein raw material with an ethanol solution containing oligosaccharides, and then perform enzymatic hydrolysis with protease.

10. The use according to claim 9, characterized in that, The concentration of the ethanol solution is 20-50% (v / v), preferably 30-40% (v / v); based on the volume of the ethanol solution, the addition amount of the oligosaccharides is 8-35% (w / v), preferably 10-30% (w / v).

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