WO2021039681A1

WO2021039681A1 - Method for fractionating soybean proteins

Info

Publication number: WO2021039681A1
Application number: PCT/JP2020/031755
Authority: WO
Inventors: 将宏杉山; 佐本　将彦; 康生松村; 健太郎松宮
Original assignee: 不二製油グループ本社株式会社
Priority date: 2019-08-27
Filing date: 2020-08-24
Publication date: 2021-03-04
Also published as: JPWO2021039681A1

Abstract

The purposes of the present invention are to provide a method for fractionating soybean proteins, to provide a method for analyzing protein composition in soybeans, and to provide a method for evaluating a soybean species, etc. by determining the protein composition ratio in soybean seeds. According to the present invention, a large portion of soybean proteins can be fractionated by extracting the soybean proteins with the use of a solvent having a high ionic strength as a means for preventing the emulsification of non-denatured soybean proteins.

Description

Soy protein fractionation method

The present invention relates to a method for fractionating soybean protein. The present invention also relates to a method for analyzing protein composition in soybean. Furthermore, it also relates to an evaluation method for soybean varieties and the like by determining the protein composition ratio in soybean seeds. Furthermore, the present invention also relates to soybean protein products obtained by fractionation.

Soy protein is widely used to improve the physical characteristics of foods due to its unique gelling power, and its use as a highly nutritious health food material is increasing.

The stored protein of soybeans precipitates at around pH 4.5, and can be relatively easily divided into an acid-soluble protein fraction mainly composed of soluble components other than the stored protein and an acid-precipitating protein fraction mainly composed of stored protein. .. The isolated soybean protein is obtained by recovering this acid-precipitating protein fraction, and is currently widely used in the food industry.

The proteins that make up soybean protein are also classified into 2S, 7S, 11S, and 15S globulin based on the sedimentation coefficient by ultracentrifugation analysis. Of these, 7S globulin and 11S globulin are the main constituent protein components of the globulin fraction. In addition, β-conglycinin in the immunological nomenclature is substantially equivalent to 7S globulin, and glycinin is substantially equivalent to 11S globulin.

The proteins that make up soybean protein have different properties in terms of physical properties such as viscosity, coagulation, and surface activity, as well as nutritional and physiological functions. For example, 7S globulin has been reported to reduce triglyceride in blood (Non-Patent Document 1). In addition, 11S globulin has a high gelling power and is said to control the hardness and texture of tofu gel.

Furthermore, soybean-derived proteins include proteins with high affinity for polar lipids that make up biological membranes such as protein bodies and oil bodies, which account for about 35% of industrially produced isolated soybean proteins. It has been reported that it occupies (Non-Patent Documents 2 and 3).

It is important to clarify the protein composition in soybean seeds from the viewpoint of evaluation of processing suitability of soybean seeds for soybean foods and nutritional health functionality. As a method for analyzing a protein composition, there are a method of preparing an antibody of a specific protein and quantifying it by an antigen-antibody reaction, a method of evaluating it by protein staining after electrophoresis, and the like (Patent Document 1). However, the antigen-antibody reaction is based on the reaction of only a specific protein recognized by the antibody, and there is a problem that the quantitativeness and reproducibility are lowered when the reaction becomes unstable. In addition, protein staining by electrophoresis also has a problem that it is difficult to quantify when the degree of staining differs depending on the type of protein or when innumerable bands are present.

On the other hand, the protein extracted by utilizing the unique dissolution behavior of the protein was fractionated based on the protein composition, and the nitrogen transfer rate to each fraction was obtained to obtain the protein content ratio of each fraction. There is a report (Patent Document 2). This is a method that can grasp the overall protein composition, although it is an approximation, unlike the molecular species research of a specific protein. Moreover, since it is a nitrogen analysis, it is highly reliable in terms of reproducibility and quantitativeness. However, although there are cases where this method is used to report the protein composition of a part of soybean, a method of extracting and fractionating almost all the proteins in soybean seeds has not been reported so far.

The above documents and the documents shown in the present specification are incorporated in the present specification by specifying the source.

International Publication No. 2009/0845229 International Publication No. 2006/129647

The protein composition ratio contained in soybean seeds has not been clarified so far. The analysis of the crude protein mass contained in soybean seeds is the main, and although there are some analysis examples by electrophoresis, it is insufficient to determine the content ratio due to the presence of proteins that are difficult to stain such as LP. In addition, soybean varieties lacking a specific protein molecular species have been developed by breeding, etc., but there is a problem that it is difficult to accurately analyze how the protein composition has changed due to the deletion. For example, when producing a variety containing a large amount of a specific target protein in soybean, it is difficult to select and breed seeds unless it is possible to evaluate how the overall composition ratio changes due to deletion other than the target. Against this background, from the viewpoint of food processing suitability and nutritional health function, an evaluation method that can be an accurate and efficient index for evaluation and selection of soybean varieties and breeding of soybean varieties as a future target is required.

Generally, protein extraction in soybean seeds is performed by suspending in an aqueous solution. Since lipids are also present in soybean seeds, emulsification occurs at the same time that proteins are extracted. When emulsification occurs, it becomes difficult to make a difference in the dissolution behavior peculiar to proteins. There is also a method of using defatted soybeans as a raw material, but when removing lipids, some proteins are denatured, which changes the dissolution behavior and lowers the fractionation accuracy. Therefore, an accurate protein composition ratio in soybeans is obtained. It becomes difficult. There is also a method of denaturing soybean protein to change the dissolution behavior and then fractionating, but there is a problem that the extraction rate is lowered due to the denaturation of the protein.

With the conventional method, 7S, 11S, LP and whey fractions can be separated. The 7S and 11S proteins have a strong gelling power and are almost tasteless, while the LP protein shows only a weak gelling power but has a tofu-like richness. However, soybeans contain a wide variety of proteins such as stored proteins such as 7S and 11S and whey proteins, as well as polar lipid-associated proteins including oil body-associated proteins and membrane proteins. If it becomes possible to extract and further fractionate most of the proteins in soybeans, it will be possible to obtain protein materials with various functional characteristics, and it will be possible to evaluate the suitability of individual soybean proteins. , Soybean varieties can be evaluated.

One of the objects of the present invention is to provide a method for fractionating soybean protein from soybean seeds. Another object of the present invention is to provide a method for analyzing the protein composition in soybean seeds. A more specific purpose is to provide a method for evaluating soybean varieties and the like by determining the protein composition ratio in soybean seeds.

As a result of diligent research on the above-mentioned problems, the present inventors have extracted soy protein using a solvent having high ionic strength as a means for suppressing emulsification of undenatured soy protein with lipid. , Found that most of the soy protein can be fractionated. More specifically, soy protein is extracted using a solvent with high ionic strength, separated into an oil layer, an intermediate layer, and a precipitation layer by solid-liquid separation, and then the intermediate layer is acid-diluted and solid-liquid separated into an aqueous layer. It was found that the above-mentioned problems can be solved by a method including separating into a precipitation layer, diluting the aqueous layer, and then fractionating into a whey fraction and a globulin fraction by solid-liquid separation. The present invention has been completed based on the above novel findings.

That is, the present invention
(1) The following steps:
(A) Prepare a soybean raw material having a lipid content of 15% by weight or more and a water-soluble nitrogen index (NSI) of 80 or more per dry matter;
(B) A solvent having an ionic strength of 1.5 mol / L or more is added to the soybean raw material to extract the protein;
(C) The extracted protein is solid-liquid separated and separated into an oil layer (O), an intermediate layer (M), and a precipitation layer (R);
(D) Acid is added to the intermediate layer (M) to lower the pH to 6 or less;
(E) The pH-lowered intermediate layer (M) is solid-liquid separated and separated into an aqueous layer (MA) and a precipitated layer (MR);
(F) Dilute the aqueous layer (MA) with water;
(G) The diluted aqueous layer (MA) is solid-liquid separated and separated into an aqueous layer (W) and a precipitated layer (G);
Soy protein fractionation method, including
(2) Between steps (C) and (D), the following steps:
(A) A solvent having an ionic strength of 1.5 mol / L or more is added to the oil layer (O) obtained in the step (C), and the aqueous layer (OA) and the oil layer (O2) or the oil layer (O2) are separated by solid-liquid separation. ) And the sedimentation layer (OR);
(B) An aqueous solvent is added to the precipitate layer (R) obtained in the step (C) to extract the protein, and the protein is separated into an aqueous layer (RA) and an okara layer (R2) by solid-liquid separation;
(C) The intermediate layer (M), the aqueous layer (OA) and the aqueous layer (RA) are mixed to form a new intermediate layer (M'), and the intermediate layer (M') is used as the intermediate layer (M) in the subsequent steps. To use,
(1), which further includes
(3) After step (G), the following steps:
(D) Dilute the aqueous layer (W) obtained in step (G) with water;
(E) The diluted aqueous layer (W) is solid-liquid separated and separated into an aqueous layer (W2) and a precipitation layer (WG).
The method (1) or (2), further comprising
(4) The method according to any one of (1) to (3), wherein the solvent in step (B) further contains a reducing agent.
(5) The method according to any one of (1) to (4), wherein the water-soluble nitrogen index (NSI) of the step (A) is 90% or more.
(6) The method according to any one of (1) to (5), wherein the dilution of the aqueous layer in step (G) has an ionic strength of 150 to 30 mM.
(7) The method according to any one of (3) to (6), wherein the dilution of the aqueous layer in step (d) has an ionic strength of 150 to 30 mM.
(8) A method for analyzing a protein composition in soybean, which comprises measuring the protein mass of a fraction obtained by any of the methods (1) to (7).
(9) Soybeans, which include obtaining the protein composition ratio in soybean seeds by measuring the protein mass of each fraction obtained by any of the methods (1) to (7) from the soybean seed raw material. Variety evaluation method,
Regarding.

According to the present invention, soybean protein in undenatured soybean seeds containing lipid can be separated into four fractions: oil body-associated protein (OBAP), polar lipid-associated protein (PLAP), whey protein, and globulin. .. Further, the globulin can be separated into a total of 5 fractions by separating each of the 7S globulin and 11S globulin fractions. This makes it possible to know the protein composition of soybean varieties in the natural state. In addition, the present invention makes it possible to efficiently select soybean varieties suitable for the quality required for soybean products. Furthermore, the method of the present invention can also be used as a method for evaluating soybean varieties for breeding soybean varieties having a characteristic protein composition and for producing soybean varieties by genome editing. Further, by selecting suitable varieties using the present invention, it is possible to contribute to high quality of soybean products, cost reduction of raw materials, production of soybean products having new functions, and the like. Furthermore, the method of the present invention can recover almost all proteins from unmodified soybean raw materials in a fractionated state, so that soybean protein products having various functional characteristics can be obtained. It can be used for the development of various foods and drinks.

It is a figure which shows the process of Example 1. FIG. It is a figure which shows the process of Example 2. FIG. It is a figure which shows the SDS-PAGE result of each fraction fractionated by the method of Example 2 using the seed of the soybean variety Fukuyutaka as a soybean raw material. Lane M: molecular weight marker, front: initial solution sample, O: OBAP fraction, P: PLAP fraction, W: whey fraction, G: globulin fraction (7S, 11S mixture). LOX: Lipoxygenase. It is a figure which shows the SDS-PAGE result of Example 3. Lane M: Molecular weight marker, 1: Ionic strength 1.2 mol / L 2: Ionic strength 0.6 mol / L 3: Ionic strength 0.4 mol / L 4: Ionic strength 0.3 mol / L 5, 5: Ionic strength 0.24 mol / L, 6: Ionic strength 0.2 mol / L, 7: Ionic strength 0.15 mol / L, 8: Ionic strength 0.12 mol / L, 9: Ionic strength 0.09 mol / L, 10: Ionic strength 0.075 mol / L, 11: Ionic strength 0.06 mol / L, 12: Ionic strength 0.045 mol / L, 13: Ionic strength 0.03 mol / L, 14: Ionic strength 3 mol / L (without dilution). It is a figure which shows the process of Example 4. It is a figure which shows the protein composition of each variety soybean fractionated by the method of this invention.

In this specification, "food and drink" refers to all foods including beverages, and is also simply described as "food". That is, the term "food" includes beverages, unless otherwise specified as "excluding beverages".

In the present specification, the term "about" means a range of ± 10%, preferably ± 5%. In addition, the numerical value that becomes the boundary value of the range is considered to be described in the present specification.

In the present specification, "7S globulin" is also called β-conglycinin, and is generally a glycoprotein composed of three types of subunits (α', α, β), and any of the subunits is used. It may be missing. These subunits are randomly combined to form a trimer. The isoelectric point is around pH 4.8 and the molecular weight is about 170,000. Hereinafter, it may be simply abbreviated as "7S".

In the present specification, "11S globulin" is also called glycinin, and acidic subunits and basic subunits are bound by disulfide bonds to form a dimer in which 6 molecules are collected. The molecular weight is about 360,000. Hereinafter, it may be simply abbreviated as "11S".

When simply referred to as "globulin" in the present specification, it means a mixture in which 7S and 11S are not fractionated, or a general term for 7S and 11S.

In the present specification, "lipophilic Proteins" refers to a group of minor acid-precipitating soybean proteins other than 7S and 11S among the acid-precipitating soybean proteins of soybean, such as lecithin and glycolipid. It is accompanied by a large amount of polar lipids. Hereinafter, it may be simply abbreviated as "LP". This LP contains proteins showing mainly about 34 kDa, about 24 kDa, and about 18 kDa in the estimated molecular weight by SDS-polyacrylamide gel electrophoresis, lipoxygenase, γ-conglycinin, and many other miscellaneous proteins.

In the present specification, the "oil body associated protein" (Oil Body Associated Proteins) includes a large amount of oleosin contained in the membrane of the oil body, 34 kDa protein which is an allergen protein, etc. among the proteins contained as LP. A group of hydrophobic proteins that are highly associated with lipids. Hereinafter, this may be simply abbreviated as "OBAP".

In the present specification, "Polar Lipid Associated Proteins" refers to proteins contained as LP, which are more polar than OBAP and are mainly contained in cell membranes and protein body membranes. It refers to a group of proteins that are highly associated with polar lipids and contain a large amount of such substances. It is characterized by containing about 40 kDa of protein. Hereinafter, this may be simply abbreviated as "PLAP".

In the present specification, "whey protein" (Whey Proteins) refers to a group of proteins that are soluble at an acidic pH near pH 4.5. It contains about 93 kDa protein and about 54 kDa protein. Hereinafter, this may be simply abbreviated as "whey".

In the present specification, the estimated molecular weight of the protein is a molecular weight marker (product name: "Precision Plus") in SDS-PAGE using a commercially available ready-made gel (product name: "e-pagel 12.5%", manufactured by Atto Co., Ltd.). It can be calculated based on the mobility of "Protein (registered trademark) 2-color standard" manufactured by Bio-Rad.

In the present specification, "soybean product" refers to processed foods made from soybean itself, soybean protein material, processed foods to which soybean protein material is added, and the like. Specific embodiments include traditional foods such as tofu, soy milk, natto, fried tofu skin, yuba, miso, and soy sauce. Other embodiments include soybean protein materials such as soybean powder, soymilk powder, concentrated soybean protein, separated soybean protein, and texture used as raw materials for various processed foods. In addition, as another embodiment, various processed foods to which the soybean protein material is added can be mentioned. The type of processed food is not limited, but includes, for example, processed meat foods such as ham, sausage, and hamburger, health foods such as protein powder and protein beverages, plant-based cheese-like foods, and plant-based meat-like foods.

In one embodiment, the present invention provides a method for fractionating soybean protein.

<Soybean ingredients>
The soybean raw material used in this embodiment is an unmodified or low-modified greasy soybean raw material. More specifically, it is a fat-containing soybean raw material having a water-soluble nitrogen index (NSI) of about 80 or more, for example, about 83 or more, about 85 or more, about 88 or more, about 90 or more, about 93 or more, about 95 or more. .. The soybean raw material used in this embodiment is preferably a non-defatted fat-containing soybean raw material. For example, a soybean raw material having a lipid content of about 90% or more, for example, about 95% or more of the normal lipid content of a soybean variety used as a soybean raw material can be mentioned. More specifically, a fat-containing soybean raw material having a lipid content per dry matter of about 13% by weight or more, for example, about 15% by weight or more and about 20% by weight or more is preferable.

In addition, NSI can be expressed by the ratio (% by weight) of water-soluble nitrogen (crude protein) to the total amount of nitrogen based on a predetermined method, and in this specification, it is the value measured based on the following method. To do. That is, 100 ml of water is added to 2.0 g of the sample, the mixture is stirred and extracted at 40 ° C. for 60 minutes, and centrifuged at 1400 × g for 10 minutes to obtain supernatant 1. 100 ml of water is added to the remaining precipitate again, and the mixture is stirred and extracted at 40 ° C. for 60 minutes and centrifuged at 1400 × g for 10 minutes to obtain supernatant 2. The supernatant 1 and the supernatant 2 are combined, and water is further added to make 250 ml. No. After filtering with 5A filter paper, the nitrogen content of the filtrate is measured by the Kjeldahl method. At the same time, the nitrogen content in the sample was measured by the Kjeldahl method, and the ratio of nitrogen (water-soluble nitrogen) recovered as a filtrate to the total nitrogen in the sample expressed as% by weight is defined as NSI.

The form of the soybean raw material used in this embodiment is not particularly limited, and may be, for example, whole soybean, half-cracked soybean, glitz, or powder.

<Extraction from soybean ingredients>
In the fractionation method of this embodiment, the solvent used for extracting the protein from the soybean raw material preferably has an ionic strength of about 1.5 mol / L or more, for example, about 2 mol / L or more, about 2.3 mol / L or more, and about 2 Solvents of .5 mol / L or more and about 3 mol / L or more can be mentioned. The upper limit of the ionic strength is not particularly limited, and examples thereof include about 10 mol / L or less, about 7 mol / L or less, and about 5 mol / L or less.

The type of salt added to adjust the ionic strength is not particularly limited, and examples thereof include inorganic acid salts, organic acid salts, and alkali metal salts. For example, hydrochlorides, sulfates, phosphates, nitrates, acetates, sodium salts, potassium salts and the like can be mentioned. More specifically, sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium phosphate, potassium phosphate, sodium nitrate, potassium nitrate, sodium acetate, potassium acetate and the like can be mentioned.

The pH of the solvent is not particularly limited, and examples thereof include about 6 to about 10, about 7 to about 9, and about 8.

The amount of the solvent used for extraction is not particularly limited, and is, for example, about 3 to about 20 times by weight, about 4 to about 15 times by weight, about 6 to about 12 times by weight, and about 7 to about 10 times by weight of the soybean raw material. The degree can be mentioned. The larger the amount of the solvent, the higher the extraction rate of the water-soluble component and the better the separation, but if the amount is too large, concentration is required and the cost is high.

Extraction time and temperature are not particularly limited. Examples of extraction time are about 1 minute to about 10 hours, about 10 minutes to about 2 hours, for example, about 5 minutes or more, about 10 minutes or more, about 15 minutes or more, about 20 minutes or more, about 30 minutes or more, about 1 Time or more, about 90 minutes or more, and the like. Examples of the extraction temperature include about 4 to about 50 ° C., about 10 to about 30 ° C., room temperature, and the like.

At the time of extraction, stirring may be performed. The stirring speed may be, for example, about 100 to 5000 rpm.

A reducing agent may be added to the solvent used for extraction. It is considered that the addition of the reducing agent cleaves the disulfide bond of the soybean protein and further improves the extraction rate. The type of reducing agent is not particularly limited, but for example, sodium sulfite, sodium hydrogen sulfite, chlorine, nitrogen trichloride, ammonium persulfide, sodium chlorite, mercaptoethanol, N-acetyl-L-cysteine, dithiotreiol, tributylphosphine. And so on. The concentration of the reducing agent is not particularly limited, but is, for example, about 0.03%, about 0.05%, about 0.1%, about 0.3%, about 0.5%, about 0.01 to 1%, about. 0.03 to 0.5% and the like can be mentioned.

<Solid-liquid separation after extraction>
After extraction, the suspension from the soybean raw material is separated into solid and liquid by centrifugation, filtration or the like. By solid-liquid separation, the soybean extract is separated into an oil layer (O), an intermediate layer (M), and a precipitation layer (R). It is preferable to use a centrifuge as the solid-liquid separation. The conditions for centrifugation are not limited, and examples thereof include about 500 to about 1200,000 × g, about 1,000 to about 800,000 × g, about 5,000 to about 50,000 × g, and about 18,000 × g. The time for centrifugation is also not limited, and examples thereof include about 1 minute to about 20 minutes, about 1.5 minutes to about 15 minutes, about 2.5 minutes to about 10 minutes, and about 5 minutes.

<Recovery of OBAP fraction>
The oil reservoir (O) obtained by the above extraction may be recovered as it is as an OBAP fraction. In another embodiment, the oil layer (O) may be washed by adding a solvent having the ionic strength specified in the solvent used for extracting the protein from the soybean raw material. The solvent used for washing may be the same as or different from the solvent used for extracting the protein from the soybean raw material. The conditions regarding the solvent, the amount of the solvent, the washing time, the temperature, and the stirring speed are the same as those specified for the above-mentioned extraction from the soybean raw material. After cleaning, solid-liquid separation is performed. It is preferable to use a centrifuge as the solid-liquid separation. In the case of two-layer separation, the oil layer (O2) is recovered as an OBAP fraction, and the aqueous layer (OA) is mixed with the solid-liquid separated intermediate layer (M) of the extract. In the case of three-layer separation, a mixture of the oil layer (O2) and the precipitate layer (OR) is recovered as an OBAP fraction, and the intermediate layer (OA) is mixed with the intermediate layer (M) from which the extract is solid-liquid separated. To do. The conditions for solid-liquid separation are the same as those specified for extraction from soybean raw materials described above.

<Re-extraction of sedimentation layer (R)>
In one embodiment, an aqueous solvent is added to the precipitate layer (R) and re-extraction is performed. The aqueous solvent used may be water or, as described above, a salt or one containing a salt and a reducing agent. For example, the same solvent used in the first extraction may be used. The conditions of the amount of solvent, the extraction time, the temperature, and the stirring speed at the time of re-extraction are the same as the conditions specified for the above-mentioned extraction from the soybean raw material. After re-extraction, solid-liquid separation is performed, the precipitate layer (R2) is used as an okara layer, and the aqueous layer (RA) is mixed with the intermediate layer (M). It is preferable to use a centrifuge as the solid-liquid separation. The conditions for solid-liquid separation are the same as those specified for extraction from soybean raw materials described above.

<Acid deposit of intermediate layer (M) (or (M')>
The intermediate layer (M) or the intermediate layer (M) was mixed with the aqueous layer (OA) from the washing of the oil layer (O) and / or the aqueous layer (RA) from the re-extraction of the precipitate layer (R). For the new intermediate layer (M'), the ionic strength is adjusted to the ionic strength specified in the extraction from the soybean raw material as it is or by adding a salt as needed, and then an acid is added to adjust the pH. Adjust to near the isoelectric point of soybean protein, for example, about 4 to about 6, about 4 to about 5, about 4.2 to 4.8, about 4.5. In certain embodiments, the ionic strength is adjusted to be comparable to the solvent used for extraction from the soybean raw material. In another embodiment, the reducing agent may be added to the concentration specified in the extraction from the soybean raw material together with the adjustment of the ionic strength. In a more specific embodiment, the reducing agent is derived from the soybean raw material. Adjust so that it is comparable to the solvent used for the extraction of.

The acid used for acid precipitation is not particularly limited, and organic acids and inorganic acids can be appropriately used. For example, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, lactic acid and the like can be mentioned.

After acid precipitation, solid-liquid separation is performed, and both the aqueous layer (MA) (both the aqueous layer separated from the intermediate layer (M) and the intermediate layer (M') are simply referred to as "aqueous layer (MA)". The same applies to the notation of each subsequent fraction) and the precipitate layer (MR). The precipitated layer (MR) is collected as a PLAP fraction. It is preferable to use a centrifuge as the solid-liquid separation. The conditions for solid-liquid separation are the same as those specified for extraction from soybean raw materials described above.

<Whey fraction and globulin fraction fraction>
In one embodiment, the acid-precipitated aqueous layer (MA) is diluted with water to sufficiently reduce the ionic strength so that all globulin precipitates, and if necessary, the pH is set near the isoelectric point of soy protein. For example, adjust to about 4 to about 6, about 4 to about 5, about 4.2 to 4.8, and about 4.5. It is preferable to use distilled water or pure water as the water referred to here, but the water is not limited to such high-purity water, and water containing other components such as salt may be used. Good. In a more specific embodiment, the pH is adjusted to be comparable to the pH adjusted by the acid precipitation of the intermediate layer (M) or (M'). The degree of dilution can be appropriately adjusted by those skilled in the art, but the ionic strength is about 0.15 mol / L or less, for example, about 0.12 mol / L or less, about 0.09 mol / L or less, about 0.08 mol / L or less, It is preferable to dilute to about 0.06 mol / L or less, about 0.05 mol / L or less, about 0.03 mol / L or less, and about 0.15 mol / L to about 0.03 mol / L. After dilution, the aqueous layer (W) is recovered as a whey fraction and the precipitated layer (G) is recovered as a globulin fraction by solid-liquid separation. It is preferable to use a centrifuge as the solid-liquid separation. The conditions for centrifugation are not limited, and examples thereof include about 1000 to about 5000 × g, about 1200 to about 4500 × g, about 1500 to about 3000 × g, and about 1800 × g. The time for centrifugation is also not limited, and examples thereof include about 1 minute to about 20 minutes, about 1.5 minutes to about 15 minutes, about 2.5 minutes to about 10 minutes, and about 5 minutes.

<Whey fraction and 7S and 11S fractions>
In one embodiment, the acid-precipitated aqueous layer (MA) is diluted with water, the ionic strength is adjusted so that only 11S of globulin precipitates, and the pH is adjusted near the isoelectric point of soy protein if necessary. For example, adjust to about 4 to about 6, about 4 to about 5, about 4.2 to 4.8, and about 4.5. It is preferable to use distilled water or pure water as the water referred to here, but the water is not limited to such high-purity water, and water containing other components such as salt may be used. Good. In a more specific embodiment, the pH is adjusted to be about the same as the pH adjusted by the acid precipitation of the intermediate layer (M). The degree of dilution can be appropriately adjusted by those skilled in the art, and for example, the ionic strength is about 0.16 mol / L to about 0.9 mol / L, for example, about 0.2 mol / L to about 0.6 mol / L, about 0. It is preferable to dilute to about 2 mol / L to about 0.5 mol / L and about 0.24 mol / L to about 0.4 mol / L. After dilution, the aqueous layer (W) and the precipitated layer (G) are recovered by solid-liquid separation, and the precipitated layer (G) is recovered as an 11S fraction. Further, the aqueous layer (W) is diluted with a solvent such as water to reduce the ionic strength necessary and sufficient so that all 7S precipitates, and if necessary, the pH is set near the isoelectric point of soybean protein, for example, about 4. Adjust to about 6 and about 4.5. In a more specific embodiment, the pH is adjusted to be about the same as the pH adjusted by the acid precipitation of the intermediate layer (M). The degree of dilution can be appropriately adjusted by those skilled in the art, and for example, the ionic strength is about 0.15 mol / L or less, for example, about 0.12 mol / L or less, about 0.09 mol / L or less, about 0.08 mol / L. Hereinafter, it is preferable to dilute the mixture so as to be about 0.06 mol / L or less, about 0.05 mol / L or less, about 0.03 mol / L or less, and about 0.15 mol / L to about 0.03 mol / L. After dilution, the supernatant (W2) and the precipitate layer (WG) are collected by solid-liquid separation, and the supernatant (W2) is collected as a whey fraction and the precipitate layer (WG) is collected as a 7S fraction. It is preferable to use a centrifuge as the solid-liquid separation. The conditions for centrifugation are not limited, and examples thereof include about 1000 to about 5000 × g, about 1200 to about 4500 × g, about 1500 to about 3000 × g, and about 1800 × g. The time for centrifugation is also not limited, and examples thereof include about 1 minute to about 20 minutes, about 1.5 minutes to about 15 minutes, about 2.5 minutes to about 10 minutes, and about 5 minutes.

<Evaluation of protein mass of each fraction>
The protein content of each fraction obtained above (OBAP fraction, PLAP fraction, whey fraction, globulin fraction, or 7S fraction and 11S fraction instead of globulin fraction) is measured. A person skilled in the art can appropriately select the method for measuring the protein mass. In order to make a relative comparison of the protein contents of each fraction, it is preferable to sample the extract from the soybean raw material before solid-liquid separation as the initial sample. For example, the weight of each fraction is measured and then dried. A person skilled in the art can appropriately select the drying method, and examples thereof include air drying, spray drying, and freeze drying. Freeze-drying is preferable because of less heat denaturation. After measuring the weight after drying, the nitrogen content of the powder after drying is measured and multiplied by a nitrogen protein conversion coefficient of 6.25 to calculate the protein content. For the nitrogen content, for example, the Dumas method, the Kjeldahl method, or the like can be used. The protein mass ratio can be evaluated by making a relative comparison of the protein content of each fraction when the initial sample is 100% from the wet weight, the water content, and the protein content in the dry powder of each fraction.

<Evaluation of 7S and 11S amounts in the globulin fraction>
When the above globulin fraction is obtained, the composition of 7S and 11S can be determined by, for example, the staining intensity ratio obtained by subjecting the globulin fraction to SDS-PAGE according to a conventional method and performing CBB staining or the like. This is because the dyeing intensities of CBB dyeing are the same for 7S and 11S.

In another aspect, the present invention relates to a method for analyzing a protein composition in soybean, which comprises measuring the protein mass of each fraction fractionated by the above fractionation method. In a further aspect, the present invention relates to a method for evaluating soybean varieties, which comprises determining the protein composition ratio in soybean seeds by measuring the protein mass of each obtained fraction.

By analyzing the protein composition of soybeans, it is possible to determine the physical characteristics (gelling ability, flavor, etc.) suitable for the intended soybean product, and to select soybean varieties with more suitable physical characteristics. In addition, the method of this embodiment can be used for breeding soybean varieties having the intended characteristics and for producing soybean varieties by genome editing.

In still another aspect, the present invention relates to each fraction fractionated by the above fractionation method. In this aspect, not only the previously known 7S, 11S and whey fractions, but also new soybean protein materials such as OBAP fraction and PLAP fraction are provided. The obtained fraction may be used as it is as a soybean protein material, or may be subjected to a treatment such as drying.

Hereinafter, embodiments of the present invention will be described more specifically by way of examples. Unless otherwise specified, the concentration, etc. is based on weight. For example, "%" means "weight% (w / w)" unless otherwise specified.

Example 1: Fraction 1 into 4 fractions
The soybean protein was separated into 4 fractions by the following operation. The outline is shown in FIG.

1. 1. Extraction The whole soybeans that had been molted and hypocotyled (variety: Fukuyutaka, NSI 90 or more, lipid content 21%) were crushed to obtain whole soybean flour. A 9-fold amount of buffer (1 M sodium sulfate, 0.1% sodium bisulfite) was added to the soybean powder and suspended. The ionic strength of the buffer at this time is 3 mol / L. 1N NaOH was added to adjust the pH to 8.0. The suspension was stirred at room temperature for 1 hour with a stirrer (200 rpm) for extraction. A part of the extract was sampled as the initial solution. The extract was centrifuged at 18,000 × g for 10 minutes and separated into an oil layer (O), an intermediate layer (M) and a precipitation layer (R). The oil reservoir (O) was recovered as an OBAP fraction.

2. 2. Fraction of PLAP 2N HCl was added to the obtained intermediate layer (M) to lower the pH to 4.5. Then, it was centrifuged at 18,000 × g for 10 minutes, and separated into an aqueous layer (MA) and a precipitate layer (MR). The precipitated layer (MR) was recovered as a PLAP fraction.

3. 3. Whey and globulin fraction (4 fractions)
The aqueous layer (MA) was diluted 100-fold with water (w / w) to reduce the ionic strength of the solvent to 0.03 mol / L, and then the pH was adjusted to 4.5 with 0.5N HCl. Then, the mixture was centrifuged at 1800 × g for 5 minutes, and the supernatant (W) was collected as a whey fraction and the precipitate (G) was collected as a globulin fraction.

4. Evaluation of Protein Mass of Each Fraction Each fraction (OBAP, PLAP, whey, globulin) recovered above was freeze-dried and dried. The weight before and after drying was measured. The nitrogen content of the dry powder was measured by the Dumas method and multiplied by a nitrogen protein conversion coefficient of 6.25 to calculate the protein content. From the wet weight, water content, and protein content in the dry powder of each fraction, the protein content of each fraction was compared relative to each other when the initial solution sample was 100%.

5. Quantification of 7S and 11S in the globulin fraction The globulin fraction was subjected to SDS-PAGE according to a conventional method. Commercially available ready-made gel (product name: "e-Pagel 12.5%", manufactured by Ato Co., Ltd.) and molecular weight marker (product name: "Precision Plus Protein (registered trademark) 2-color standard" manufactured by Bio-Rad) were used. .. The amounts of 7S and 11S globulin were evaluated based on the staining intensity ratio of CBB staining.

Example 2: Fraction 2 into 4 fractions
The soybean protein was separated into 4 fractions by the following operation. The outline is shown in FIG.

1. 1. Extraction The whole soybeans that had been molted and hypocotyled (same as in Example 1) were crushed to obtain whole soybean flour. A 9-fold amount of buffer (1 M sodium sulfate, 0.1% sodium bisulfite) was added to the soybean powder and suspended. The ionic strength of the buffer at this time is 3 mol / L. 1N NaOH was added to adjust the pH to 8.0. The suspension was stirred at room temperature for 1 hour with a stirrer (200 rpm) for extraction. A part of the extract was sampled as the initial solution. The extract was centrifuged at 18,000 × g for 10 minutes and separated into an oil layer (O), an intermediate layer (M) and a precipitation layer (R).

2. 2. Oil layer cleaning To the obtained oil layer (O), 4 times the amount of the buffer was added, and the mixture was stirred with a stirrer at room temperature for 10 minutes. Centrifugation was carried out at 18,000 × g for 10 minutes, and the mixture was separated into an oil layer (O2), an aqueous layer (OA) and a precipitation layer (OR). The oil layer (O2) and the precipitate layer (OR) were combined to form an OBAP fraction, and the aqueous layer (OA) was mixed with the intermediate layer (M).

3. 3. Precipitation layer re-extraction A 9-fold amount (w / w) of water was added to the precipitate layer (R) to suspend it, and the mixture was stirred at room temperature for 10 minutes with a stirrer. The suspension was centrifuged at 18,000 xg for 10 minutes and separated into an aqueous layer (RA) and a precipitate layer (R2). The aqueous layer (RA) was mixed with the intermediate layer (M).

4. Fractionation of PLAP Decreased by adding water during re-extraction of the precipitate layer to the obtained new intermediate layer (M') (mixture of intermediate layer (M), aqueous layer (OA), and aqueous layer (RA)). Sodium sulfate and sodium bisulfite were added to adjust the salt concentration so that the ionic strength was the same as in "1. Extraction". Add 2N HCl to lower the pH to 4.5, centrifuge at 18000 xg for 10 minutes, separate into aqueous layer (MA) and precipitate layer (MR), and recover the precipitate layer (MR) as a PLAP fraction. did.

5. Whey and globulin fraction (4 fractions)
The aqueous layer (MA) was diluted 100-fold with water (w / w) to reduce the ionic strength of the solvent to 0.03 mol / L, and then the pH was adjusted to 4.5 with 0.5N HCl. Then, the mixture was centrifuged at 1800 × g for 5 minutes, and the supernatant (W) was collected as a whey fraction and the precipitate layer (G) was collected as a globulin fraction.

6. Evaluation of Protein Mass of Each Fraction Each fraction (OBAP, PLAP, whey, globulin) recovered above was freeze-dried and dried. The weight before and after drying was measured. The nitrogen content of the dry powder was measured by the Dumas method and multiplied by a nitrogen protein conversion coefficient of 6.25 to calculate the protein content. From the wet weight, water content, and protein content in the dry powder of each fraction, the protein content of each fraction was compared relative to each other when the initial solution sample was 100%.

7. Quantification of 7S and 11S in the globulin fraction The globulin fraction was subjected to SDS-PAGE according to a conventional method. Commercially available ready-made gel (product name: "e-Pagel 12.5%", manufactured by Ato Co., Ltd.) and molecular weight marker (product name: "Precision Plus Protein (registered trademark) 2-color standard" manufactured by Bio-Rad) were used. .. The amounts of 7S and 11S globulin were evaluated based on the staining intensity ratio of CBB staining.

FIG. 3 shows the SDS-PAGE results of each fraction and the initial solution sample fractionated from the soybean seeds of the cultivar Fukuyutaka by the method of Example 2 above. A 24 kDa band indicating oleocin can be confirmed in the OBAP fraction. As for the globulin fraction, bands corresponding to 7S and 11S and their subunits can be confirmed, and the amount of 7S and 11S globulin can be calculated from the intensity ratio thereof.

Example 3: Examination of ionic strength at the time of fractionation of whey and globulin In Example 2 and 5. As shown in the table below, the ionic strength at the time of dilution during the fractionation of whey and globulin (4 fractions) is fractionated without dilution and adjusted between 1.2 mol / L and 0.03 mol / L. , The obtained whey fraction was subjected to SDS-PAGE and CBB staining was performed. The results are shown in FIG.

From FIG. 4, it was confirmed that most of 7S and 11S were transferred from the whey fraction to the globulin fraction in lanes 7 to 13, that is, when the ionic strength was 0.15 mol / L or less. On the other hand, in lanes 1 to 6 (ionic strength 0.2 mol / L or more), most of 7S was retained in the whey fraction. On the other hand, 11S gradually began to shift from the whey fraction to the globulin fraction by dilution, and most of 11S moved to the globulin fraction in lane 5 (ionic strength 0.24 mol / L).

Example 4: Fractionation into 5 fractions Soybean protein was separated into 5 fractions by the following operation. The outline is shown in FIG.

1. 1. Extraction-4. Up to the fractionation of PLAP, fractionation was performed in the same procedure as in Example 2 above.

5. Fractionation of whey and globulin The aqueous layer (MA) was diluted 15-fold with water (w / w) to reduce the ionic strength of the solvent to 0.2 mol / L, and then the pH was adjusted to 4.5 with 0.5N HCl. Adjusted to. Then, the mixture was centrifuged at 1800 × g for 5 minutes, and the precipitate (G) was collected as an 11S fraction. Then, the aqueous layer (W) was diluted 2.22 times with water (w / w) to further reduce the ionic strength of the solvent to 0.09 mol / L, and then the pH was adjusted to 4.5 with 0.5N HCl. did. Then, the mixture was centrifuged at 1800 × g for 5 minutes, and the aqueous layer (W2) was recovered as a whey fraction and the precipitate layer (WG) as a 7S fraction.

6. Evaluation of Protein Mass of Each Fraction Each fraction (OBAP, PLAP, 7S, 11S, whey) recovered above was freeze-dried and dried. The weight before and after drying was measured. The nitrogen content of the dry powder was measured by the Dumas method and multiplied by a nitrogen protein conversion coefficient of 6.25 to calculate the protein content. From the wet weight, water content, and protein content in the dry powder of each fraction, the protein content of each fraction was compared relative to each other when the initial solution sample was 100%.

By this method, the protein composition of soybean samples could be evaluated by measuring the nitrogen content.

Example 5: Difference in protein composition depending on soybean varieties In addition to Fukuyutaka used in Example 1, each variety of Sato no smile, Tachinagaha, and 11S-deficient soybean was fractionated by the method of Example 3 to determine the composition ratio. It was. The results are shown in FIG. It was shown that the composition of the five protein fractions differs depending on the soybean variety. The result was that the 11S-deficient soybean had a larger 7S, whey, and PLAP fraction than other varieties. It was suggested that when 11S, which is a stored protein of soybean and occupies most of the soybean protein, is deficient, only 7S, which is the other stored protein, does not necessarily increase. Fukuyutaka, Sato no Smile, and Tachinagaha are all varieties that are widely distributed in Japan, but there are differences not only in the ratio of 7S and 11S but also in the amount of whey and OBAP. As described above, it is considered that the present invention can clarify the difference in protein composition between soybean varieties, and can be used for selection of varieties having the required quality and evaluation of new varieties produced by breeding.

Claims

The following steps:
(A) Prepare a soybean raw material having a lipid content of 15% by weight or more and a water-soluble nitrogen index (NSI) of 80 or more per dry matter;
(B) A solvent having an ionic strength of 1.5 mol / L or more is added to the soybean raw material to extract the protein;
(C) The extracted protein is solid-liquid separated and separated into an oil layer (O), an intermediate layer (M), and a precipitation layer (R);
(D) Acid is added to the intermediate layer (M) to lower the pH to 6 or less;
(E) The pH-lowered intermediate layer (M) is solid-liquid separated and separated into an aqueous layer (MA) and a precipitated layer (MR);
(F) Dilute the aqueous layer (MA) with water;
(G) The diluted aqueous layer (MA) is solid-liquid separated and separated into an aqueous layer (W) and a precipitated layer (G);
How to fractionate soy protein, including.
Between steps (C) and (D), the following steps:
(A) A solvent having an ionic strength of 1.5 mol / L or more is added to the oil layer (O) obtained in the step (C), and the aqueous layer (OA) and the oil layer (O2) or the oil layer (O2) are separated by solid-liquid separation. ) And the sedimentation layer (OR);
(B) An aqueous solvent is added to the precipitate layer (R) obtained in the step (C) to extract the protein, and the protein is separated into an aqueous layer (RA) and an okara layer (R2) by solid-liquid separation;
(C) The intermediate layer (M), the aqueous layer (OA) and the aqueous layer (RA) are mixed to form a new intermediate layer (M'), and the intermediate layer (M') is used as the intermediate layer (M) in the subsequent steps. To use,
The method according to claim 1, further comprising.
After step (G), the following steps:
(D) Dilute the aqueous layer (W) obtained in step (G) with water;
(E) The diluted aqueous layer (W) is solid-liquid separated and separated into an aqueous layer (W2) and a precipitation layer (WG).
The method according to claim 1 or 2, further comprising.
The method according to any one of claims 1 to 3, wherein the solvent in the step (B) further contains a reducing agent.
The method according to any one of claims 1 to 4, wherein the water-soluble nitrogen index (NSI) of the step (A) is 90% or more.
The method according to any one of claims 1 to 5, wherein the dilution of the aqueous layer in step (G) has an ionic strength of 150 to 30 mM.
The method according to any one of claims 3 to 6, wherein the dilution of the aqueous layer in step (d) has an ionic strength of 150 to 30 mM.
A method for analyzing a protein composition in soybean, which comprises measuring the protein mass of a fraction obtained by the method according to any one of claims 1 to 7.
A soybean variety including obtaining a protein composition ratio in soybean seeds by measuring the protein mass of each fraction obtained by the method according to any one of claims 1 to 7 from a soybean seed raw material. Evaluation method.