JPH11266791A - Polypeptide mixture, emulsifier and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a polypeptide mixture usable in fields of a food, a cosmetic, a medicine and the like, and further to obtain an emulsifier and to provide its production method. SOLUTION: This polypeptide mixture contains >=20% polypeptide derived from soybean glycinin based on a crude protein, and consists essentially of a protein having an N-terminal amino acid sequence of the formula; Phe-Leu- Glu-His-Ala-and about 9,000 molecular weight (measured by SDS-polyacrylamide electrophoresis). The method for producing the polypeptide mixture comprises concentration of the polypeptide to a concentration of >=20% of the crude protein by fractionating the polypeptide formed by subjecting the soybean glycinin to degradation by protease and having the N-terminal amino acid sequence of the formula; Phe-Leu-Glu-His-Ala-and about 9,000 molecular weight (measured by SDS-polyacrylamide electrophoresis), at pH 4-6.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a polypeptide mixture, an emulsifier, and a method for producing the same, which can be used in the fields of foods, cosmetics, pharmaceuticals, and the like.

[0002]

2. Description of the Related Art Conventionally, emulsions have been produced by using natural or synthetic emulsifiers such as glycerol fatty acid ester, phospholipid, sorbitan fatty acid ester and sucrose fatty acid ester as emulsifiers, or protein such as soy protein, milk protein and wheat protein. Based emulsifiers have been used. These commonly used emulsifiers are not suitable for the production of food emulsions from the viewpoint of flavor, or protein emulsifiers generally have weak emulsifying power and need to be used in combination with other emulsifiers. Was.

[0003] In recent years, natural emulsifiers, especially protein emulsifiers, have attracted attention because of their emphasis on health and safety. For milk proteins, a method using a polypeptide having a specific amino acid sequence (JP-A-58-174232) and A specific fraction obtained from buttermilk (JP-A-8-51934) is known, and a specific partial decomposition product of wheat protein (JP-A-64-14274) is known. I have. For soybean protein, a method of enzymatic degradation under specific conditions (JP-A-56-26171, JP-A-57-16674, JP-A-6-197788)
It has been known. Further, a method using a glycinin acidic subunit which is a component of soybean protein (Japanese Patent Application Laid-Open No. 63-36748) and a method using a glycinin basic subunit (Japanese Patent Application Laid-Open No. 9-23837) are also known. However, it is essential that all of them are separated into subunit components, and their emulsifying power is insufficient in an acidic region.

[0004]

SUMMARY OF THE INVENTION In view of the above circumstances, an object of the present invention is to provide a polypeptide mixture, an emulsifier, and a method for producing the same which can be used in the fields of foods, cosmetics, pharmaceuticals, and the like.

[0005]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a specific polypeptide obtained by allowing a protease to act on soybean protein, particularly glycinin, has excellent emulsifying power. Thus, the present invention has been completed.

That is, one aspect of the present invention is a polypeptide mixture containing soybean glycinin-derived polypeptide in an amount of 20% or more of the crude protein. In particular, the soybean glycinin-derived polypeptide has an N-terminal amino acid sequence Phe-Leu -Glu-His-Al
It is preferably a polypeptide mixture containing a- as the main component a polypeptide having a molecular weight of about 9000 (by SDS-polyacrylamide electrophoresis). Another aspect of the present invention is an emulsifier containing the above-mentioned polypeptide mixture as an active ingredient. The other one of the present invention relates to a method for producing the above-mentioned polypeptide mixture, and an N-terminal amino acid sequence Phe-Leu-Glu-His-Ala generated by decomposing soybean glycinin with a protease.
-It is characterized in that a polypeptide having a molecular weight of about 9000 (by SDS-polyacrylamide electrophoresis) is fractionated at pH = 4 to 6 to concentrate the polypeptide to 20% or more of the crude protein. And a method for producing a polypeptide mixture.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The polypeptide mixture of the present invention comprises:
N-terminal amino acid sequence Phe-Leu-Glu-His-Ala-, produced by decomposing soybean glycinin with protease, molecular weight about 9000
Polypeptide mixtures that are (by SDS-polyacrylamide electrophoresis) are suitable.

[0008] The polypeptide mixture of the present invention has an N-terminal amino acid sequence Phe-Leu-Glu-His-Ala- produced by protease degradation of soybean glycinin, and a molecular weight of about 9000.
This is a polypeptide mixture in which the polypeptide (by SDS-polyacrylamide electrophoresis) is concentrated to 20% or more of the crude protein. It is convenient and preferable to concentrate the polypeptide at a pH of 4 to 6, preferably at a pH of 4.5 to 5.5. Other known methods, for example, gel filtration, ion exchange chromatography, isoelectric focusing, adsorption, and other separation techniques can be used in combination.

The emulsifier of the present invention contains the above-mentioned polypeptide as an active ingredient, and usually contains at least 20%.
If it is less than that, the function as an emulsifier is inferior.

[0010] The polypeptide of the present invention can be produced by using a soybean protein containing glycinin as a substrate and decomposing it with a protease. The polypeptide mixture of the present invention can be prepared from soybean protein containing glycinin, preferably by a known method (for example, the method of Thanh and Shibasaki; J. Agric. Food Chem., 2).
Glycinin is fractionated or concentrated in advance as a substrate, and a protease is used in a concentration of 0.001 to 15% for a 0.5% to 15% solution thereof and a protease for a substrate solid content of 0.001 to 15%.
1%, preferably in the range of 0.01-0.5%,
5 to 4 hours, preferably 10 minutes to 90 ° C., preferably 30 to 60 ° C., pH = 1 to 5, preferably pH = 2 to 4.
It can be carried out by reacting for 2 hours. If the reaction time is too long, the amount of the polypeptide having a molecular weight of about 9000 decreases, and the emulsifying performance decreases. The protease used here is pH = 1 ~
5, preferably all proteases exhibiting activity at pH = 2-4 are suitable, and commercially available enzyme preparations such as animal-derived pepsin and cathepsin and a series of microorganism-derived aspartic proteases can also be used. Pepsin is preferably used as an enzyme capable of producing a large amount of a polypeptide having the above properties.

To separate or concentrate the desired polypeptide from the reaction solution after the completion of the enzymatic reaction, pH fractionation is simple and preferred, but other known methods such as gel filtration, ion exchange chromatography, and isoelectric point It is possible to perform the separation by a combination of separation techniques such as electrophoresis and adsorption. If necessary, further sterilization and drying are performed.

The polypeptide mixture of the present invention can be effectively used in the preparation of an oil-in-water emulsified composition containing water and oils and fats in which oil droplets are emulsified and dispersed. The oil used in the preparation of the oil-in-water emulsion composition is not particularly limited as long as it forms an oil-in-water emulsion, and various oils and fats can be used. Fats, fish oil, soybean oil, rapeseed oil, coconut oil, palm oil and the like, and their hardened oils, fractionated oils, transesterified oils, etc., as well as silicone oils, perfume oils, machine oils, petroleum fractions, etc.

The composition ratio of the aqueous phase / oil phase of the oil-in-water emulsion composition obtained by using the polypeptide mixture of the present invention is not particularly limited, but is preferably 2/8 to 9/1, and more preferably 3/7 to 8.
It is preferable to prepare so as to have a ratio (weight ratio) of / 2. The amount of the polypeptide mixture of the present invention varies depending on the composition ratio of the water phase relative to the oil phase of the emulsified composition and the type of the oil phase.
It is preferably in the range of 05 to 5% by weight.

The polypeptide mixture of the present invention may be used in combination with a surfactant which has been conventionally used for the purpose of stabilizing emulsification. For example, natural or synthetic emulsifiers such as glycerol fatty acid ester, phospholipid, sorbitan fatty acid ester, sucrose fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene sorbitan ester, alkylamine derivative, alkyl alcohol, alkyl sugar derivative and the like can be mentioned. it can. Further, proteins and polysaccharides other than the polypeptide emulsifier of the present invention may be added for the purpose of imparting viscoelasticity by assisting emulsification. Examples of these proteins include soy protein, wheat protein,
Examples thereof include proteins derived from animals and plants, such as milk protein, egg white, egg yolk, blood protein, fish meat protein, animal meat protein, and degradation products thereof. It is optional to use a known pH stabilizer or protein solubilizer, for example, inorganic phosphates, organic acids, EDTA, and the like.

The preparation of an emulsified composition using the polypeptide mixture of the present invention can be carried out by a known emulsification or homogenization method. The temperature of emulsification may depend on the nature of the oil phase component, but is generally from 10 to 100 ° C, preferably from 20 to 90 ° C, as long as the function of the polypeptide which is the main component of the emulsifier is not deteriorated.

The polypeptide mixture of the present invention may be used in the preparation of an oil-in-water emulsion composition, for example, emulsified foods such as creams, mayonnaise, dressings, pastes, emulsified flavors, enteral nutrition, cosmetic emulsions Etc. can be used.

[0017]

EXAMPLES Hereinafter, the embodiments of the present invention will be described in detail with reference to examples, but the technical scope of the present invention is not limited by these.

Test Example 100 ml of a 2% by weight solution of glycinin obtained by the method of Thanh and Shibasaki was adjusted to pH 2 and 0.05% per solid weight.
Of pepsin (manufactured by Sigma) was added, and the enzyme reaction was carried out at 37 ° C. and 55 ° C. Each reaction solution was adjusted to pH 4.5, and the centrifuged supernatant was freeze-dried to obtain a freeze-dried product. The lyophilized product was analyzed by SDS-electrophoresis, and the content of polypeptide having a molecular weight of about 9000 (determined by densitometer) (expressed in the amount of crude protein) and emulsifying activity (J. Agric. Food Chem., 26, 716 (1978)) Was evaluated and described in the following table. Emulsifying activity is pH =
4. 1 ml of soybean oil was added to 3 ml of a sample solution (0.5% by weight) adjusted to pH = 5.5 and pH = 7, an emulsion was prepared using an ultrasonic disperser, and diluted 2000-fold with a 0.1% SDS solution to obtain a solution. Turbidity (absorbance at 500 nm) was measured. It can be seen that the greater the content of the polypeptide having a molecular weight of about 9000, the better the emulsifying power and the relatively short reaction time is preferable.

Table II-1 Reaction conditions Polypeptide Emulsifying power (absorbance at 500 nm) Temperature Time Content (%) pH = 5.5 pH = 7 ────── ────── ──────────── 37 ℃ 0.5 hr 33 0.58 0.63 37 ℃ 2 hr 25 0.48 0.55 37 ℃ 22 hr 7 0.24 0.39 55 ℃ 0.5 hr 28 0.43 0.63 55 ℃ 2 hr 12 0.27 0.35 55 ℃ 22 hr 4 0.08 0.15 ──────────────────────── ───────

Example 1 100 ml of a 2% by weight solution of glycinin obtained by the method of Thanh and Shibasaki was adjusted to pH 2 and 37 ° C.
0.05% pepsin (manufactured by Sigma) was added, and an enzyme reaction was performed for 30 minutes. The reaction solution was adjusted to pH 4.5, and the centrifuged supernatant was adjusted to pH 6, and lyophilized to obtain 1 g of a lyophilized product.
40 ml of a 1% by weight solution (pH 6) of the lyophilized product was subjected to isoelectric focusing using a preparative isoelectric focusing apparatus (trade name: Rotophore, manufactured by Bio-Rad). Fractions migrated in the fractions having a pH of 4.5 to 5.5 were collected and freeze-dried to obtain a polypeptide mixture (200 mg).

When the above polypeptide mixture was analyzed by SDS-electrophoresis, the polypeptide having a molecular weight of about 9000 occupied 80% (quantification by densitometer).
Approximately 9,000 molecular weight band from SDS-electrophoresed gel
(Polyvinylidine difluoride) Transferred to a membrane and examined the amino acid sequence of 5 residues from the N-terminal of these polypeptides with a protein sequencer, it was Phe-Leu-Glu-His-Ala- It was a polypeptide derived from the subunit.

Example 2 and Comparative Example 1 The emulsifying power of the polypeptide mixture obtained in Example 1 was evaluated by a turbidity method (see the method of J. Agric. Food Chem., 26, 716 (1978)). That is, 0.25 ml of soybean oil was added to 1 ml of a polypeptide solution (0.05% by weight) adjusted to pH = 4, pH = 5.5 and pH = 7.
Was added thereto, and an emulsion was prepared using an ultrasonic disperser. The emulsion was diluted 500 times with a 0.1% SDS solution, and the turbidity of the solution was measured (absorbance at 500 nm). As a comparison, the emulsifying power of the freeze-dried product of the pepsin reaction solution of Example 1 (content of polypeptide having a molecular weight of 9000, 16%; Comparative Example 1) was evaluated in the same manner as described above.

Table II-2 Emulsifying power (absorbance at 500 nm) pH = 4 pH = 5.5 pH = 7 ───────────────────────────── Polypeptide (Example 2) 0.50 0.50 0.20 Lyophilized reaction solution (Comparative Example 1) 0.10 0.03 0.04 ─────────────────────────────

The polypeptide had a high emulsifying power.

Example 3 and Comparative Examples 2, 3 and 4 The preparation of a mayonnaise-like dressing was attempted using the polypeptide mixture of Example 1 as an emulsifier. For comparison, a lyophilized product of the reaction solution described above (Comparative Example 2), JP-A-63-36748
The glycinin acidic subunit prepared by the method of JP-A-9-23837 (Comparative Example 3) and the glycinin basic subunit prepared by the method of JP-A-9-23837 (Comparative Example 4) were compared as samples. For the preparation of the dressing, the following ingredients except for the salad oil were mixed and then emulsified while adding the salad oil to prepare a mayonnaise-like dressing.

Table 3 (Mixed table of mayonnaise-like dressing) 60 Salad oil 60 parts Vinegar 15 parts Sample 2 parts Seasoning 4 parts Spice 1 part Water 18 parts ────── ─────

Table 4 Average particle size Example 3 (polypeptide emulsifier of the present invention) 2 μm Comparative Example 2 (lyophilized reaction solution) 10 μm Comparative Example 3 (glycinin acidic subunit) 70 μm Comparative Example 4 (glycinin basic subunit) Separation ────────────────── ────────

Only the product of the present invention showed a mayonnaise-like shape, and the others were only separated or soft. A similar effect was also observed in the comparison of particle diameters, and it was found that the product of the present invention could obtain sufficient quality as a mayonnaise-like dressing.

Example 4 and Comparative Examples 5, 6 and 7 The preparation of a coffee cream-like emulsion was attempted using the polypeptide mixture of Example 1 as an emulsifier. For comparison, a lyophilized product of the reaction solution described above (Comparative Example 5), JP-A-63-36748
The glycinin acidic subunit prepared by the method of JP-A-9-23837 (Comparative Example 6) and the glycinin basic subunit prepared by the method of JP-A-9-23837 (Comparative Example 7) were compared as samples. Coffee cream was prepared by emulsifying the following formulation at 60 ° C. with an ultrasonic disperser.

Table 5 (combination table of coffee cream-like emulsion) 硬化 Hardened rapeseed oil 30 parts Sample 4 parts Water 66 parts ───────────

Commercial instant coffee (Nestlé) 30
g and 50 g of sugar are dissolved in 1 L of water (pH is adjusted to 7).
About 10 ml of the above coffee cream-like emulsion was added to 100 ml of the coffee liquid heated to ~ 85 ° C, and the mixture was stirred and heated at 120 ° C for 10 minutes in an autoclave. The emulsified state after heating was observed.

FIG.─6 ──────────────────────────Emulsified state──────────────── ────────── Example 3 (polypeptide emulsifier of the present invention) Good Comparative Example 5 (lyophilized product of reaction solution) Slightly separated Comparative Example 6 (glycinin acidic subunit) Completely separated Comparative Example 7 Glycinin basic subunit) Completely separated ──────────────────────────

It can be seen that only the product of the present invention can maintain a good emulsified state and is excellent in heat resistance.

[0034]

As described above, according to the present invention, it is possible to provide a polypeptide mixture obtained from soy protein which can be used in the fields of foods, cosmetics, pharmaceuticals and the like.

[0035]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Phe Leu Glu His Ala 15

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 14/415 C07K 14/415 // A23C 11/06 A23C 11/06 A23F 5/24 A23F 5/24 A23L 1/24 A23L 1 / 24 A (72) Inventor Wataru Kugimiya 4-3, Kinokudai, Taniwahara-mura, Tsukuba-gun, Ibaraki Prefecture Inside the Tsukuba Research and Development Center, Fuji Oil Co., Ltd. (72) Kumiko Hoshino 4-3-1 Kinokudai, Yawahara-mura, Tsukuba-gun, Ibaraki Prefecture Fuji Oil Co., Ltd. Tsukuba R & D Center

Claims (5)

[Claims] 1. A polypeptide mixture containing at least 20% of a polypeptide derived from soybean glycinin in a crude protein. 2. The polypeptide derived from soybean glycinin is N-type.
2. The polypeptide mixture according to claim 1, comprising a polypeptide having a terminal amino acid sequence of Phe-Leu-Glu-His-Ala- as a main component. 3. The polypeptide derived from soybean glycinin has a molecular weight of about 9000 (by SDS-polyacrylamide electrophoresis).
3. The method according to claim 1, wherein the polypeptide is a main component.
A polypeptide mixture as described. 4. An emulsifier comprising the polypeptide mixture according to claim 1 as an active ingredient. 5. The N-terminal amino acid sequence formed by protease degradation of soybean glycinin is Phe-Leu-Glu-His-Ala-,
Further, by fractionating a polypeptide having a molecular weight of about 9000 (by SDS-polyacrylamide electrophoresis) at pH = 4 to 6, the polypeptide is concentrated to 20% or more of the crude protein. Method for producing a mixture of polypeptides.