JPH11221024A - Polypeptide, polypeptide mixture and emulsifier - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an emulsifier with excellent emulsifying power, enabling a stable emulsion to be prepared in the field such as of foods, cosmetics and medicines and the like. SOLUTION: This emulsifier consists of/or contains a polypeptide with a molecular weight of <=8,000 (determined by SDS-polyacrylamide electrophoresis) having an N-terminal amino acid sequence: Asn-Phe-Leu-Ala-Gly-Ser-Gln-Asp- Asn-Val-Ile-Ser-Gln-Ile-Pro-Ser-Gln-Val-Gln-Glu-Leu-Ala-Phe which is obtained, for example, by making a protease act on soybean β-conglycinin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a polypeptide which can be used in the preparation of an emulsion in the fields of foods, cosmetics, pharmaceuticals and the like, and an emulsifier using the polypeptide.

[0002]

2. Description of the Related Art Soybean protein has been used as an excellent food material because it has not only high nutritional value but also various functional properties such as emulsifying property, gel forming property and water retention property. Conventionally, in the production of emulsions, natural or synthetic emulsifiers such as glycerol fatty acid ester, phospholipid, sorbitan fatty acid ester and sucrose fatty acid ester, or protein-based emulsifiers such as soy protein, milk protein and wheat protein are used as emulsifiers. However, these commonly used synthetic emulsifiers are not suitable for the production of food emulsions from the viewpoint of flavor, and furthermore, protein-based emulsifiers generally have weak emulsifying power, and require the use of other emulsifiers in combination. .

[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), 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)
Etc. are known.

[0004]

SUMMARY OF THE INVENTION In view of the above circumstances, an object of the present invention is to provide an emulsifier having excellent emulsifying power and capable of preparing a stable emulsion in the fields of foods, cosmetics and pharmaceuticals.

[0005]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have found that soybean proteins, particularly β-
Obtained by allowing protease to act on conglycinin,
The present inventors have found that a specific polypeptide is excellent in emulsifying power and can prepare a stable emulsion, and have completed the present invention.

[0006] That is, the present invention relates to a method wherein the N-terminal amino acid sequence is As
n-Phe-Leu-Ala-Gly-Ser-Gln-Asp-Asn-Val-Ile-Ser-Gln-
Ile-Pro-Ser-Gln-Val-Gln-Glu-Leu-Ala-Phe- with molecular weight 8
000 or less polypeptide. Further, the present invention, the N-terminal amino acid sequence is Asn-Phe-Leu-Ala-Gly-Ser-Gln-Asp-As
n-Val-Ile-Ser-Gln-Ile-Pro-Ser-Gln-Val-Gln-Glu-Leu-
A polypeptide mixture containing 30% or more of Ala-Phe- and a polypeptide having a molecular weight of 8000 or less. Further, the present invention, the N-terminal amino acid sequence is Asn-Phe-Leu-Ala-Gly-Ser-Gln-Asp-As
n-Val-Ile-Ser-Gln-Ile-Pro-Ser-Gln-Val-Gln-Glu-Leu-
An emulsifier containing Ala-Phe- and a polypeptide having a molecular weight of 8000 or less. N-terminal amino acid sequence is Asn-Phe-Leu-Ala-Gl
y-Ser-Gln-Asp-Asn-Val-Ile-Ser-Gln-Ile-Pro-Ser-Gln-
An emulsifier containing 30% or more of a Val-Gln-Glu-Leu-Ala-Phe-polypeptide having a molecular weight of 8000 or less is preferable.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The polypeptide of the present invention can be obtained as a specific polypeptide by allowing a protease to act on soybean protein, particularly β-conglycinin which is a so-called 7S fraction. Alternatively, the peptide can be synthesized by amino acid synthesis. The molecular weight of the polypeptide is 8000
The following (by SDS-polyacrylamide electrophoresis) is appropriate.

[0008] The N-terminal amino acid sequence is Asn-Phe-
Leu-Ala-Gly-Ser-Gln-Asp-Asn-Val-Ile-Ser-Gln-Ile-Pr
o-Ser-Gln-Val-Gln-Glu-Leu-Ala-Phe-.

[0009] The polypeptide mixture of the present invention contains the polypeptide in an amount of 30% or more. The emulsifier of the present invention contains the above-mentioned polypeptide, and its content is suitably 30% or more. Below this, the function as an emulsifier is inferior.

An example of a method for producing the polypeptide, polypeptide mixture and emulsifier of the present invention will be described.

The polypeptide of the present invention is prepared by adding an oil or fat to a hydrolyzate obtained by allowing a protease to act on β-conglycinin to prepare an emulsion, and concentrating the polypeptide into the emulsion. By removing oils and fats from the resulting emulsion, the polypeptide can be separated and produced.

The polypeptide of the present invention can be decomposed with a protease using soybean protein containing β-conglycinin as a substrate. The protease used here is
There is no particular limitation, and papain, pepsin, trypsin, subtilisin and the like can be used, but papain is preferably used as an enzyme capable of producing a large amount of a polypeptide having the above-mentioned properties.

The polypeptide used as the emulsifier of the present invention is a soybean protein containing β-conglycinin, preferably a known method (eg, the method of Thanh and Shibasaki; J. Agri
c. Food Chem., 24, 1117 (1976) can be exemplified) and β-conglycinin fractionated or concentrated is used as a substrate.
For a 0.5% to 15% solution, the protease is added in a range of 0.001 to 1%, preferably 0.01 to 0.5%, based on the substrate solid content, and at 20 to 90 ° C, preferably 30 to 80 ° C. Stay
The reaction can be carried out by reacting at pH = 5 to 10, preferably at pH = 6 to 9, for 5 minutes to 2 hours, preferably for 10 minutes to 1 hour. still,
The reaction conditions are optimal temperature, optimal pH and stable pH of the enzyme agent used.
And the production amount of the target polypeptide.

After completion of the enzymatic reaction, to separate or concentrate the desired polypeptide from the reaction solution, pH fractionation, gel filtration, ion exchange chromatography, isoelectric focusing,
Known separation methods such as an adsorption method can be used in combination, and sterilization and drying are performed if necessary.

The emulsifier of the present invention contains the above-mentioned polypeptide (preferably at least 30% by weight).

The emulsifier of the present invention can be used effectively in the preparation of an oil-in-water emulsion 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 oil phase relative to the water in the oil-in-water emulsion composition obtained by using the emulsifier of the present invention is not particularly limited, but is preferably 2/8 to 9/1, and more preferably 3/7 to 8/2. Ratio (weight ratio)
It is preferable to prepare so that The addition amount of the polypeptide emulsifier 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, but is usually 0.01 to 10% by weight, preferably 0.05 to 5% by weight based on the total amount. It is preferably within the range.

The polypeptide emulsifier of the present invention may be used in combination with a surfactant 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 emulsifier of the present invention can be carried out by a known emulsification or homogenization method. The temperature of emulsification may depend on the properties 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 reduced.

The polypeptide emulsifier 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.

[0021]

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.

Example 1 Thanh and Shibasaki, J. Agric. Food Chem., 24, 1117 (197
6 ml of a 2% by weight solution of soybean β-conglycinin obtained according to the method of 6) was adjusted to pH 7 and 70 ° C.
01% papain (manufactured by Sigma) was added, and an enzyme reaction was performed for 30 minutes. The reaction solution was heated at 100 ° C for 5 minutes to inactivate the enzyme, adjusted to pH 5, and the centrifuged supernatant was lyophilized to obtain 200 mg of a lyophilized product. 0.5% by weight solution of lyophilizate (pH
5) 13.3 ml of soybean oil was added to 40 ml, and an emulsion was prepared with an ultrasonic dispersing machine. The emulsion was centrifuged, separated into an aqueous layer and an emulsified layer, and the emulsified layer was recovered. The emulsified layer was washed with a chloroform-methanol (2: 1) solvent and air-dried to obtain 70 mg of a solid (polypeptide mixture). (3.5% yield)

When the above polypeptide mixture was analyzed by SDS-electrophoresis, a polypeptide having a molecular weight of 5,000 to 6,000 was found.
It accounted for 50% (quantification by densitometer). When these 5000 to 6000 bands were transferred to a PVDF membrane from the gel subjected to SDS-electrophoresis, the N-terminal amino acid sequences of these polypeptides were examined with a protein sequencer.
Both are Asn-Phe-Leu-Ala-Gly-Ser-Gln-Asp-Asn-Val-Il
e-Ser-Gln-Ile-Pro-Ser-Gln-Val-Gln-Glu-Leu-Ala-Phe-
The polypeptide was composed of

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, soybean oil was added to 1 ml of a 0.05% by weight solution (pH 7) at a concentration of 0.1%.
Add 25 ml and prepare an emulsion with an ultrasonic disperser, 0.1% SDS
After diluting 500 times with the solution, the solution turbidity was measured (absorbance at 500 nm). For comparison, the pH of the papain reaction solution of Example 1 was
5 The emulsifying power of the dried supernatant (hereinafter referred to as Comparative Example 1) was evaluated in the same manner as described above.

Emulsifying power (absorbance at 500 nm) Polypeptide mixture (Example 2) 0.35 pH5 dried supernatant (Comparative example 1) 0.18 The polypeptide mixture had high emulsifying power.

Example 3 A polypeptide having 23 residues corresponding to the N-terminal amino acid sequence of the polypeptide mixture obtained in Example 1 was synthesized using a peptide synthesizer. Using this synthetic peptide, the emulsifying power was evaluated by the method of Example 2 (except that 1 ml of a 0.02% by weight solution (pH 7) was used), and the emulsifying power (absorbance at 500 nm) was 0.75. Thus, 23 residues corresponding to the N-terminal amino acid sequence were essential for high emulsifying power.

[0027]

According to the present invention, foods, cosmetics,
In the field of pharmaceuticals and the like, polypeptides, polypeptide mixtures and emulsifiers that can be used in preparing emulsions can be provided.

[0028]

[Sequence list] SEQ ID NO: 1 Sequence length: 23 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asn Phe Leu Ala Gly Ser Gln Asp Asn Val 1 5 10 Ile Ser Gln Ile Pro Ser Gln Val Gln Glu 11 15 20 Leu Ala Phe 21 23

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI B01F 17/30 B01F 17/30 C07K 14/42 ZNA C07K 14/42 ZNA

Claims (4)

[Claims] (1) the amino acid sequence of the N-terminal is Asn-Phe-Leu-Ala-Gl;
y-Ser-Gln-Asp-Asn-Val-Ile-Ser-Gln-Ile-Pro-Ser-Gln-
Val-Gln-Glu-Leu-Ala-Phe- and a polypeptide having a molecular weight of 8000 or less. 2. The N-terminal amino acid sequence is Asn-Phe-Leu-Ala-Gl
y-Ser-Gln-Asp-Asn-Val-Ile-Ser-Gln-Ile-Pro-Ser-Gln-
A polypeptide mixture containing 30% or more of Val-Gln-Glu-Leu-Ala-Phe- and a polypeptide having a molecular weight of 8000 or less. 3. The method according to claim 1, wherein the N-terminal amino acid sequence is Asn-Phe-Leu-Ala-Gl.
y-Ser-Gln-Asp-Asn-Val-Ile-Ser-Gln-Ile-Pro-Ser-Gln-
An emulsifier containing Val-Gln-Glu-Leu-Ala-Phe- and a polypeptide having a molecular weight of 8000 or less. 4. The method according to claim 1, wherein the N-terminal amino acid sequence is Asn-Phe-Leu-Ala-Gl.
y-Ser-Gln-Asp-Asn-Val-Ile-Ser-Gln-Ile-Pro-Ser-Gln-
An emulsifier containing 30% or more of Val-Gln-Glu-Leu-Ala-Phe-polypeptide having a molecular weight of 8000 or less.