JPH0510129B2 - - Google Patents

Description

[Detailed description of the invention]

(Technical Field) The present invention relates to a method for efficiently producing a protein-based emulsifier that is less irritating to the skin, has a strong emulsifying effect, and has moisturizing properties. (Background Art) Generally, emulsifiers are widely used in cosmetics, foods, and the like. Examples of emulsifiers used in cosmetics include anionic surfactants and nonionic surfactants. However, anionic surfactants cause skin irritation, and nonionic surfactants also have drawbacks in that they do not have sufficient emulsifying properties or cause some skin irritation. Furthermore, since this type of emulsifier cannot impart water retention properties to creams, etc., moisturizing agents such as glycerin are currently added, but this changes the emulsion stability and texture. There is a problem. In addition, sugar esters and monoglycerides of fatty acids are used as emulsifiers in foods, but these emulsifiers do not have a very strong emulsifying effect, and a large amount of emulsifying agent must be used to achieve sufficient emulsifying effect. It has the disadvantage that it must be done. Therefore, there is a strong desire for emulsifiers that are less irritating to the skin, have a strong emulsifying effect, and have moisturizing properties. Prior to this application, the present inventors proposed a method for producing a protein-based emulsifier by bonding the carboxy terminus of a hydrophilic protein decomposition product with the amino group of an amino acid alkyl ester p-toluenesulfonate through an amide bond. [Patent Application No. 201823 (1982) (Japanese Patent Application Laid-Open No. 1983-94124)]. This protein-based emulsifier is less irritating to the skin, has a strong emulsifying effect, and has moisturizing properties. However, when the amino acid alkyl ester p-toluenesulfonate used in this method reacts with hydrophilic proteins in the presence of endopeptidase, it has poor solubility in the solvent, a buffer-acetone mixture, and is particularly The longer the alkyl group becomes, the more difficult it becomes to form a homogeneous system. Therefore, the reaction yield of protein-based emulsifiers has been unsatisfactory. In addition, in the above reaction, the amino acid alkyl ester p-toluenesulfonate used as a raw material is manufactured by removing unreacted impurities after the ester synthesis reaction is completed.
It is purified by repeated recrystallization with solvents such as acetone. However, unreacted amino acids, p-
It is difficult to sufficiently remove impurities such as toluenesulfonic acid, and they remain as impurities. If an amino acid alkyl ester p-toluenesulfonate containing such impurities is used as a grafting agent, the conversion rate to the desired protein-based emulsifier will be low, and the impurities will be mixed into the resulting protein-based emulsifier. Therefore, the protein-based emulsifier had to be washed many times with hot acetone or the like in the final step. In order to solve these problems, as a result of further research, the present inventors treated amino acid alkyl ester-p-toluenesulfonate with alkali or under salting-out conditions to obtain free amino acids. When alkyl esters are obtained and applied to protein grafting reactions, the amino acids and
- The problem of removing unreacted impurities such as toluenesulfonic acid and the problem of solubility in the reaction system are improved, and a protein-based emulsifier can be obtained in a good yield;
Furthermore, the inventors discovered that an odorless protein-based emulsifier can be obtained by dialysis, and have completed the present invention. (Object of the Invention) Therefore, an object of the present invention is to provide a method for efficiently producing a protein-based emulsifier that is less irritating to the skin, has a strong emulsifying effect, and has moisturizing properties. Other objects and advantages will become apparent from the description below. (Disclosure of the Invention) The above object is to react an amino acid and an aliphatic alcohol under p-toluenesulfonic acid catalyst, and then
A method for producing a protein-based emulsifier, characterized in that the amino group portion of an amino acid alkyl ester represented by the following general formula obtained by desalting in an alkaline medium is bonded with an amide bond to the carboxyl terminal of a hydrophilic protein decomposition product, and this This is achieved by a method for producing a protein-based emulsifier, which is characterized in that the reaction product obtained by the method is further dialyzed. R ₁・OR ₂ ...(1) (However, in the formula, R ₁ and R ₂ are the same as above.) In the above general formula (1), the amino acid residue represented by R ₁ means the carboxyl group of the amino acid ( −
COOH) minus OH

It means [formula]. The amino acid ester represented by the above general formula (1) according to the present invention includes, for example, highly hydrophobic amino acids having a linear alkyl group such as glycine, alanine, and phenylalanine, valine, leucine, isoleucine, Amino acids with highly hydrophobic but branched alkyl groups such as norleucine and proline, amino acids other than those listed above such as lysine, ornithine, and histidine, etc. The carboxyl group of amino acids such as myristyl alcohol, pentadecyl alcohol, cetyl alcohol, Compounds substituted with an alkoxy group of alcohols such as alcohols having 14 to 22 carbon atoms such as 2-hexyldecanol, heptadecyl alcohol, stearyl alcohol, isostearyl alcohol, aracyl alcohol, 2-octyldodecanol, behenyl alcohol, etc. These are listed as preferred. The alkaline medium used in the present invention includes:
Preferably, an aqueous NaOH or KOH solution is used. Alkali concentration is preferably 0.01N to 5N,
More preferably, it is 0.05N to 2N. Further, the alkali treatment is preferably carried out by salting out in the presence of salts. Salts for salting out include alkali metal salts, ammonium salts, etc. Specific examples include sodium chloride, potassium chloride, potassium carbonate, potassium phosphate, ammonium sulfate, sodium sulfate, etc. Potassium is preferred. The salt concentration in salting out is usually in the range of 0.5% to 25%, preferably 1% to 10%. The amino acid alkyl ester-p-toluenesulfonate mentioned above is a solid, but the free amino acid alkyl ester obtained in this way is in an oily state or a solid state with a low melting point, and is a hydrophilic protein as described below. During the reaction, it has extremely good solubility in the buffer-acetone mixture used as the reaction solvent and can react in a homogeneous system, so the reaction efficiency with hydrophilic proteins is greatly improved. Examples of hydrophilic proteins applicable to the present invention include casein, gelatin, sericin, soluble collagen, zein, serum albumin, lactalbumin, and ovalbumin, which may be used alone or in combination. Among these proteins, proteins other than sericin are sold as reagents. Furthermore, sericin can be obtained by hot water extraction from cut cocoons and freeze-drying. In addition, endopeptidases used in enzymatic reactions have the ability to cleave proteins from their central parts, and include serine proteases, thiol proteases, carboxyl proteases,
Includes metalloproteases and other endopeptidases. Among these, it is most preferable to use thiol protease. Examples of the thiol protease include papain, cathepsin B, bromelain, chymopapain, cathepsin L, yeast proteinase B, cathepsin S, and TZ-peptidase, which may be used alone or in combination. The protein emulsifier can be obtained by reacting the above-mentioned amino acid alkyl ester with a hydrophilic protein in the presence of the above-mentioned endopeptidase. The enzyme reaction product thus obtained is dialyzed after the endopeptidase activity is inactivated under acidic conditions. Favorable results can be obtained when dialysis is carried out using a holofiber type ultrafiltration membrane, particularly a holofiber type ultrafiltration membrane housed in a circular housing. Materials for the holofiber type ultrafiltration membrane include cellulose acetate, cellulose, polyacrylonitrile, ethylene vinyl alcohol, polyvinyl alcohol, and cuprofan membrane (manufactured by Enka, Germany).
etc., and preferably cellulose, polyacrylonitrile, cuprofan membranes are selected. When dialyzing and concentrating using the holofiber type ultrafiltration membrane, the enzyme reaction product is preferably 25
It is preferable to keep the temperature at 35°C to 45°C, more preferably 35°C to 45°C. By performing dialysis and concentration using this holofiber type ultrafiltration device, deodorization can be completely performed, and desalination and concentration can be performed in a very short time. Furthermore, the subsequent drying process can be carried out economically and quickly because the water content is reduced in the concentration process, and the desired odorless protein emulsifier can be processed quickly and economically. And you can get it in large quantities. (Effects of the Invention) In the present invention, since the amino acid alkyl ester p-toluenesulfonate is once desalted, the desired product can be obtained in high yield. In addition, this protein-based emulsifier has a typical surfactant structure, with the hydrophilic group consisting of a protein that inherently has strong hydrophilicity and the hydrophobic group consisting of a hydrophobic amino acid alkyl ester ( The front end of the hydrophobic group is bonded to the rear end of the hydrophilic group, and no extra hydrophobic group is bonded to the middle portion, etc.), so it has strong emulsifying power. Furthermore, since this emulsifying effect is hardly affected by temperature due to the polymer structure of the protein moiety, this emulsifier does not cause a decrease in the emulsifying effect, especially at low temperatures. Since the hydrophilic group portion has a relatively large molecular weight (about 500 or more, usually several thousand), the entire emulsifier has a large molecular weight and hardly irritates the skin. Moreover, this emulsifier in particular has sufficient moisturizing properties because the hydrophilic group portion essentially consists of proteins that have moisturizing properties. Therefore, when this is used in cosmetics, especially creams, sufficient water retention can be imparted without using conventional humectants such as polyhydric alcohols. In addition, the protein-based emulsifier dialyzed by the method of the present invention is odorless, so it can be used without any discomfort, and even when flavored, the original scent of the fragrance will not be lost. It became possible to add incense. As described above, the above protein-based emulsifiers cause less irritation to the skin, etc., have strong emulsifying effects, and have moisturizing properties, so when used in cosmetics, for example, excellent effects can be obtained. . Furthermore, when this is used as an emulsifier for foods, the amount of emulsifier used can be reduced due to its strong emulsifying effect. Furthermore, since most of the constituent parts of this emulsifier are derived from natural products, no problem will occur even if the emulsifier is repeatedly ingested. Furthermore, the odorless protein-based emulsifier can be ingested without causing any discomfort to the sense of taste or smell, which is important in foods. The present invention will be specifically explained below with reference to Examples. Example 1 An amino acid alkyl ester was produced as described below, and then subjected to an enzymatic reaction. (Synthesis of amino acid alkyl ester) Collect 0.05 mol of leucine as an amino acid and add p-toluenesulfonic acid monohydrate.
0.055 mol was collected, and further 0.075 mol of myristyl alcohol was collected as an alcohol, benzene as a solvent was added thereto, the mixture was thoroughly stirred and mixed, and the mixture was heated to reflux at the reflux temperature of benzene to proceed with the esterification reaction. In this case, since water produced as the esterification reaction progressed came out in an azeotropic state with benzene, the reaction was completed in about 5 to 10 hours while removing it from the reaction system. After cooling to room temperature, the reaction solution was washed three times with a 3% NaCl solution containing 0.3N NaOH to perform desalting. Thereafter, the benzene layer was collected and thoroughly washed with 3% salt water until it became neutral. After washing lightly with water, the benzene layer was collected and dried over anhydrous sodium sulfate overnight. After drying, the benzene layer was collected by filtration, and the solvent was removed from this benzene solution using an evaporator to obtain a purified oily substance of free leucine myristyl ester. (Enzyme reaction) Dissolve 120 g of gelatin, a hydrophilic protein, in 480 ml of 1M carbonate buffer (PH9.0), and add 120 g of gelatin to this
ml of acetone was added, immersed in a 37°C hot water bath, and thoroughly stirred to homogenize. Next, 39.3 g of leucine myristyl ester obtained as described above (an amount of 1 mol per 1000 g of gelatin) was added thereto, and the mixture was sufficiently stirred to homogenize. In this case, the amino acid alkyl ester such as leucine myristyl ester is preferably added at a ratio of 1 mol to 1000 g of hydrophilic protein such as gelatin in terms of reaction efficiency. Then,
In addition, L-cysteine hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.)
1.05 g was added, and further 60 mg of endopeptidase papain (manufactured by Sigma) was added and reacted for 15 minutes with stirring. Then, the overall pH was adjusted to 2 using 2N hydrochloric acid to stop the reaction. The reaction solution was approximately 1.5. (Purification) The above reaction solution was kept at about 40°C and filtered into a holofiber type ultrafiltration device (Cuprofan membrane, fiber inner diameter 200μ, membrane thickness 11μ, effective membrane area 1.5m ² ) for about 10 minutes.
The liquid was fed at a flow rate of ml/min, and the external liquid was warm water at about 40°C at a flow rate of about 1/min. After dialysis, the internal pressure of the same holofiber type ultrafiltration device was set to approximately 2Kg/cm ²
The dialysis solution was then concentrated under the same conditions as above, and approximately 700 ml was finally obtained. This concentrated solution was dried using a spray dryer to obtain about 85 g of an odorless protein-based emulsifier. Example 2 An amino acid alkyl ester is produced as follows, and then subjected to an enzymatic reaction. (Synthesis of amino acid alkyl ester) Collect 0.05 mol of glycine as an amino acid and p-toluenesulfonic acid monohydrate.
0.055 mol was collected, and further 0.075 mol of stearyl alcohol was collected as alcohol, benzene as a solvent was added thereto, the mixture was thoroughly stirred and mixed, and the mixture was heated to reflux at the reflux temperature of benzene to proceed with the esterification reaction. In this case, since water produced as the esterification reaction progressed came out in an azeotropic state with benzene, the reaction was completed in about 7 hours while removing it from the reaction system. Next, after cooling the reaction solution to room temperature,
Add 300ml of 3% NaCl solution containing 0.5NNaOH,
Desalination was carried out by alkali treatment under salting-out conditions. The alkali treatment was repeated three times. Thereafter, the benzene layer was collected and thoroughly washed with 3% saline. The subsequent steps were carried out in the same manner as in Example 1 to obtain purified free glycine stearyl ester oil. (Enzyme reaction) 70 g of zein was used in place of 120 g of gelatin, and this was dissolved in 480 ml of 1M carbonate buffer. Then,
Add 120ml of acetone to this, immerse it in a hot water bath at 35°C, stir it to make it homogeneous, and then add the glycine-stearyl ester that was previously melted at 50°C.
19.0 g was added (an amount of 1 mol per 1000 g of zein) and stirred to homogenize. Then, to this, L-
Add 1.05g of cysteine hydrochloride (manufactured by Wako Pure Chemical Industries),
Furthermore, 45 mg of endopeptidase papain (manufactured by Sigma) was added and reacted for 15 minutes with stirring. Then, the overall pH was adjusted to 2 using 2N hydrochloric acid to stop the reaction. Approximately 1.5 as reaction solution
It became. (Purification) The above reaction solution was kept at about 40°C, and about 10 ml/ ^approx .
The liquid is pumped at a flow rate of 1 min, and the external liquid is warm water at about 40°C.
The liquid was delivered at a flow rate of /min. After dialysis, the internal pressure of the same holofiber type ultrafiltration device was set to about 2 Kg/cm ² and the dialysis solution was concentrated under the same conditions as above to finally obtain about 600 ml. This concentrated solution was dried using a spray dryer to obtain about 63 g of an odorless protein-based emulsifier. In Examples 1 and 2, the dialysis efficiency in the dialysis process using a holofiber type ultrafiltration device was measured using a tabletop digital conductivity meter (Denki Kagaku Keiki Co., Ltd. A0-6).
The results of the study and comparison based on the conductivity of the solution using
Shown in the table.

[Table] As shown in Table 1, even after just one dialysis, the conductivity of the external solution was almost the same as that of tap water, and the expected dialysis efficiency was obtained. (Evaluation of deodorizing effect) The deodorizing effect was determined by smell using the reaction solution before ultrafiltration treatment as a control. It was conducted by 10 experts and evaluated using the following evaluation criteria. 1: Strong odor 2: Slight odor 3: No odor As a result, the deodorizing effects of Examples 1 and 2 were evaluated as shown in the table below.

[Table] From the same table, it was observed that the holofiber type ultrafiltration membrane had a cleansing and deodorizing effect. As described above, according to the present invention, a protein-based emulsifier that is less irritating to the skin, has a strong emulsifying effect, and has moisturizing properties can be efficiently obtained. The protein emulsifier can be completely deodorized by dialysis and concentration.

Claims (1)

[Scope of Claims] 1. The amino group moiety of an amino acid alkyl ester represented by the following general formula obtained by reacting an amino acid and an aliphatic alcohol in the presence of a p-toluenesulfonic acid catalyst and then desalting in an alkaline medium, A method for producing a protein-based emulsifier, characterized by attaching an amide bond to the carboxyl terminal of a hydrophilic protein decomposition product. R ₁ ·OR ₂ [In the formula, R ₁ represents an amino acid residue and R ₂ represents an alkyl group having 14 to 22 carbon atoms. ] 2 After reacting an amino acid and an aliphatic alcohol under the catalyst of p-toluenesulfonic acid, the amino group portion of an amino acid alkyl ester represented by the following general formula obtained by desalting in an alkaline medium is converted into a hydrophilic protein decomposition product. 1. A method for producing a protein emulsifier, which comprises attaching an amide bond to the carboxyl terminal of a protein, and then dialyzing the resulting reaction product. R ₃ ·OR ₄ [In the formula, R ₃ represents an amino acid residue, and R ₄ represents an alkyl group having 14 to 22 carbon atoms. 3. The method for producing a protein emulsifier according to claim 2, wherein the amino acid is glycine, alanine, or leucine. 4. The method for producing a protein-based emulsifier according to claim 2 or 3, wherein the hydrophilic protein decomposition product is casein, gelatin, sericin, soluble collagen, zein, serum albumin, lactalbumin, or ovalbumin. 5. The method for producing a protein emulsifier according to any one of claims 2 to 4, wherein the dialysis is performed using a holofiber ultrafiltration membrane. 6. A patent in which dialysis is performed using a holofiber ultrafiltration membrane housed in a circular housing under heat retention at 25°C to 50°C after inactivating the endopeptidase activity in the reaction product under acidic conditions. A method for producing a protein emulsifier according to any one of claims 2 to 5.