JPH0576334B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing an emulsifier using zein, which is a protein contained in corn kernels. The present invention opens the way to the effective use of zein, which has not been effectively utilized in the past. [Prior Art] It has been known that proteins such as gelatin, casein, and albumin are used as emulsifiers, but twein has no emulsifying activity, and no attempts have been made to use thein as a raw material for emulsifiers. do not have. [Problems to be Solved by the Invention] Tzein is a protein contained in approximately 5% of corn kernels. And from the point of view of efficient use of corn, the use of zein is also required,
The fact that tzein is not soluble in water and lacks necessary amino acids such as lysine and lilliptophan are obstacles to the effective use of tzein. Thus, conventionally, zein had no effective use, but the present invention solves the problem of opening the way to the effective use of zein by producing an emulsifier using this zein. [Means for solving the problem] As a result of research on the effective use of tzein, the present inventors found that when tzein and phosphatidic acid are mixed in an alkaline aqueous solution with a pH of 12 or higher, a stable tzein-phosphatidic acid complex is formed. This complex has emulsifying power and dissolves in water at pH 4.0 or higher, and becomes an almost transparent aqueous solution at pH 7.0 or higher. It was discovered that the partially hydrolyzed product of this complex has emulsifying power at pH 2.0 or higher, and has high emulsifying activity and emulsifying capacity over a wide range of pH. The present invention is based on the above discovery, and the present invention provides a tzein-phosphatidic acid complex, which is characterized in that the tzein-phosphatidic acid complex is formed by mixing tzein and phosphatidic acid in an alkaline aqueous solution with a pH of 12 or higher. The present invention is a method for producing an emulsifier, and the present invention involves mixing tzein and phosphatidic acid in an alkaline aqueous solution with a pH of 12 or higher to form a tzein-phosphatidic acid complex, and adding protease to the resulting complex at a pH of 7 to 9. This is a method for producing an emulsifier, which is characterized by partially hydrolyzing the emulsifier. In the present invention, any alkaline aqueous solution having a pH of 12 or higher can be used, but for example, 0.1N NaOH aqueous solution, KOH aqueous solution, etc. are preferably used. And in the present invention
The reason for using an alkaline aqueous solution with a pH value of 12 or higher is that tzein will not dissolve in an aqueous solution with a PH value lower than 12, and a tzein-phosphatidic acid complex will not be formed even if tzein and phosphatidic acid are mixed. Next, phosphatidic acid is obtained by, for example, washing commercially available soybean lecithin with cold acetone (e.g., -20°C) to remove neutral lipids, and then treating the acetone-washed lecithin with phospholipase D (manufactured by Boehringer Mannheim). It can be obtained by doing this, but it can also be obtained commercially. To mix tzein and phosphatidic acid in an alkaline aqueous solution with a pH of 12 or higher, tzein and phosphatidic acid may be added and mixed in an alkaline aqueous solution with a pH of 12 or higher. They may be added and mixed, or solutions obtained by separately dissolving tzein and phosphatidic acid in an alkaline aqueous solution with a pH of 12 or higher may be mixed. The mixing ratio of tzein and phosphatidic acid in an alkaline aqueous solution having a pH of 12 or higher is preferably 20 to 100 parts by weight of phosphatidic acid per 100 parts by weight of tzein. When tzein and phosphatidic acid are mixed in an alkaline aqueous solution with a pH of 12 or higher, preferably by stirring, a tzein-phosphatidic acid complex is formed. In this case, it may be heated to an appropriate temperature, but
Typically, the complex is formed at room temperature. The liquid in which the tzein-phosphatidic acid complex is formed in this way can be used as an emulsifier after being adjusted to the desired pH of the emulsion, for example, a pH of 4 to 7. In addition, in order to obtain the zein-phosphatidic acid complex from the solution in which the complex was formed, the complex is precipitated at a pH of 2.0 or lower, and the precipitate is collected by an appropriate means such as centrifugal precipitation. The complex can be obtained by dialysis or other washing with water and drying, if necessary. This complex can be used as an emulsifier as it is or by dissolving it in water. Next, the Tzein obtained as described above −
Partial hydrolysis of the phosphatidic acid complex by protease is performed by mixing tzein and phosphatidic acid in an alkaline aqueous solution with a pH of 12 or higher to form a tzein-phosphatidic acid complex.
The tzein-phosphatidic acid complex separated from the above solution may be prepared as an aqueous solution with a pH of 7 to 9 and then treated with protease. Suitable examples of proteases used for this purpose include pronase E (Kaken Chemical Co., Ltd.), trypsin (Sigma Co., Ltd.), α-chymotrypsin (Sigma Co., Ltd.), and papain (Sigma Co., Ltd.). The action of protease is usually carried out for about 2 to 24 hours at room temperature or at a temperature appropriate for the action of the protease used. To stop the reaction, e.g. boil the solution in a boiling bath for 10
This can be done by heating for about a minute. Partial hydrolysis of the tzein-phosphatidic acid complex obtained by the action of protease as described above has emulsifying power at pH 2.0 or higher, and has high emulsifying activity and emulsifying solution over a wide range of pH. , is an excellent emulsifier. Here, consideration regarding the expression of emulsifying properties of tzein according to the present invention is as follows. Zein contains about 50% hydrophobic amino acids and has a high α-helical content of 50%, so it has a rigid structure and tends to aggregate in water. The emulsifying properties of proteins require surface hydrophobicity and flexible structure of the protein. Although tzein is a highly hydrophobic protein that dissolves in alcohol and chloroform-methanol mixture, it does not exhibit any emulsifying properties. In other words, in order for tzein to exhibit emulsifying properties, it is thought that its hard structure must be broken. Tzein and phosphatidic acid have a pH of 12
When mixed in the above strong alkaline aqueous solution, it becomes a complex with emulsifying properties (Tzein does not exhibit emulsifying properties even when complexed with phosphatidylcholine or phosphatidylethanolamine) , suggesting the possibility of denaturation of tzein by strong alkali and retention of a flexible structure by binding of phosphatidic acid. The flexible structure of the zein-phosphatidic acid complex is also thought to be due to the fact that this complex is susceptible to the action of proteases. Furthermore, the fact that the emulsifying properties of the zein-phosphatidic acid complex in the acidic region were improved by the protease treatment suggests that partial hydrolysis of tzein is accompanied by an improvement in solubility. [Effects of the Invention] According to the present invention, tzein, which has hitherto not been effectively utilized, is dissolved in water and has a pH of 7.0.
An emulsifier consisting of a tzein-phosphatidic acid complex which becomes an almost transparent aqueous solution and has an emulsifying power at a pH of 4.0 or higher is thus obtained.Furthermore, according to the present invention, by partially hydrolyzing this complex with protease. The present invention is useful because it provides an emulsifier that has emulsifying power at pH 2.0 or higher and has high emulsifying activity and emulsifying capacity over a wide range of pH. Note that the emulsifier according to the present invention can be suitably used as an emulsifier for foods, cosmetics, medicines, and the like. [Example] The present invention will be explained in more detail with reference to Examples below. Example 1 After dissolving 20 g of soybean lecithin (manufactured by Wako Pure Chemical Industries, Ltd.) in 30 ml of chloroform, 500 g of cold acetone (-20°C) was dissolved.
ml dropwise while stirring to remove the precipitated phospholipids.
The phospholipids were collected by centrifugal precipitation at ×g and dried under vacuum to obtain 15 g of phospholipids. Add 750 ml of diethyl ether to 15 g of this phospholipid. 0.4M acetate buffer (PH5.6)
Add 300 ml, 300 ml of 0.2MCaC ₂ , 900 ml of distilled water, and 100 units of phospholipase D (derived from cabbage, manufactured by Boehringer Mannheim), react with stirring at 20°C for 10 hours, and then add 150 ml of N-HCl, 2000 ml of diethyl ether, and 500 ml of ethanol. The ether layer was collected, concentrated and dried to obtain 10.5 g of phosphatidic acid. 3 mg of Zwein (manufactured by Hanui Chemical Co., Ltd.) and 3 mg of phosphatidic acid obtained as above were mixed with 0.1N-NaOH.
It was dissolved in 1.5 ml of an aqueous solution (PH12) and mixed to form a zein-phosphatidic acid complex. After adjusting the pH of this liquid to 7.0 with N-HCl, 0.25 ml of soybean oil was added thereto, and the emulsifying activity was examined by ultrasonication as described below. As a result, the emulsifying activity is
It was 0.32. For ultrasonic processing, a microchip is attached to an ultrasonic processing device (sonifier manufactured by Branson), and the output is
Emulsification was performed by ultrasonication at 30 W for 1 minute at 20°C, and the resulting emulsion was diluted 2000 times with 1% sodium dodecyl sulfate aqueous solution and emulsified with a turbidity at a wavelength of 600 nm using a spectrophotometer. It was made active. The higher the emulsifying activity value, the greater the emulsifying power. For comparison, 0.25 ml of soybean oil was added to 1.5 ml of distilled water containing 3 mg of Zwein (manufactured by Hanui Kagaku Co., Ltd.) and subjected to ultrasonication in the same manner as above, but no emulsifying activity was observed. In addition, 3 mg of Zwein (manufactured by Hanui Chemical Co., Ltd.) and 3 mg of phosphatidic acid were added to 1.5 ml of distilled water.
When 0.25 ml of soybean oil was added and sonicated in the same manner as above, no emulsifying activity was shown. From these results, Tzein is 0.1N−NaOH
(PH12) When mixed with phosphatidic acid in an aqueous solution, it forms a zein-phosphatidic acid complex, exhibits emulsifying activity, and can be used as an emulsifier. Example 2 10 g of Zwein (manufactured by Hanui Chemical Co., Ltd.) and 10 g of phosphatidic acid obtained as described in Example 1 were added.
It was dissolved in 500 ml of 0.1N-NaOH aqueous solution (PH12), and the mixed solution was adjusted to pH 2.0 with N-HCl to form a precipitate of the tzein-phosphatidic acid complex. This precipitate was collected by centrifugation at 1000 x g and dried to form the tzein-phosphatidic acid complex.
Obtained 12g. 12 g of this complex was dissolved in 50 ml of distilled water and dialyzed against 10 ml of distilled water. This dialysis fluid is used as an emulsifier as it is or after being dried. Example 3 The protein concentration of the dialyzed fluid obtained in Example 2 was measured by Bradford's method, and diluted with distilled water so that the protein concentration was 0.2% (by weight) [Bio-Rad]. A protein measurement kit manufactured by Rad) was used. Add this diluted aqueous solution to 0.1N−
Adjust the pH to 8.3 with NaOH, add 2 units of pronase E (manufactured by Kaken Kagaku Co., Ltd.) per 1 ml of protein, react at 37°C for 2 hours, and then heat at 100°C for 10 minutes to stop the reaction. A partial hydrolyzate of -phosphatidic acid complex was obtained. The pH of the partial hydrolyzate of this zein-phosphatidic acid complex was adjusted to 2 to 7 with 0.1N HCl, and the emulsifying activity was measured in the same manner as described in Example 1. The results are shown in Table 1. Also,
Table 1 also shows the emulsifying activity of the tzein-phosphatidic acid complex and casein at pH 3 to 7.

[Table] Next, the pH of the above partial hydrolyzate of tzein-phosphatidic acid was adjusted to 2 to 2 with 0.1N-HCl.
Table 2 shows the results of measuring the emulsification capacity. Table 2 also shows the emulsifying capacity of the tzein-phosphatidic acid complex and casein at pH 3 to 7. The emulsification capacity is determined by measuring the amount of oil that can be emulsified by adding 0.25 ml or more of soybean oil to a pH-adjusted sample solution, and measuring the amount of soybean oil that will cause a continuous oil phase to appear. The amount of oil was converted into the emulsification capacity of the sample per gram of protein. The higher the emulsifying capacity value, the more fats and oils can be emulsified.

[Table] From the results in Tables 1 and 2, the partial hydrolyzate of the tzein-phosphatidic acid complex obtained as described above has high emulsifying activity and emulsifying capacity over a wide range of pH. I understand.

Claims (1)

[Scope of Claims] 1. A method for producing an emulsifier comprising a tzein-phosphatidic acid complex, which comprises mixing tzein and phosphatidic acid in an alkaline aqueous solution with a pH of 12 or higher to form a tzein-phosphatidic acid complex. 2. Mix tzein and phosphatidic acid in an alkaline aqueous solution with a pH of 12 or higher to form a tzein-phosphatidic acid complex, and add the resulting complex to a pH of 7.
A method for producing an emulsifier, characterized by partially hydrolyzing it by allowing protease to act at the ~9-position.