JP3195466B2 - Method for producing tyrosine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing tyrosine, and more particularly, to a method for producing tyrosine for efficiently collecting tyrosine from soy sauce cake.

[0002]

2. Description of the Related Art In general, demand for tyrosine is increasing year by year as an intermediate in the field of pharmaceutical production. Conventionally, as a method for producing tyrosine, a direct fermentation method using a bacterium such as Corynebacterium has been known. Further, as a recent technique, it has been proposed to extract tyrosine from the debris of a vegetable protein decomposed product used as a raw material of a seasoning (Japanese Patent Application Laid-Open No. Sho 60-6).
No. 1553). Such a method comprises the following steps:
That is, this is a method in which a suspension containing an amino acid such as tyrosine and calcium citrate as an organic acid salt as main components is adjusted to be alkaline to remove insoluble calcium citrate and the like, and then tyrosine is crystallized and separated.

[0003]

However, these methods are not always suitable for mass production of tyrosine, and development of a method for mass-producing high-quality tyrosine simply and inexpensively is awaited. By the way, Fukuma is 19
In 1978, as a result of hydrolysis of soy sauce cake by-produced by an old brewing method and analysis of amino acids, it was revealed that the cake contains about 2.3% of tyrosine.
Pp. 69, 1978). Apart from that, there is no technical literature on tyrosine in soy sauce cake. Accordingly, an object of the present invention is to provide a method for producing tyrosine that can be easily and mass-produced with high yield of high-purity tyrosine.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, first, soy sauce cake by-produced in the production of soy sauce substantially contains organic acid salts such as calcium citrate. Not as a component, but not protein-binding tyrosine such as proteins and oligopeptides,
Non-binding, that is, free tyrosine is usually 0.07 to
It was found that it was contained in an extremely large amount of 0.21%, and 4 to 6%. Thus, the fact that soy sauce cake is suitable as a tyrosine source for mass production, the tyrosine-containing precipitate which can be easily obtained by concentrating the hot water extract of soy sauce cake at pH 12
By the above, impurities are insoluble, only tyrosine is selectively dissolved, high purity, it can be obtained in good yield, furthermore, by hydrogen peroxide treatment, decolorized uncolored high purity We have obtained new knowledge that tyrosine can be obtained.

[0005] That is, the present invention has achieved the above-mentioned object based on the above-mentioned findings. The present invention concentrates a hot water extract of soy sauce cake to form a tyrosine-containing precipitate, and adjusts the tyrosine-containing precipitate to pH 12 or more. It is another object of the present invention to provide a method for producing tyrosine, which comprises crystallizing and collecting tyrosine after removing insolubles by the method. The method for producing tyrosine as described above, wherein decolorization treatment with hydrogen peroxide is carried out in any step from the hot water extraction to crystallization and collection of tyrosine.

Hereinafter, a method for producing tyrosine according to the present invention will be described in detail. The soy sauce lees used in the method for producing tyrosine according to the present invention is lees produced as a by-product during normal soy sauce production such as the present brewing method and the new brewing method, and those having a large free tyrosine content are preferably selected. And it does not matter whether or not it is dried. The hot water extract of soy sauce cake is obtained as a tyrosine-containing solution after removing soy sauce cake with hot water and removing residues. And 60 to 90% of the tyrosine in the soy sauce cake is recovered in the hot water extract.

Specifically, for example, it is obtained as follows. First, the above-mentioned soy sauce cake is appropriately ground as required.
For example, for 100 g of dried soy sauce cake having a water content of 3 to 15%, 500 to 5000 ml of water is added. After sufficient stirring, heat at a temperature of 60-120 ° C. for 6 minutes to 2 hours.
Next, the residue is filtered, centrifuged, etc. to obtain a hot water extract in the range of 260 to 2600 ml. Before hot water extraction, 5
It is preferable to wash with soy sauce cake with a 0 to 100% alcohol solution, and the soy sauce cake washing removes flavone and polysaccharide,
Increase the recovery and purity of tyrosine crystals.

[0008] The hot water extract may be concentrated by a usual conventional operation, and there is no particular limitation. For example, it is as follows. It is preferable to concentrate the extract to about 1/10 to 7/10 using a flash evaporator or the like under a reduced pressure at a temperature of 30 to 100 ° C. The concentrate is brought to a temperature of 0 to 20 ° C.
Upon standing for 1 to 40 hours, a tyrosine-containing precipitate forms. This precipitate contains free tyrosine in soy sauce cake and impurities other than tyrosine, does not contain calcium citrate,
Collect by centrifugation, filtration, etc. Incidentally, 70 to 90% of tyrosine in the hot water extract is recovered in this precipitate.
And the precipitate contains 40-60% of tyrosine.

As an important step of the method for producing tyrosine according to the present invention, the above precipitate is mixed with an alkaline solution,
With twelve or more, tyrosine is dissolved and other insolubles are removed. The tyrosine in the precipitate is almost dissolved in the solution by this operation, and the contaminants are removed as insolubles.

Specifically, for example, it is as follows.
The tyrosine-containing precipitate is suspended in water or a solution containing a water-soluble organic solvent, and the pH is adjusted with an alkali (solution or solid).
12 or more, preferably 12.5 to 13.5. Further, the final pH is 12 or more, preferably 12.5 to 13.
In order to reach 5, the tyrosine may be dissolved by directly adding an alkaline aqueous solution or an alkaline solution containing a water-soluble organic solvent to the tyrosine-containing precipitate. The liquid volume of the water or water-soluble organic solvent-containing solution and the alkaline aqueous solution or aqueous organic solvent-containing alkaline solution to be added to the tyrosine-containing precipitate is 1 to 50 ml, preferably 5 to 40 ml, per gram of the wet weight of the precipitate.

Examples of the water-soluble organic solvent include alcohols such as methanol, ethanol, propanol and isopropanol, acetone, dioxane and the like, and these can be used alone or in combination. The concentration of the water-soluble organic solvent may be 80% or less, preferably 60% or less. Alkali increases the pH of the solution to 1
Any substance capable of increasing the number to two or more may be used, and is preferably a reagent grade such as sodium hydroxide, potassium hydroxide, ammonium hydroxide and the like. PH of the tyrosine-containing precipitate
When it is 12 or more, the temperature is 0 to 40 ° C, preferably 10 to
Mix at 30 ° C. Further, the mixture is left standing or stirred at the same temperature for 10 minutes to 1 hour. Thus, tyrosine is sufficiently dissolved in the solution, but impurities are not dissolved and become insoluble. This insoluble matter is removed by ordinary filtration, centrifugation or the like.

[0012] Tyrosine in the precipitate is almost 100% soluble in the solution when the pH is 12 or more. However, in an alkaline solution having a pH of less than 12, tyrosine is not sufficiently dissolved in the solution. There are many. Therefore, at pH 12 or higher, tyrosine can be produced from soy sauce cake at a recovery rate of 35% or higher, and at pH 12 or lower, the recovery rate decreases.

Crystallization and collection of tyrosine from the above-mentioned tyrosine-containing solution can be carried out according to a conventional method. For example,
Using an acid such as hydrochloric acid, sulfuric acid, or acetic acid, the pH of the solution is adjusted to 6 to
Adjust to 10 and let stand at a temperature of 0-10 ° C. The generated crystals are re-crystallized from hot water as needed. Then, usually, it is collected by filtration with a filter or the like and dried under reduced pressure to obtain 90% or more high-purity tyrosine. The recovery of crystalline tyrosine by this crystallization operation is 80-90% as that from the tyrosine-containing precipitate.

In this manner, high-purity tyrosine can be obtained by the method for producing tyrosine according to the present invention.
Furthermore, uncolored tyrosine can be obtained by combining this method with decolorizing treatment with hydrogen peroxide.
This decolorization treatment is applied to the tyrosine-containing substance in any steps from crystallization and collection of tyrosine from the hot water extract of soy sauce cake. Among them, a hot water extract, a solution in which tyrosine is dissolved at a pH of 12 or more, a solution in which insolubles are removed from the solution, and an aqueous solution of tyrosine crystals are preferable, and particularly preferable.

This processing is specifically as follows, for example. A final concentration of 0.01 to 30% of a hydrogen peroxide solution containing 0.3 to 30% of hydrogen peroxide in the above-mentioned tyrosine-containing material is defined as hydrogen peroxide.
10%, preferably added to be 0.1 to 2%,
Decolorize the tyrosine-containing material at a temperature of 0 ° C. to room temperature for 30 minutes to 2 hours. After the decolorization treatment, the following step operation in the tyrosine production method may be performed. By the addition of this decolorizing treatment, 90 or more crystals can be produced by an L value representing the degree of coloring of the crystal (the crystal is directly measured for the L value with a colorimetric colorimeter (manufactured by Nippon Denshoku Industries Co., Ltd.)).

[0016]

EXAMPLES Hereinafter, the method for producing tyrosine according to the present invention will be specifically described with reference to examples. The present invention is not limited to the following embodiments. Example 1 4000 g of a 95% ethanol solution was added to 500 g of dried soy sauce cake having a water content of 9% (containing 5.5% of tyrosine, that is, 22.2 g in total), and the temperature was 70 ° C. for 1 hour.
After stirring, the ethanol-soluble portion was removed by filtration to obtain ethanol-washed soy sauce cake. Add 7.5 liters of water to this soy sauce cake and add 3
Boil for 0 minutes. Next, the boiled product was filtered and the residue was removed to obtain 3.90 l of a hot water extract (containing 15.4 g of tyrosine).

The hot water extract was concentrated to 1/3 with a flash evaporator at a temperature of 80 ° C. under reduced pressure, and allowed to stand at a temperature of 4 ° C. overnight to form a tyrosine-containing precipitate. Centrifugation (1
20.5 g of this tyrosine-containing precipitate (containing 10.84 g of tyrosine) was collected at 0000 rpm for 10 minutes. The precipitate was divided into five equal parts, and for each of the four samples,
123 ml of water was added for suspension, and the pH of the suspension was adjusted to 10, 12, 13, and 14 at a temperature of 25 ° C. with stirring using 6N sodium hydroxide solution and solid sodium hydroxide, respectively. In addition, stir at the same temperature for 30 minutes,
Tyrosine in the precipitate was dissolved in an aqueous alkaline solution. Next, the insoluble material is centrifuged (10,000 rpm, 10 minutes).
And a tyrosine-containing solution was obtained. Table 1 shows the tyrosine recovery rate (A) for the tyrosine-containing precipitate of the tyrosine-containing solution at this time.

Further, each tyrosine-containing solution is added with 20% hydrochloric acid to form p.
It was adjusted to H9 and allowed to stand at room temperature for 1 hour to crystallize tyrosine. The crystals were collected on a filter and washed twice with water. Further, the crystals were recrystallized twice from hot water and dried under vacuum to obtain tyrosine crystals. Table 1 shows the tyrosine recovery rate (B) of the tyrosine crystals with respect to the soy sauce cake at this time, and the purity thereof. The quantification of tyrosine was determined by high performance liquid chromatography (HPLC) (Hitachi L-8500, manufactured by Hitachi Keisoku Engineering Co., Ltd.), and the purity was determined by elemental analysis.

[0019]

[Table 1]

From Table 1, it can be seen that the hot water extract of soy sauce cake can be concentrated to sufficiently collect tyrosine. Furthermore, when the tyrosine-containing precipitate is adjusted to pH 12 or higher, tyrosine with higher purity can be obtained at a higher recovery rate than in the case of pH 10, which proves to be useful for industrial production.

(Example 2) One sample obtained by dividing the tyrosine-containing precipitate obtained in Example 1 into five equal parts was adjusted to pH 13 in the same manner as in Example 1, and the insoluble matter was removed by centrifugation. The resulting tyrosine-containing solution was divided into two equal parts. To one of the samples, 0.615 ml of a 30% hydrogen peroxide solution was added, and left at room temperature for 1 hour to perform a decolorizing treatment.
Tyrosine crystals were obtained in the same manner as in Example 1. About the other sample, a tyrosine crystal was obtained in the same manner as described above except that the decolorization treatment was not performed. Table 2 shows the result of measuring the L value of the tyrosine crystal at this time.

[0022]

[Table 2]

From Table 2, it can be seen that uncolored tyrosine crystals having an excellent L value can be obtained by the decolorizing treatment with hydrogen peroxide.

[0024]

According to the method of the present invention, high-purity tyrosine can be easily and efficiently produced by using soy sauce cake by-produced during the production of soy sauce.
Furthermore, a non-colored product can be produced, which is extremely effective as an inexpensive mass production method of tyrosine.

──────────────────────────────────────────────────の Continuing on the front page (72) Katsumi Yuasa, Inventor 339 Noda, Noda-shi, Chiba Kikkoman Corporation (72) Inventor Hayami Ito 1-1-1, Kawasaki-cho, Akashi-shi, Hyogo Kawasaki Heavy Industries Akashi Factory (72) Inventor Masao Hayashiya 1-1, Kawasaki-cho, Akashi-shi, Hyogo Prefecture Inside the Akashi Plant of Kawasaki Heavy Industries, Ltd. (72) Inventor Hiroyuki Mizukami 1-1-1, Kawasaki-cho, Akashi-shi, Hyogo Kawasaki Heavy Industries, Ltd. (56) References JP-A-60-61553 (JP, A) "Journal of the Japan Shoyu Institute," No. 4, pp. 68-69 (1978) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07C 229/36 C07C 227/42 CA (STN)

Claims (2)

(57) [Claims] 1. A hot water extract of soy sauce cake is concentrated to form a tyrosine-containing precipitate, and the tyrosine-containing precipitate is adjusted to pH 1
2. A method for producing tyrosine, comprising crystallizing and collecting tyrosine after removing insolubles by 2 or more. 2. The method for producing tyrosine according to claim 1, wherein the steps from the extraction with hot water to the crystallization and collection of tyrosine include a decolorization treatment step with hydrogen peroxide.