JP2685424B2 - Protamine production method - 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 protamine. More specifically, the present invention
Easy and high-purity extraction of protamine from fish Miko,
The present invention relates to a method for producing protamine that can be purified.

[0002]

2. Description of the Related Art Protamine is a basic protein having a molecular weight of 4000 to 8500 present in mature sperm nuclei of fish such as herring and salmon and vertebrates such as chicken, and in recent years, it has been used as a drug or food. , Has attracted attention as an antibacterial agent for cosmetics and cleaning products. As a method for industrially producing this protamine, generally, a method is known in which the testis (shirako) of a fish is ground, and protamine is extracted from the ground product with mineral acid. Since various proteins contained in blood, mucus, or algae are extracted together as impurities, conventionally, an extraction method or a purification method of a mineral acid extract solution for obtaining high-purity protamine containing no impurities is required. Various proposals have been made.

[0003] For example, a method has been proposed in which shirako is directly extracted with a mineral acid without grinding, and as a method for purifying a mineral acid extract, a salting-out method by adding an alkali agent, There have been proposed a precipitation method by addition of condensed phosphate, a precipitation method using an organic solvent such as ethanol, a method of acting an enzyme, a method of ion exchange or electrodialysis after releasing protamine.

However, it is difficult to efficiently extract and purify protamine by these methods.
In order to produce high-purity protamine, considerable effort and cost have been required. For example, even when protamine is extracted without grinding millet, basic impurity proteins such as histones are extracted at the same time, and they interfere with the subsequent purification process, making it difficult to obtain high-purity protamine. It was

Therefore, there has been a demand for the development of a new protamine production method capable of producing protamine in a high purity by a simple method. The present invention has been made in view of the circumstances as described above, improves the drawbacks of the conventional method, and can easily and selectively extract protamine from the agar of fish, and histone etc. in the extract. Impurities such as protein, blood, and mucus components are not mixed in,
Moreover, it aims at providing the manufacturing method of the high quality protamine which can also refine | purify the mineral acid extract of protamine to high purity.

[0006]

Means for Solving the Problems The present invention is to solve the above-mentioned problems by producing protamine characterized in that, when protamine is extracted from fish milts, the milts are washed in advance with dilute mineral acid. Provide a way.

[0007]

As described above, the production method of the present invention is for extracting protamine from fish milts. However, the above-mentioned washing treatment method allows selective extraction of protamine. It is possible to preliminarily wash the spores of fish using a dilute mineral acid as a washing liquid, and then to extract protamine with high purity by an arbitrary method such as extraction with a mineral acid.

In this washing treatment, impurities such as histones, impurities such as blood and mucus components are selectively extracted into dilute mineral acid, and the impurities are sufficiently washed out from the algae. It is preferable that the agar to be subjected to the washing treatment is ground so that the contaminants attached or contained therein can be sufficiently washed and removed. There are no particular restrictions on the type of white roe, and the white roe which has been conventionally used as a raw material for producing protamine such as salmon, herring, rainbow trout, and tuna can be widely used.

As the dilute mineral acid used as the washing liquid, it is preferable to use a dilute mineral acid of about 0.05 to 0.2 N, although it depends on the kind of Miko. Of course, this concentration is not limited, but it is difficult to sufficiently wash out impurities with a dilute mineral acid of less than 0.05 N, while it is not possible to sufficiently wash it. In addition, protamine is also extracted, resulting in loss of protamine and reduced productivity, which is not preferable.

The amount of the dilute mineral acid to be used is preferably determined so that the total amount of the acid (normality of the acid N × the amount of the acid used 1) is sufficient, depending on the type and amount of the Miko. It is preferable that the capacity is about 1 to 3 times the capacity of Miko. Also,
The washing treatment can be easily performed by mixing and stirring such a dilute mineral acid with Miko and separating and removing the washing liquid. In this case, the mixing and stirring time (washing time) depends on the kind of Miko and the dilute ore. Appropriately determined according to the normality of acid and the amount used so that high cleaning efficiency can be realized. In general, a sufficient washing treatment can be achieved within a few hours.

As described above, it is preferable that the cleaning conditions are set so that the mutual relationships such as the kind of white mite, the normality and usage amount of the dilute mineral acid, and the cleaning time are suitable as a whole. As such a condition, it is possible to exemplify that 1N volume of 0.2N hydrochloric acid is used with respect to the ground product of salmon or herring and other fish spores, and the washing time is 1 hour.

The method for separating and removing the washing liquid from which impurities have been extracted after washing can be carried out by a conventional method such as centrifugation or filtration. After the washing treatment is performed on the white cabbage in this manner, protamine is produced by extraction from the washed product (that is, the washed white cabbage). This makes it possible to obtain a highly pure protamine extract in which the contents of impurities such as histones and impurities such as blood and mucus components are remarkably reduced.

The extraction method in this case is not particularly limited, and a conventional extraction method using a mineral acid can be used. The extraction conditions in this case are normality and amount of the mineral acid used and extraction time. Is appropriately set so as to enhance the extraction efficiency of protamine. Although the extraction conditions depend on the type of Miko, the amount of 1N mineral acid used for Miko such as salmon and herring is twice as much, and the extraction time is 1 hour.

When setting the extraction conditions, the extraction time should not be set longer than necessary and is usually within several hours. Even when extraction is performed using a dilute mineral acid of about 0.2 N to 0.5 N, the amount of extraction is halved if the extraction takes too long. The extracted protamine can be further converted into a free form by desalting and concentrating and drying by a conventional method such as electrodialysis, if necessary, and a desired salt form such as a hydrochloride, a sulfate or a nitrate can be formed by a conventional salt forming method. Can be

As described above, according to the method of the present invention, protamine can be selectively extracted with high purity from the larva of fish by subjecting it to a washing treatment. The mineral acid extract of protamine can be extracted by an appropriate method. It may be further purified. Examples of the purification method in this case include a conventionally known salting-out method by adding an alkaline agent, a precipitation method by adding a condensed phosphate, a precipitation method by an organic solvent such as ethanol, a method for acting an enzyme, and protamine. A method such as ion exchange or electrodialysis after liberation of the is appropriately used in combination.

The method proposed by the inventor of the present invention for adding an acid-soluble cationic polymer flocculant to a mineral acid extract and separating and removing impurities can also be appropriately used. As the acid-soluble cationic polymer flocculant, those which become polycations when dissolved in an acid solution can be widely used. For example, chitosan, polyaminoalkylmethacrylate, polyethyleneimine, halogenated polydiallylammonium, ionene-based coagulant and the like can be used. Especially, chitosan can be preferably used.

These polymer flocculants can be used by a conventional flocculation and separation method. For example, when chitosan is used, chitosan is added to an extract of protamine with an arbitrary mineral acid, the mixture is stirred and mixed, the pH is adjusted to about 8 to 10, and impurities are precipitated together with chitosan. Separate and remove by centrifugation. In the present invention, in the case of producing a higher purity protamine, the purification method for the mineral acid extract from the washed algae, particularly the acid-soluble cationic polymer aggregation proposed by the present inventor, is used. It is desirable to apply a purification method using agents. That is, first, shirako is subjected to the washing treatment with the above-mentioned dilute mineral acid, then protamine is extracted from the washed product with a mineral acid, and this purification method is applied to the mineral acid extract solution of the protamine.

Even in this case, the extraction method of protamine with a mineral acid can be a conventional method. In addition, it is also possible to form a desired salt form such as a free form, a hydrochloride, a sulfate or a nitrate after the purification by a conventional method. Hereinafter, the present invention will be specifically described based on examples.

[0019]

EXAMPLES Example 1 In order to examine suitable washing conditions for Mikoshi, the following (i) to (iii) were prepared for three kinds of Mikoshi with protamine contents of 8%, 5% and 2.5%, respectively. ), And the components in those extracts were detected by electrophoresis.

(I) 1N hydrochloric acid, extraction treatment with 1 volume for 1 hour. (Ii) Extraction treatment with 0.2N hydrochloric acid and 1.5 times volume for 1 hour. (iii) Extraction treatment with 0.2N hydrochloric acid and 1.5 times volume for 1 hour, and then extraction treatment with 1N hydrochloric acid and 1 volume for 1 hour. As a result, for any kind of white spores with different protamine contents, impurities such as histones were detected along with protamine in the extract of (i), and most of the protamine in the extract of (ii). A considerable amount of histones and other impurities were detected without being detected, and in the extract of (iii), only protamine was detected without detecting impurities such as histones.

As is clear from the comparison of (ii) and (iii) with respect to (i), in order to obtain a highly pure protamine extract containing no contaminants such as histones, extraction treatment with 1N hydrochloric acid was performed. It was confirmed that it was effective to extract and remove impurities by washing with 0.2N hydrochloric acid before the treatment. Example 2 In order to examine suitable washing conditions or extraction conditions for Shirako, the extraction treatment was performed by changing the type of Shirako, the normality and amount of hydrochloric acid used as an extraction solvent, and the extraction time as follows. The recovery rate of protamine extracted in the extract is
It measured by PLC and the impurity protein component was detected by the electrophoresis method. The HPLC standard is Salmine su manufactured by Sigma, manufactured by Nakarai Tesque, and manufactured by Wako Pure Chemical Industries.
lfate, Salmine chloride, Salmine free base and Clup
Eine sulfate was used.

(I) Shirako: Protamine content 8%, 5
%, 2.5% (ii) Normality of hydrochloric acid: 0.2N, 0.5N, 1N, 2N (iii) Amount of hydrochloric acid used (volume ratio to Miko): 1 time, 2
2 times, 5 times (iv) Extraction time ... 1 hour, 2 hours, 20 hours The results of HPLC measurement are shown in FIG. 1 and FIG. 2 for each agar of protamine content 8%, 5% and 2.5%. And FIG.

As a result, although the recovery of protamine extracted in hydrochloric acid varied depending on the type of algae, it showed almost the same behavior with respect to the normality of hydrochloric acid, the amount used and the extraction time. That is, in any of the sardines, the recoveries of protamine were almost equal when using 1N and 2N hydrochloric acid, and remarkably lowered when using 0.2N. Particularly, 0.2N hydrochloric acid was used at 1-fold volume. As for the product, protamine was not detected at all in the extract.

From these results, it was found that dilute mineral acid of 0.2 N or less is suitable for washing treatment of Miko, and mineral acid having a normality higher than 0.2 N is suitable for extraction treatment. Further, when 0.5N or less, the recovery rate of protamine largely depends on the amount of hydrochloric acid used, but when it is 1N or more, the dependency becomes small, so the extraction treatment for securing a stable recovery rate. It has been found that it is preferable to use a mineral acid of about 1N or more.

Regarding the extraction time, as is clear from FIG. 2, there is no great difference in the recovery rate of protamine within a few hours, but it may be because protamine is hydrolyzed after a long-time treatment of about 20 hours. , The recovery rate is significantly reduced,
It was also found that the treatment conditions were not favorable. Example 3 1 kg of salmon shirako (protamine content 7%) was ground,
After adding 1 L of 0.2 N hydrochloric acid, the mixture was stirred and washed, and the washing solution was removed by centrifugation (7000 G, 5 minutes).

Next, 2 L of 1N hydrochloric acid was added to the residue, and the mixture was stirred and centrifuged to collect a supernatant, which was neutralized to obtain a protamine extract. When the components of this protamine extract were analyzed, the solid content was 7.44% and the protamine content was 2.8.
The content was 5%, the content of NaCl was 4.01%, and the purity of protamine (protamine content / (solid content-NaCl content)) was 83.2%.

The purity of this protamine was remarkably higher than that of the comparative example not subjected to the washing treatment described later. Example 4 In the same manner as in Example 3, 1 Kg of salmon roe was washed with 1 L of 0.2 N hydrochloric acid, protamine was extracted with 2 L of 1 N hydrochloric acid, and centrifuged to collect a supernatant.

To this supernatant, 2.5 kg of chitosan was added to adjust the pH and the protamine extract was purified. When the components of this purified protamine extract were analyzed, the solid content was 6.90%, the protamine content was 2.68%, the NaCl content was 4.05%, and the purity of protamine was 94%. COMPARATIVE EXAMPLE In the same manner as in Example 3, 1 kg of salmon shirako was ground and 1
2 l of N hydrochloric acid was added, and the mixture was stirred, centrifuged and the supernatant was collected and neutralized to obtain a protamine extract.

When the components of this protamine extract were analyzed, the solid content was 8.77% and the protamine content was 2.65.
%, NaCl content 3.78%, the purity of protamine was only 53.1%. The protamine purity of this comparative example, which was not subjected to washing treatment or purification treatment, was significantly inferior to that of the above-mentioned examples. Example 5 Salmon sardine that had been frozen and stored (protamine content 7%)
1 kg was thawed and after grinding, the rind was removed. 0.2 to this
1 l of N hydrochloric acid was added, and the mixture was stirred for 1 hour for washing, and the washing solution was removed by centrifugation (7000 G, 5 minutes).

Next, 2 l of 1N hydrochloric acid was added to the residue, the mixture was stirred for 1 hour, centrifuged and the supernatant was collected to obtain a protamine extract. 0.2 to 1.5 liters of this protamine extract
150 ml of 1% chitosan (Flownac made by Kyowa Yushi Kogyo Co., Ltd.) dissolved in hydrochloric acid of N was added, pH was adjusted to 8 to 10 for coagulation and precipitation, centrifugation was performed to collect a supernatant, and a protamine extract 1.7 l of purified liquid was obtained.

Next, using this purified solution, (i) protamine sulfate, (ii) protamine hydrochloride, and (iii)
Free protamine was prepared and their purity was measured. (I) Protamine sulfate 100 ml of 10% sulfuric acid per 1 liter of purified protamine extract
And 5 l of ethanol were added, and the mixture was cooled to 5 ° C and then centrifuged (7
(000 G, 5 minutes) and the residue was collected and further dried to obtain 36 g of protamine sulfate. The purity of this protamine sulfate was 94%. (Ii) Protamine Hydrochloride 5 liters of ethanol was added to 1 liter of the purified protamine extract solution, cooled to 5 ° C. and centrifuged (7000 G, 5 minutes) to collect the residue, which was further dried to obtain 35 g of protamine hydrochloride. The purity of this protamine hydrochloride was 93%. (Iii) Free protamine Purified protamine extract 1 l was adjusted to pH 13, desalted and dialyzed by electrodialysis (Asahi Glass Co., Ltd. Du-ob type, 30 V, 5 hr) until almost no current flowed, and further concentrated and dried. 27.5 g of free protamine was obtained. The purity of this free protamine was 95%. Example 6 Salmon Miko that had been frozen and stored (protamine content 4%)
Protamine sulfate, protamine hydrochloride and free protamine were produced in the same manner as in Example 5 except that 1 Kg was used and 1.5 L of 0.2 N hydrochloric acid was used.

As a result, 18 g of protamine sulfate, 18 g of protamine hydrochloride and 15 g of free protamine were obtained. Example 7 Sheep of frozen herring (protamine content 4
%) 1 Kg was treated in the same manner as in Example 5 to obtain 1.7 l of a purified protamine extract.

Next, this purified solution was treated with an anion exchange resin (Duolite A-162 manufactured by Sumitomo Chemical Co., Ltd.), and ethanol 5 was added.
l, added, cooled to 5 ° C., and then centrifuged (7000 G, 5
And the residue was collected. The residue was redissolved in 100 ml of warm water and 1 l of ethanol was added to obtain a precipitate. This was dried to obtain 20 g of free protamine. The purity of this free protamine was 98%, which was extremely high purity.

[0034]

Industrial Applicability According to the method of the present invention, protamine can be easily and selectively extracted from the alga of fish and the protamine extract can be highly purified. It is possible to produce high-quality protamine which contains almost no impurities and is extremely high in purity and useful for pharmaceuticals.

[Brief description of the drawings]

FIG. 1 is a correlation diagram between the concentration of hydrochloric acid and the recovery rate, which shows the recovery rate of protamine from agar which has a protamine content of 8%.

FIG. 2 is a correlation diagram between the concentration of hydrochloric acid and the recovery rate, which shows the recovery rate of protamine from agar which has a protamine content of 5%.

FIG. 3 is a correlation diagram between the concentration of hydrochloric acid and the recovery rate, which shows the recovery rate of protamine from agaricus having a protamine content of 2.5%.

Claims (3)

(57) [Claims] 1. A method for producing protamine, comprising the steps of: before extracting protamine from fish milts, wash the milts with dilute mineral acid in advance. 2. The method for producing protamine according to claim 1, wherein the dilute mineral acid is 0.05 to 0.2N. 3. The method for producing protamine according to claim 1 or 2, wherein the dilute mineral acid is used in an amount of 1 to 3 times the amount of the fish roe.