JPH0636750B2 - Method for concentrated preparation of milk ganglioside - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention is a method for advantageously concentrating a high content ganglioside from ganglioside originally contained in milk using various dairy substances as starting materials. Regarding

(Prior art and its problems) In recent years, biological research on gangliosides has become popular, and research on gangliosides in the fields of medicine and pharmacy such as nerve function, canceration, viral infection, differentiation, proliferation, hormone sceptor, etc. becomes important. It has become to. In particular, ganglioside has been attracting attention as a unique active substance localized on the cell surface.

Ganglioside is a substance known to be present in large amounts in the brain of animals, but is also present in milk in relatively large amounts.

For milk ganglioside, see Nagai et al.
27 , 182-185 (1985)), and its preparation method has recently been applied for a patent as a neutralizing agent for toxins (JP-A-60-72819). This method is a method for obtaining a fat globule membrane from buttermilk, but the protein content is high, and ganglioside is not obtained at a high concentration.

In addition to this, at present, there is no known method for obtaining a high concentration of ganglioside, and it has been considered difficult to prepare milk ganglioside on an industrial scale, and it is not easy.

Therefore, it has been desired to develop a method for efficiently and advantageously concentrating ganglioside contained in a milk substance to obtain a high concentration of ganglioside.

It is an object of the present invention to provide a method for efficiently concentrating and preparing ganglioside from a milk substance containing ganglioside as a starting material.

(Means for Solving Problems) The present invention is characterized in that a dairy substance containing milk ganglioside is reacted with a proteolytic enzyme to decompose the protein, and the resulting proteolytic solution is filtered to concentrate the ganglioside. And

The filtration is preferably performed by an ultrafiltration method or a gel filtration method. Besides this, a dialysis method can also be used.

In the present invention, examples of the dairy substance used as a starting material include milk, buttermilk obtained from milk, whey, skim milk, and the like.

These dairy substances contain proteins, lactose, lipids, etc. in addition to gangliosides, and therefore these substances must be removed.

To remove proteins, proteolytic enzymes are used to decompose peptides or amino acids. Examples of this proteolytic enzyme include trypsin, pepsin, chymotrypsin, papain, takadiastase, and Bacillus subtilis protease. The reaction conditions are, for example, 3% within the range of pH 2-10 and temperature 20-60 ° C by adding proteolytic enzyme to 0.1 to 10% by weight with respect to the protein concentration in the raw material milk substance.
It is preferable to carry out the reaction in about 100 hours.

In this way, the solution obtained by decomposing the protein in the milk substance is heated at 90 to 100 ° C. for several minutes to inactivate the enzyme.

Next, this solution is treated by an ultrafiltration method, a gel filtration method, a dialysis method or the like to obtain a concentrated ganglioside.

The ultrafiltration method is a molecular-level filtration method that screens large solute molecules from small solute molecules and solvent molecules. To concentrate gangliosides by this method, an apparatus equipped with an ultrafiltration membrane is used. Using this device, gangliosides are concentrated in the concentration tank, and peptides, amino acids, lactose, and other low-molecular substances decomposed by proteolytic enzymes are removed from the eluate. The ultrafiltration membrane has a molecular weight cut off of 2,000 to 500,000, such as dialysis membrane or DD.
GR-51P manufactured by S company can be used.

In the gel filtration method, a solution containing a high molecular solute and a low molecular solute is added to a column of gel particles swollen with a solvent, the solvent is allowed to flow, and the high molecular solute and the low molecular solute to be eluted later are separated. Is the way. In this method, the proteolytic solution is passed through a gel filtration column to obtain concentrated gangliosides in the void fraction. As the gel, commonly used ones such as Sephadex, biogel, agarose gel and the like are used.

The concentrated solution thus obtained is collected and dried to obtain a high content ganglioside powder.

The ganglioside content in the powder obtained as described above is
Although it depends on the dairy substance, it is usually contained in an amount of 0.5 to 5%. In order to further increase the purity of ganglioside, a conventionally known method, for example, a method of adsorbing ganglioside on an ion exchange resin, eluting it, and then separating and purifying by silica gel chromatography to obtain a single molecular species, etc. Can be used.

(Effect of the invention) The present invention is a novel method for concentrating milk ganglioside contained in a milk substance, that is, degrading a protein using a proteolytic enzyme, and concentrating the protein by an ultrafiltration method, a gel filtration method, a dialysis method, or the like. Since the method of preparation was used, gangliosides could be efficiently and advantageously concentrated and prepared, and a high concentration of gangliosides could be prepared on an industrial scale.

It should be noted that ganglioside generally forms a complex with a protein, and an organic solvent containing alcohol is essential for cutting off the result.

However, in the present invention, it has become possible to concentrate and prepare without the need for these alcohols.

(Examples) Hereinafter, the present invention will be specifically described with reference to Examples.

Example 1 0.5 kg of a solution prepared by dissolving 20 kg of buttermilk powder in 180 water
Bacillus subtilis protease is added to the solution at pH 7.6 at a temperature of 40 ° C for 15
After reacting for a time to decompose the protein, it was heated at 90 ° C. for 10 minutes to inactivate the enzyme.

Then, this solution was added to a membrane area of GR-51P (manufactured by DDS) of 0.
Using an ultrafiltration device (LAB-20 type module, manufactured by DDS) equipped with a 36 m ² filtration membrane, temperature 40 ° C., flow rate 15 /
Minutes, the mixture was filtered at an average pressure of 0.6 MPa to concentrate the ganglioside. After the concentration was completed, the concentrated liquid was collected from the concentration tank and freeze-dried to obtain 1400 g of powder.

The powder thus obtained contained 2.0% gangliosides.
% (However, converted to sialic acid).

Example 2 To milk 50, 150 g of trypsin was added, pH 7.6, temperature 40 ° C.
After decomposing the protein for 24 hours at 90 ° C., the enzyme was inactivated by heating at 90 ° C. for 10 minutes.

Then, this solution was passed through a gel filtration column (60 × 100 cm) packed with HW-55 (manufactured by Toyo Soda), and a void fraction in which ganglioside was concentrated was collected and freeze-dried to obtain 1200 g of a powder. Got

The powder thus obtained contained 0.5% gangliosides.
% (However, converted to sialic acid).

Example 3 1 kg of high-content ganglioside powder prepared according to Example 1 and Example 2 was mixed with a chloroform-methanol mixture (1:
10) of 1) was added, and the mixture was stirred at room temperature for 30 minutes and extracted.

Next, the chloroform-methanol mixed solution was collected and passed through an ion exchange resin (DEAE-Toyopearl acetate type) 1 to adsorb ganglioside, and then the resin was washed with a chlorohelm-methanol mixed solution (1: 1) 2 and then 0 .
Gangliosides were eluted with 5 molar sodium acetate-methanol solution 4. After concentrating the eluate and removing methanol, water 5 was added to the ultrafiltration membrane (GR-61pp, DDS
Desalination and concentration of ganglioside were performed.

The concentrated solution containing ganglioside was dried with an evaporator, and then passed through a silica gel column (4.0 × 50 cm) to adsorb ganglioside, and then sequentially washed with chloroform 1 and chloroform-methanol mixed solution (4: 1) 1, followed by chloroform: The ganglioside was eluted with a mixed solution 2 of methanol (2: 8). The eluate was evaporated to dryness with an evaporator to obtain 15 g of white powder.

The purity of the thus-obtained powder ganglioside was 90% or more (resorcinol method) by thin film chromatography.

Claims (2)

[Claims] 1. A method for concentrating milk ganglioside, which comprises concentrating the ganglioside by treating a milk substance containing milk ganglioside with a proteolytic enzyme to decompose the protein and treating the resulting proteolytic solution. 2. The method for concentrated preparation of milk ganglioside according to claim 1, wherein the treatment is carried out by an ultrafiltration method, a gel filtration method or a dialysis method.