JPH06199900A - Production of mucin - Google Patents

Abstract

PURPOSE:To readily obtain a high-purity mucin containing not fats by using saliva directly collected from bovine suborbital gland as a raw material. CONSTITUTION:A mucin is produced by separating the mucin from saliva of bovine suborbital gland. For example, bovine saliva is treated by cetavlon method to give a crude mucin hardly containing lipid, etc. The prepared crude mucin is further purified by ultrafiltration to give a high-purity mucin from which mucin fragments having <=100,000 molecular weight are removed. According to a preferable condition, a more purified mucin can be obtained by precisely filtering the crude mucin before the ultrafiltration.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention separates mucin from saliva directly collected from bovine submandibular gland as a raw material with high purity,
It relates to a method for producing fat-free mucin.

[0002]

BACKGROUND OF THE INVENTION Salivary gland mucin is a salivary gland of mammals such as bovine, equine, porcine and ovine, namely, submandibular gland,
It is a glycoprotein that is biosynthesized and secreted in the parotid gland and sublingual gland, and has a unique consistency depending on the type of animal. It is a macromolecule containing a large amount of sialic acid and having a molecular weight of about 100,000 to 1,000,000. is there.

It is known that mucin has an excellent moisturizing action and emulsifying action and is highly safe for the human body. In recent years, focusing on the characteristics of these mucins, cosmetics,
Research is being conducted on its use as a moisturizer for pharmaceuticals (especially external skin preparations) and as an emulsifier in various industrial fields such as foods, cosmetics, pharmaceuticals, quasi-drugs, toiletries and the like.

The method for separating and purifying salivary mucins has been studied in detail up to the present, mainly using bovine, ovine and porcine submandibular glands, and the following methods have been widely used.

After homogenizing the submandibular gland from which fat and connective tissue have been removed, an appropriate solvent (water, 0.1M NaCl,
0.05M phosphate buffer (pH 7.0, etc.) 2-3
The mixture is added in an amount twice and the mixture is stirred and extracted for 5 to 10 hours while keeping the mixture neutral. The insoluble matter was removed from the extract through several layers of gauze, and the filtrate was centrifuged at 10,000 xg for 20 minutes to obtain a transparent solution. The residue on gauze is similarly extracted several times, and the whole extract is mixed and purified. For purification, the extract is acidified (p
H3 to 2.5), the mucin clot method, which utilizes the property that mucin binds and precipitates with other proteins, ammonium sulfate fractionation method, alcohol fractionation method in the presence of Ba ²⁺ and Ca ²⁺ , setablon (Cetyltrimethylammonium bromide), cetylpyridinium chloride (CPC), or other such precipitation method using a quaternary amine positive surfactant, and finally a means such as dialysis was adopted.

Of the above-mentioned purification methods, the precipitation method using setablon or CPC (hereinafter referred to as the setablon method) is currently considered to be the most effective.

According to the setablon method, when a 5-10% solution of setablon is dropped into the submandibular gland extract, mucin containing sialic acid forms a complex with setablon and precipitates. The precipitate is concentrated to a high concentration of neutral salt, for example 50% (w /
When it is dissolved in the CaCl ₂ solution of v) and alcohol fractionation is performed, mucin is precipitated at a concentration of 50-60%. The precipitated mucin is collected by centrifugation, dialyzed and dried to obtain a mucin dehydrated powder.

The yield of mucin is 0.2 of the fresh weight of the submandibular gland.
.About.0.5%, and the obtained mucin was a single component by electrophoresis or ultracentrifugation.

[0009]

However, according to the above-mentioned conventional method, fat invading the submandibular gland tissue is also extracted, and it is difficult to sufficiently remove it. Mucin without fat could not be easily obtained.

[0010] The present invention provides a method for obtaining a high-purity, fat-free mucin using the submandibular saliva by inserting a cannula into a bovine submandibular canal and directly collecting saliva. Has an aim.

[0011]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have found that bovine submandibular saliva can be easily collected as follows. And if you use this submandibular saliva that has never been known as a raw material for mucin production,
We have found that mucin without fat can be easily obtained.

That is, the submandibular skin of bovine is incised, the submandibular duct is exposed near the epidermis of the oral cavity, and a slight incision is made.
A cannula is introduced there through an incision in the skin, and is inserted toward the submandibular side from the opening of this conduit. On the other hand, the cut wound of the skin is closed while fixing the cannula with adhesive tape.
The tip of the cannula is extended to an appropriate length, a receiver is placed on the tip, and the submandibular saliva that spontaneously falls is collected. Add saliva to the collected saliva and store it in a cool place. here,
As the preservatives, usual preservatives, such as benzoic acids, paraoxybenzoic acids, dehydroacetic acids, sorbic acids, phenols, alcohols, halogens, glutaraldehydes, β-propiolactones, or the like, or Although they can be used in any combination, they are preferably odorless or those having an odor that can be easily removed in a subsequent step, and paraoxybenzoic acids are particularly preferable.

The collected bovine saliva is then subjected to a mucin separation / purification treatment according to a conventional method. As the separation / purification method, any of the methods described in the above-mentioned prior art or any other applicable method can be used.For example, by treating bovine saliva with the setablon method, almost all lipids and the like are contained. No crude mucin is obtained.

The obtained crude mucin can be further purified by ultrafiltration to obtain highly pure mucin from which mucin fragments having a molecular weight of 100,000 or less have been removed. Conventionally, it was thought that those with high viscosity and insoluble matter (fibrous substance) such as mucin were not suitable for purification by ultrafiltration, but when purification of salivary gland mucin was performed by ultrafiltration, Practically, there is no problem of clogging of the filtration membrane, and the molecular weight is 1
Mucin fragments of less than 0,000 and impurities are removed, and the desired mucin having a molecular weight of 100,000 or more can be efficiently obtained.

Here, ultrafiltration means an operation of separating a suspended substance from a dispersion medium by filtration using a dialysis semipermeable membrane or a gel membrane. The ultrafiltration method using a semipermeable membrane for dialysis is one in which various compounds existing in a dispersed state in a liquid are filtered out according to the size of the passage hole of the membrane. Further, in the ultrafiltration method using a gel membrane, by applying pressure to an aqueous solution of various compounds in contact with the membrane, the solvent water is diffused by exchanging with a large amount of water molecules retained in the gel membrane, Eventually it will flow out of the membrane. At this time, the low molecular weight compound existing in the solvent also diffuses into the gel film along with water and is filtered out of the film,
The polymer compound having large particles has a very low diffusion rate in the gel film as compared with the low molecular compound and is practically hardly filtered, and as a result, the low molecular compound and the polymer compound are filtered. In addition, the gel film is classified into a flat plate type and a tube type. Ultrafiltration is a method suitable for concentrating a solution such as protein under very mild conditions.

The ultrafiltration used in the present invention is a so-called gel filtration ultrafiltration. The gel film used has a molecular weight of 10
A flat plate-shaped one or a tube-shaped one may be used as long as the substances can be separated in about 10,000 times. The flat gel film that can be used in the present invention includes YM100
(Manufactured by Amicon) and the like. As the tubular gel film, H1P100, H10P100, H10X1
00 (above made by Amicon), SM14669 (made by Sartorius), Pellicon PTHK (made by Millipore) and the like.

The applied pressure is usually 0.5 to 7 kg / c
m ² , preferably in the range of 1 to ³ kg / cm ² . The flow rate is usually 1 to 10 liters / minute, preferably 2 to 5 liters / minute.

The concentration of the crude mucin solution to be filtered is usually 0.05 to 1%, preferably 0.1 to 0.4%.

Examples of the ultrafiltration device that can be used in the present invention include those manufactured by Amicon, Inc., Diaflow Hollow Fiber System DC30, and those manufactured by Dow, Millipore, Sartorius, Asahi Kasei and Bioengineering. .

According to a preferred embodiment of the present invention, the crude mucin is subjected to microfiltration prior to ultrafiltration,
More purified mucin can be obtained.

The term "microfiltration" as used herein means clarification filtration for trapping suspended matter with high precision, and specifically, it is made of synthetic polymer such as cellulose derivative, polyamide, nylon, etc. This is an operation of forming a multilayer film (membrane filter) having a mesh of, and using it to clarify the solvent. Before performing microfiltration, it is called prefiltration, and filtration using a filter aid such as Celite,
Alternatively, it is a common practice to extend the life of the microfiltration membrane by a treatment such as filtration using a coarse mesh filter.

When performing microfiltration, the pore size of the filter which can be used in the present invention is usually 0.2 to 8.0 μm.
m, preferably 0.45 to 1.2 μm. Therefore,
The filter that can be used in the present invention is Ultipore N ₆₆ NN.
Examples include XZP (manufactured by Pall), CWSC01TP3 (manufactured by Millipore), SM5322406D1PH (manufactured by Sartorius), and the like. Examples of the microfiltration device include those manufactured by Pall, Millipore, and Sartorius.

When performing microfiltration, it is preferable to dilute the concentration of crude mucin to 0.1 to 0.4% (w / v).
By performing microfiltration prior to ultrafiltration, mucin having a molecular weight of 100,000 or more can be purified and concentrated more efficiently.

If microfiltration is not performed, ultrafiltration using a tubular gel membrane may cause clogging, so it is preferable to use a flat gel membrane. Hereinafter, the present invention will be further described with reference to examples.

[0025]

【Example】

Example 1 Bovine submandibular saliva was collected, 0.15% of methyl paraoxybenzoate was added as a preservative, and the mixture was stored in a cool place. 10 liters of this submandibular gland saliva in an extractor with a cooling device,
A 10% (w / v) CPC aqueous solution was added dropwise with stirring at 5 to 10 ° C to cause precipitation. The dropping is continued until no precipitation occurs. The precipitates were collected, 50% (w / v) calcium chloride aqueous solution was added and stirred, and ethanol was added to this solution to recover precipitates in the ethanol concentration range of 60 to 75% (v / v). An appropriate amount of ethanol was added to this to remove CPC and dehydrate to obtain an ethanol precipitate (crude mucin).

Example 2 Crude mucin (corresponding to 10 liters of submandibular saliva) and water 30
Add 0 ml to dissolve, and insoluble matter remains (dialysis tube (Visking dialysis tube C-150)
The mold was filled in a mold having an inner diameter of 100 mm) and dialyzed against water. After dialysis, the in-membrane liquid was taken, insoluble matter was removed by rough filtration, and fine insoluble matter was removed using a precision filtration device (manufactured by Pall Co., using a filter having a hole diameter of 1.2 to 0.45 μm). At this time, the mucin concentration was about 0.2%. Next, the filtrate was centrifuged at 11,000 × g for 20 minutes to remove insoluble matter, and freeze-dried to obtain 10 g of submandibular gland mucin. This mucin has a sialic acid content of 0.71%, a protein content of 0.82%,
The sialic acid / protein ratio was 0.87.

Example 3 4 liters of water was added to and dissolved in crude mucin (equivalent to 10 liters of submandibular saliva), insoluble matter was removed by coarse filtration, and a microfiltration device (manufactured by Pall Ltd., hole diameter 1.2) was used. .About.0.45 .mu.m filter was used) to remove finer insoluble matter. At this time, the mucin concentration was about 0.2%. Next, an ultrafiltration device (Diaflow Hollow Fiber System DC4 type manufactured by Amicon, using cartridge HIP100-2) was used to remove those having a molecular weight of 100,000 or less and salts. The pressure during ultrafiltration is 0.7 kg / cm ² (10 p
si) and the flow rate was 200 ml / min. The filtrate was centrifuged at 11,000 × g for 20 minutes to remove insoluble matter and freeze-dried to obtain 8 g of submandibular mucin. This mucin has a sialic acid content of 0.71% and a protein content of 0.7.
The ratio was 8% and the sialic acid / protein ratio was 0.91.

[0028]

EFFECT OF THE INVENTION By using submandibular saliva that has never been known as a raw material for producing mucin, mucin having high purity and containing no fat can be easily obtained.

Claims (3)

[Claims] 1. A method for producing mucin, comprising separating mucin from bovine submandibular saliva. 2. The method according to claim 1, wherein the separated mucin is further purified by ultrafiltration. 3. The microfiltration is performed before the ultrafiltration.
The described method.