JPH02218620A - Production of active ingredient for disease in oral cavity - Google Patents

Abstract

PURPOSE:To obtain an active ingredient for preventing periodontosis, dental caries, etc., by adding a polysaccharide to a vitellus fraction of egg prepared from a domestic fowl immunized against a bacterium in the oral cavity as an antigen, aggregating lipid and separating. CONSTITUTION:A domestic fowl is immunized against the whole cell or a cell component of bacterium in the oral cavity, a vitellus fraction (antibody is accumulated and exists) obtained from the domestic fowl is collected, the fraction is diluted with a 5-100mM buffer solution at pH5-9, a solution part thereof is collected by a means such as centrifuging, is blended with a polysaccharide such as dextran sulfate, carrageenan, xanthan gum, etc., existing lipid is aggregated, separated and removed to give an active ingredient having <=5wt.% lipid content.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing an active ingredient that is incorporated into an oral composition, 9 foods, etc., and is used to prevent oral diseases. Then, the active fraction is efficiently fractionated from the egg yolk fraction of poultry eggs that have been immunized with whole cells or cell components of oral bacteria as antigens, and active ingredients that prevent oral diseases such as periodontal disease and dental caries are obtained. Regarding how to get .

[Prior art and problems to be solved by the invention] Periodontal disease and caries, such as periodontitis and alveolar pyorrhea, are said to be two major oral diseases.The causes of these oral diseases include, for example, the former. In the latter case, bacteria such as Bacteroides gingivalis, and in the latter case, bacteria such as Streptococcus mutans are said to colonize and proliferate in the oral cavity. Therefore, prevention of oral diseases requires the prevention of these bacteria. It is effective to suppress colonization in the oral cavity.

Conventionally, from this point of view, oral compositions have been prepared using antibodies in blood or milk obtained by immunizing mammals with whole cells or cell components of periodontal disease-causing bacteria such as Bacteroides gingivalis or Streptococcus mutans. It is known that the above-mentioned bacteria can be incorporated into foods to suppress colonization of the oral cavity and prevent oral diseases (Japanese Patent Application Laid-open No. 14291-1989).
5. Japanese Patent Publication No. 60-38327. Japanese Patent Publication No. 60-38329
゜Japanese Patent Publication No. 61-112028. JP-A-61-11202
9゜Unexamined Japanese Patent Publication No. 61-112030. Japanese Patent Publication No. 61-2890
24° Japanese Patent Publication No. 62-417. Japanese Patent Publication No. 61-277632
Publication No.).

In addition, from the viewpoint of incorporation into oral compositions, whole bacterial cells or bacterial cell components of oral bacteria can be used as an active ingredient that can effectively suppress the colonization of oral bacteria and prevent periodontal disease and caries. It has also been previously proposed to use eggs of poultry immunized with this as an antigen (Japanese Patent Application No. 63-205109, 20511).
No. 0).

However, this egg-derived active ingredient has antibodies accumulated and present in the egg yolk, but the egg yolk contains a considerable amount of lipids, and lipids are the component 1 necessary for stabilizing antibodies. For example, it has been found that it is undesirable to incorporate such lipids into oral compositions as they are, since they act as inhibitors of the stabilizing effects of proteins, peptides, and saccharides, and therefore it is necessary to remove such lipids. In this case, lipids can be removed by column chromatography or the like, but a simple purification and fractionation method suitable for industrial production is desired.

[Means and effects for solving the problem] As a result of various studies in order to meet the above-mentioned needs, the present inventors have discovered (1) oral bacteria, particularly Bacteroides gingivalis, Bacteroides intermedius, and Actinomyces visco-
The antigen is one or more whole bacterial cells or bacterial cell components of Haemophilus actinomycetemcomitans, Fusibacterium nucleatum, Iconella corrodens, Streptococcus minitans, Streptococcus sanguis, etc. When poultry is immunized,
Antibodies accumulate and exist in the yolk of the obtained eggs, and these antibodies have excellent effects as active ingredients for preventing oral diseases, but as mentioned above, this yolk fraction contains a considerable amount of lipids. It has been found that this fact inhibits the stability of antibody combination when antibody-containing egg yolk is blended into oral compositions, etc., and that lipids account for 5% of the active ingredient containing the antibody.
(% by weight, the same applies hereinafter) or less, it was found that the stability of the combination of the antibody was not inhibited. For this reason, we investigated a simple method to reduce the lipid content to 5% or less, and found that adding polysaccharides to the egg yolk fraction caused the lipids in the egg yolk to aggregate, and when the aggregated lipids were removed, the lipids A purified fraction with 5% or less concentrated antibodies can be obtained, and this purified fraction has high agglutination activity against antigens, and is therefore highly effective in suppressing the colonization of oral bacteria. It has been found that the compound can be stably incorporated into compositions for use, etc., and can effectively exhibit its effects even after long-term storage.

The present invention will be explained in more detail below.

The method for producing an active ingredient for preventing oral diseases according to the present invention includes:
Oral bacteria such as Bacteroides gingivalis, Bacteroides intermedius, Actinomyces viscosus, Haemophilus actinomycetemcomitans, Fusibacterium nucleatum, Iconella corrodens, Streptococcus mutans, Streptococcus sanguis, etc. A species or two or more whole bacterial cells or bacterial cell components are used as antigens, and polysaccharides are added to the egg yolk fraction of eggs obtained from poultry immunized with the antigen to aggregate the lipids in the egg yolk fraction. A fraction with a lipid content of 5% by weight or less is collected after separating and removing aggregates.

Here, Bacteroides gingivalis, Bacteroides intermedius, Actinomyces visco
For Bacteroides gingivalis, Bacteroides intermedius, etc., all bacterial cells such as Haemophilus actinomycetemcomitans, Fusibacterium nucleatum, and Iconella corrodens are cultured using Todohepit broth supplemented with hemin and menadione. For other bacteria, trypticase soy broth is used as a medium, and the grown bacteria are cultured and pretreated according to known methods, such as washing and formalin treatment, and used as antigens. It is possible. In addition, the fimbriae and capsule of these bacteria are used as bacterial body components, and these fimbriae antigens and capsule antigens are separated from the bacterial body according to known methods. be able to.

Note that any known strains of these bacteria can be used; for example, those provided by Microtechnical Research Institute, American Type Culture Collection (ATCC), Forsyth Dental Center in Boston, etc. Bacterial strains, bacterial strains isolated from periodontal disease lesions, etc. may be used.

On the other hand, Streptococcus mutans, Streptococcus sanguis, etc. can be cultured and pretreated using known methods, such as using the external dialysis solution of BHI medium as a culture medium, washing the grown bacteria, and then treating them with formalin. Can be used. In this case, Streptococcus mutans is classified as serotype C, which is abundant in the human oral cavity.
Preferably, those belonging to As such Streptococcus mutans bacteria, NCTC10449゜In
gbritt, OMZ70. Examples include JC-2.

In addition, as Streptococcus sanguis, NCT
C10556, NCTC10558 and 5SH-83,
Examples include KIH-T.

Bacterial body components of Streptococcus mutans, Streptococcus sais, etc. include cell wall fractions and fibrous structure fractions of these bacteria.

Examples include the glucosyltransferase (GTF) fraction of the bacterium and the protein antigen fraction of the bacterium.

In this case, the cell wall fractions of Streptococcus mutans and Streptococcus sanguis were determined using the method of Bleiweig et al.
iol, t88, 1198-1200.1964), using a Braun cell disrupter, with a diameter of 0.17~
Crushing is carried out using 0.18 glass beads,
It is possible to use a product prepared by treating the obtained cell wall with trypsin to remove proteins mixed in the cell wall, washing with distilled water, and freeze-drying.

The fibrous structure fraction was prepared by J. Van Houte (J, V
(Arch, 0ra1.
Bio,,Top 6. 1131-1141゜1971)
According to this, 5% sucrose is added to BHI dialysis medium, cultured under anaerobic conditions, and 3 times the amount of ethanol is added to the centrifuged culture supernatant to collect the precipitate. can. Furthermore, the GTF fraction was determined using the method of Inoue et al. (Microbial Aspects of de
tal caries Vol, m.

6 6 5-6 8 2. 1 9 7 6 [In
formation Ratrfeval Inc.]
), Streptococcus mutans, Streptococcus sanguis, etc. were inoculated into BHI dialysis medium, and after growth, the bacterial cells were removed by centrifugation. Ammonium sulfate was added to the supernatant, and the 40% ammonium sulfate fraction was precipitated to 50 mM. A product prepared by dialyzing it with a phosphate buffer and using a concentrated solution can be used. Also.

The protein antigen fraction was determined by the method of Lahner et al. (J, General Microbiology
, 122, 217-225.1981),
Cultured in BHI dialysis medium and centrifuged, the supernatant obtained was
% ammonium sulfate, collect the precipitate,
This precipitate was subjected to DE-52 column chromatography in the presence of 6M urea, and the protein antigen fraction was further dissolved in physiological saline, and after dialysis, gel filtration was performed using Sepharose CL6B to obtain a fraction. can be used.

In the present invention, when poultry is immunized using the antigen, the poultry to be immunized is a chicken or a duck.

Quail etc. are used. In addition, poultry can be immunized with antigens alone or with a combination of two or more. In this case, the usual methods include subcutaneous or intramuscular immunization with an adjuvant, oral immunization with feed or water, etc. method can be adopted.

Accordingly, in the present invention, polysaccharides are added to the egg yolk fraction of eggs obtained from poultry that has been immunized in this way to perform a lipid aggregation treatment.

Here, when treating the egg yolk fraction with a polysaccharide, as a pretreatment, a pH 5-9. Concentration 5-10
It is preferable to adopt a method of diluting the egg yolk fraction using a 0mM buffer, suspending the egg yolk fraction in these buffers, and then collecting the solution portion by means such as centrifugation. It is recommended to add polysaccharides to the solution part thus obtained. Note that 0.001 to 0.1% of NaN can be added as a preservative to the above buffer solution. Furthermore, the above-mentioned egg yolk fraction can be suspended in the buffer solution by sufficiently stirring, but the stirring temperature is 4 to 30°C.
The solution part (
When obtaining supernatant), centrifugation is performed at 8000 x g ~ 16 oo
oxg. However, of course, these conditions can be selected as appropriate and are not limited thereto.

As the polysaccharide, dextran sulfate, carrageenan, gum arabic, xanthan gum, various pectins, sodium alginate, etc. can be used. In addition, the amount of polysaccharide added was 1000 d4. pair 0.1~1
It is preferable to add these polysaccharides dissolved in the above buffer, but in this case, the polysaccharides may be dissolved at a concentration of 0.1 to 15% by weight/volume. can.

After adding the polysaccharide, it is preferable to stir the liquid.
This allows the lipids to be sufficiently aggregated. Further, if necessary, a calcium salt such as calcium chloride, calcium carbonate, calcium phosphate, etc. can be added to this to further promote aggregation.

The active ingredient fraction of the present invention can be obtained by removing the precipitate from the liquid in which flocculation and precipitation have occurred by centrifugation, etc., and collecting the solution portion (supernatant). In this case, in the solution part.

Furthermore, the concentration of ammonium sulfate is 5~
It is preferable to carry out a fractional precipitation treatment in which the concentration is 40% by weight/volume and the resulting precipitate is collected by means such as centrifugation.The above centrifugation operation is not limited to It can be carried out at ~16000Xg.

The fraction of the present invention thus obtained (the solution part if the fractional precipitation treatment with ammonium sulfate etc. cannot be carried out, and the precipitation part if the above-mentioned fractional precipitation treatment is subsequently carried out) is then preferably ion-exchanged. After thorough dialysis with water or distilled water to remove salts and filter sterilization, it can be stored frozen, freeze-dried, etc.

The active ingredient produced according to the present invention is disclosed in Japanese Patent Application No. 63-2
As described in No. 05109 and No. 205110, it can be used by blending it into oral compositions, foods, etc. However, the active ingredient of the present invention has lipids removed by the above-mentioned operation, and the lipid is not contained in the active ingredient. Although it is contained in an amount of 5% or less, such a lipid content does not become a factor that inhibits the stable incorporation of antibodies into compositions, and therefore the effect of the active ingredient of the present invention remains unchanged even after long-term storage. It also performs well.

〔Effect of the invention〕

According to the present invention, an active ingredient with excellent formulation stability can be obtained by a very simple operation without employing precise fractionation means such as column chromatography, and is suitable for industrialization.

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to the following examples. [Example 1] Eggs obtained by immunizing chickens with all the oral bacteria shown in Table 1 were treated according to the following steps. , a purified fraction was obtained from egg yolk.

1. The yolk was separated from the white, and the corpus luteum membrane was cut and removed. 1
Weigh 00 mQ of yolk and add 400 mQ of Tris buffer (T
BS: 0.15M NaCQ, 10mM Tris-HCl (
pH 7.2), diluted with 0.1% NaNz).

2. The solution was stirred at room temperature for 30 minutes.

3. This yolk suspension was heated at 14,000×g at 20°C.
The mixture was centrifuged for 30 minutes to obtain a supernatant.

4. TB S 50d containing 7.5% w/v dextran sulfate was added to the supernatant and stirred gently for 10 minutes.

5, then IMCaCQ was added, and the solution was allowed to stand for 30 minutes to allow precipitation of excess dextran sulfate.

6. The 1!1 suspension was centrifuged to obtain a supernatant.

7. Furthermore, the operation of adding saturated ammonium sulfate solution to this supernatant for salting out, and centrifuging to obtain a precipitate was repeated three times.

8. This precipitate was sufficiently dialyzed against ion-exchanged water or distilled water to remove salts, and was transiently sterilized with 0.45 Ilm, and then this sample was freeze-dried.

As a result of the above treatment, about 300 μm of a water-soluble purified fraction (active ingredient of the present invention) having a lipid content of about 3 μm was obtained from 100 μm of yolk.

Next, after dispersing the freeze-dried products of various bacteria in physiological saline to a concentration of 1/-, 50/- were placed on a plate for agglutination, and purified fractions diluted to the respective concentrations were added. The agglutination effect of bacteria was measured. The aggregation activity results are shown in Table 1.

From the results shown in Table 1, it was found that the active ingredient of the present invention exhibits high aggregation activity because lipids have been removed.

[Example 2] Eggs obtained by immunizing chickens with Streptococcus mutans fibrous structures were treated according to the following steps.

1. The yolk was separated from the white, and the corpus luteum membrane was cut and removed. 1
00+d yolk was weighed and added to 400- citric acid-physiological saline (CBS: 0.15M NaCQ).

10mM citrate buffer (pH 7.5), 0.1% Na
diluted with N.

2. This solution was stirred at room temperature for 30 minutes. 3. This yolk suspension was stirred at 14000×g at 20°C for 30 minutes.
Centrifuge for a minute to obtain a supernatant.

CB550 containing 4.5% w/v γ-carrageenan
m1 was added to the supernatant and stirred gently for 10 minutes.

5. The solution was left for 30 minutes.

6. The suspension was centrifuged to obtain a supernatant.

7. Further, a saturated ammonium sulfate solution was added to this supernatant for salting out, and the mixture was centrifuged to obtain a precipitate. This operation was repeated twice.

8. This precipitate was sufficiently dialyzed against ion-exchanged water or distilled water to remove salts, and sterilized by 0.45-filtration, and then the sample was freeze-dried.

As a result of the above processing, the amount of fat is approximately 1■ from 100aQ of yolk.
Approximately 250 liters of water-soluble purified fraction (active ingredient of the present invention) was obtained.

Next, a test for inhibiting colonization of Streptococcus mutans in the oral cavity was conducted using a toothpaste with the following formulation containing the purified antibody obtained by the above method. The storage stability of the toothpaste used was also investigated.

The experimental method and results are shown below.

IILjLILILLL (Formulation example 1) Dicalcium phosphate dihydrate 50% glycerin 20% carboxymethylcellulose 1% sodium lauryl sulfate 1.5% myristoyl jetanolamide 0.5% fragrance
1.0% saccharin
0.1% Dextranase 0.01% Table 2 Egg anti-Streptococcus mutans fibrous structure 0.1% Total 100.0% Hamsters (5 weeks old, male) were divided into - groups of 5 and each Streptococcus mutans NCTC10449
Inoculate 10' of each strain.

After 3 days, brush the teeth with toothpaste containing each active ingredient (0.1 g), and after 1 week and 4 weeks, rub the dentition in the hamster's mouth with a sterilized cotton ball and soak it in a small amount of physiological saline to uniformly spread the bacteria. to be dispersed. A certain amount of the solution is spread on a BHI plate medium, and the total number of bacterial cells and the number of Streptococcus mutans colonies are counted. Streptococcus
The number of S. mutans bacteria was expressed as the number of bacteria per 10,000 total bacteria. The results are shown in Table 2.

Toothpaste 1: In prescription example 1, egg anti-streptococcus
Streptococcus mutans fibrous structures removed Toothpaste 2: Toothpaste of Prescription Example 1 Toothpaste 3: In Prescription Example 1, egg anti-streptococcus
Blood A, egg anti-Streptococcus mutans fibrous structure purified antibody (less than 5% lipid) B, chicken egg anti-Streptococcus mutans fibrous structure A toothpaste containing the above antibody was produced according to Formulation Example 1 of structural antibody fraction (lipid 20% or more), and a storage test was conducted at 40° C. for one month.

The activity was measured using an enzyme antibody method using whole cells of Streptococcus mutans. When the antibody titer immediately after production is taken as 100%.

The activity was determined after storage at 40°C for one month. The results are shown in Table 3.

From Table 3 and above, it was confirmed that the purified antibody can be incorporated into tooth brushing and exhibit its effectiveness, and that the purified antibody is stable even during tooth brushing when stored at 40° C. for one month.

[Example 3] Eggs obtained by immunizing chickens with the Bacteroides gingivalis capsular antigen were treated according to the following steps.

1. The yolk was separated from the white, and the corpus luteum membrane was cut and removed. 1
Weigh 00d of yolk and add 400d of phosphoric acid-physiological saline solution (
PBS: 0.15M NaCQ.

20mM phosphate buffer (pH 7.0), 0.1% NaN
).

2. The solution was stirred at room temperature for 30 minutes.

3. This yolk suspension was heated at 14,000×g at 20°C.
The mixture was centrifuged for 30 minutes to obtain a supernatant.

PBS50- containing 4.5% w/v xanthan gum
was added to the supernatant and stirred gently for 10 minutes.

5. The solution was left for 60 minutes.

6. The suspension was centrifuged to obtain a supernatant.

7. Further, a saturated ammonium sulfate solution was added to this supernatant for salting out, and the mixture was centrifuged to obtain a precipitate. This operation was repeated twice. The supernatant was thoroughly dialyzed against ion-exchanged water or distilled water to remove salts and sterilized by 0.45-filtration, and then the sample was freeze-dried.

As a result of the above treatment, approximately 230 μm of a water-soluble purified fraction (active ingredient of the present invention) having a lipid content of 1.5 μm was obtained from 100 μm of the yolk.

Next, a test for inhibiting the colonization of Bacteroides gingivalis in the oral cavity was conducted using a toothpaste with the following formulation containing the purified antibody obtained by the above method. In addition, 1. A storage stability test was conducted on the toothpaste used. The experimental method and results are shown below.

Kurou 1 both (prescription example 2) Anhydrous Kei Wi 30% glycerin 30% sorbitol
20% carboxymethylcellulose 1.0% sodium lauryl sulfate 2.0% myristoylgetanolamide 0.6% fragrance
1.0% saccharin
0.1% ethanol
2.0% Sodium fluoride Anti-Bacteroides gingivalis capsule-purified antibody 0.10% 0.1% Total 100.0% Golden hamsters (8 weeks old, male) were placed in - group of 6 to 7 animals, and lower jaw After ligating the first molar with cotton thread (No. 50), Bacteroides gingivalis 381-R'1X10
A bacterial solution of 0.1 cells/1llI was inoculated into the hamster's oral cavity. Thirty minutes after the bacterial inoculation, 1% of the hen egg anti-Bacteroides gingino (squirrel capsular antigen purified antibody) was added according to Prescription Example 2.
The dentition was brushed 20 times using a periodontal brush with the containing toothpaste.

The period was 4 weeks. The results are shown in Table 4.

Table 4 Table 5 A, Purified egg anti-Bacteroides gingivalis capsular antigen antibody (less than 5% lipid) B, Anti-hen egg anti-Bacteroides gingivalis capsular antigen antibody (20% or more lipid) Contains the above antibodies according to Formulation Example 2 A toothpaste was manufactured and a storage test was conducted at 40°C for 1 month.

The activity was measured using the enzyme antibody method using whole cells of Bacteroides gingivalis.

40°C when the antibody titer immediately after production is taken as 100%.

The activity was determined after storage for one month. The results are shown in Table 5.

From the above, it was confirmed that the purified antibody can be incorporated into toothpaste and exhibit its effectiveness, and that the purified antibody is stable even during toothpaste when stored at 40° C. for one month.

[Example 4] Eggs obtained by immunizing chickens with a protein antigen of Streptococcus mutans were treated according to the following steps.

1. The yolk was separated from the white, and the corpus luteum membrane was cut and removed. 1
Weighed the 00- yolk and added it to 400d of Tris buffer (TBS).
: 0.15M NacQ, 20mM Tris-HCl (pH
6,9), 0.1% NaN, ).

2. The solution was stirred at room temperature for 30 minutes.

3. This yolk suspension was heated at 14,000×g at 20°C.
The mixture was centrifuged for 30 minutes to obtain a supernatant.

TBS50- containing 4.5% w/v gum arabic was added to the supernatant and stirred gently for 10 minutes.

5. The solution was left for 30 minutes.

6. The suspension was centrifuged to obtain a supernatant.

7. Saturated ammonium sulfate solution was added to this supernatant for salting out, and the mixture was centrifuged to obtain a precipitate. This operation was repeated twice.

The supernatant was thoroughly dialyzed against ion-exchanged water or distilled water to remove salts, filter sterilized at 0.45 pm, and then the sample was freeze-dried.

As a result of the above treatment, about 290 µm of a water-soluble purified fraction (active ingredient of the present invention) with a lipid content of about 2 µm was obtained from 100 d of yolk.

Appearing person: Laion Co., Ltd. Agent: Patent Attorney Takashi Kojima

Claims (1)

[Claims] 1. Polysaccharides are added to the yolk fraction of eggs obtained from poultry that has been immunized with whole cells or bacterial cell components of oral bacteria as antigens, and the lipids in the yolk fraction are aggregated to form this aggregate. A method for producing an active ingredient for preventing oral diseases, which comprises collecting a separated and removed fraction having a lipid content of 5% by weight or less.