JPS5850962B2 - Oral hygiene composition - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an oral hygiene composition containing a protein-based antibacterial substance A produced by Streptococcus L-1 bacteria, and more specifically, Streptococcus spp. isolated from the human oral cavity. A novel bacteriocin-like protein-based antibacterial substance A produced by Streptococcus L-1 bacteria obtained by mutating a bacterium belonging to The present invention relates to an oral hygiene composition such as a toothpaste containing the following.

Conventionally, bactericides have been incorporated into oral hygiene compositions such as toothpastes for the purpose of preventing caries, etc., but conventional bactericides, especially synthetic bactericides, are harmful to living organisms. Due to the high risk of harmful effects, their use is becoming increasingly restricted.

For this reason, studies are underway to find natural substances that have a bactericidal effect and do not have any negative effects on living organisms. No substance with antibacterial activity has been produced or isolated, and no substance has been found that can be incorporated into oral hygiene compositions as an effective bactericidal agent.

The present inventor has created a mutant strain of a Streptococcus bacterium and is currently investigating its various properties. They isolated and purified this substance, and examined its properties. The present invention was made as a result of the discovery that a bacteriocin-like substance is produced that acts against oral cavity bacteria such as the cariogenic bacterium Streptococcus mutans. By incorporating protein-based antibacterial substance A produced by Streptococcus L-1 bacteria, which has strong antibacterial activity, as a disinfectant, it prevents oral infections, especially tooth decay, and improves oral hygiene by inhibiting plaque formation. The purpose of the present invention is to provide an oral hygiene composition that is highly safe and can promote the use of synthetic disinfectants and antibiotics.

The present invention involves culturing Streptococcus L-1 bacteria obtained by mutating a bacterium belonging to the genus Streptococcus isolated from the human oral cavity, and producing bacteriocins isolated from this culture that This is an oral hygiene composition containing a protein-based fungal substance A having an antibacterial effect against endogenous bacteria.

The Streptococcus L-1 bacteria used in the present invention can be obtained by treating Streptococcus bacteria isolated from the oral cavity using a commonly used mutant strain production method (mutagenesis method).

Streptococcus L-1, which is a mutant strain of Streptococcus bacteria discovered by the present inventor, has been deposited with the Microbial Research Institute, and its accession number is No. 3220.

To explain the mycological properties of the above-mentioned Streptococcus L-1 bacteria, this Streptococcus L-1 bacteria is non-motile, a 1.0-1.2μ coccus, and a streptococcus in which several to dozens of bacteria are chained together. It is.

Furthermore, the above L-1 bacteria is a non-spore-forming bacteria that is Durham positive and is facultatively anaerobic.

Generally, the above-mentioned L-1 bacteria does not grow on an agar plate unless the plate is placed in an anaerobic state, but when it is left standing in liquid culture, it grows on the bottom.

1-(IN) shows the growth state of the L-1 bacteria in each medium.

The growth media on which the L-1 bacterium grows relatively well are Trypticase Soy medium, BHI (Brain-Bart Infusion) medium, Myeteis salivarius medium, Todhevit medium, and Gum medium.

The L-1 bacteria has no pleomorphism, motility, anti-acidity, pigment production, or gelatin liquefaction ability, and weakly coagulates lidmus milk.

In addition, the physiological properties of the above-mentioned Streptococcus L-1 bacterium include nitrate reduction, hydrogen sulfide production, indole production, starch hydrolysis, VP test, growth when ammonium dihydrogen phosphate is used as the sole nitrogen source, and catalase. , urease, oxidase, denitrification reaction, citric acid utilization, and nitrate utilization are each negative, and the MR test and 0-F test are each positive.

The growth pH range of the L-1 bacteria is 5 to 8,
It is facultatively anaerobic.

Furthermore, regarding the fermentability of sugars, as shown in the first QV bale, inosit, starch, glycerin, and D-xylose were negative, and L-arabinose, D-glucose, and
D-mannose, D-fructose, D-galactose, maltose, sucrose, lactose, trehalose, D-sorbitol, D-mannite, raffinose, and cellobiose are each positively fermented and produce organic acids.

Furthermore, as shown in Table 1-(5), no gas production was observed for any of the above sugars.

Tables 1-(0, 1-(■), and 1-(IV) below summarize the morphology, growth state, and physiological properties of Streptococcus L-1.

Next, various properties showing the characteristics of the above-mentioned L-1 bacteria are listed.

O Aesculin decomposes and turns blackish brown.

O shows no hippuric acid degrading activity.

Does not exhibit O-arginine degrading activity.

O Sodium chloride tolerance: Does not grow in the presence of 6.5% NaCl.

O temperature resistance: Grows in the range of 15°C to 50°C, but 3
Grows faster at 0'C to 40C.

Growth on plain heart infusion agar medium supplemented with O sucrose: under normal anaerobic conditions (95 steamed rice N2.5
Grows well on rice cake C02).

Streptococcus mutans, to which the L-1 bacterium belongs, has a weak property of synthesizing polysaccharides from sucrose, and therefore forms amorphous colonies on the medium.

0 Hemolysis for sheep blood: Hemolysis is γ-hemolysis,
No hemolysis.

The above microorganism, namely Streptococcus L-1,
Although it is a member of the genus Streptococcus that is not described in the Vershis Manual of Determinative Bacteriology, 7th edition (1957), it is classified according to Facklam's classification method (R, R, Facklam).

J, of clinical Microbiolo
gy, Vol 5゜A2. Feb, (977)
), it belongs to Streptococcus and Streptococcus mutans species because it utilizes -7 and lactose and does not decompose hippuric acid.

This L-1 bacterium was found to be novel due to differences in the antibacterial spectrum against the indicator bacterium of the new antibacterial substance A obtained by culturing the above-mentioned 11 bacteria, as described below. It was thought that it was something.

Further, the new antibacterial substance A used in the present invention can be obtained by culturing the above-mentioned Streptococcus L-1 bacteria by a fermentation method.

The method for isolating and purifying this new antibacterial substance A is a method of pure extraction and separation of proteins from a suitable growth liquid medium, and an example thereof will be explained below.

First, Staphylococcus L1 bacteria was cultured in advance in 10 TIl of trypticase soy liquid medium under anaerobic conditions, and this was inoculated into 107 trypticase soy liquid medium. ℃ and culture for 1-3 days.

Next, centrifugation (for example, io, ooo rotation for 15 minutes) is applied to this culture solution to obtain a culture supernatant.

After adjusting the pH to around 7 by adding 500 g of ammonium sulfate per 11 parts of the culture supernatant, the culture supernatant was kept at a low temperature for one day.

Since a precipitate is then formed, this precipitate is collected by centrifugation (for example, at 15,000 rpm for 30 minutes), redispersed in a small amount of water, and dialyzed to desalt it.

The crude antibacterial substance obtained by desalting in this way is 10-
After removal of anionic substances through DEAE-cellulose equilibrated with 3 molar phosphate buffer, then 10-3
It was adsorbed onto CM-cellulose equilibrated with a molar pH 71J phosphate buffer, thoroughly washed with the same phosphate buffer, and then the CM-cellulose was dispersed with sodium chloride at a final concentration of 1 molar. The antibacterial substance A is eluted.

Furthermore, this solution was dialyzed and desalted, and the desalted product was
Purified antibacterial substance A is obtained by freezing at 0°C and then freeze-drying.

In addition, in the manufacturing process of this purified antibacterial substance A, commonly used techniques such as application of electrodialysis method in desalting operation, appropriate combination of ion exchange carriers, and filtration collection of bacterial cells and precipitates are used in combination or alternatively. You can also.

Further, by utilizing the fact that the antibacterial substance A has an affinity for the bacterial cells of the genus Streptococcus, a single concentration purification method can be adopted.

To explain this point below, Streptococcus
Sangis ATCC10557 or ATCC105
58 bacteria were collected in advance by a conventional culture method, and after heat sterilization, the bacterial bodies of Streptococcus sanguis were thoroughly washed with water, and this was added to the desalted crude antibacterial substance and thoroughly stirred. Post-centrifugation operation (e.g. i
o, ooo rotation for 20 minutes) to collect bacterial cells,
Furthermore, this bacterial cell was suspended in 1 mol of saline solution and antibacterial substance A was added.
was eluted and centrifuged again (e.g. 10,000
Rotate for 20 minutes) to obtain a supernatant.

The supernatant is then dialyzed and desalted, then frozen at -20°C and dried to obtain purified antibacterial substance A.

The physicochemical properties of the antibacterial substance A used in the present invention produced as described above are as shown in Table 1. It is colored by ninhydrin and biuret color reaction, and is applied to SDS acrylamide gel for proteins. From the behavior of electrophoresis, it was confirmed that the protein had a molecular weight of about 5,000 to 15,000.

This antibacterial substance A is a colorless to white neutral substance that is soluble in water but insoluble in ethanol and acetone.

The melting point is difficult to measure because the antibacterial substance A is a protein.

In addition, the antibacterial substance A has a high molecular weight (5000 to 15
000), elemental analysis was difficult.

Therefore, instead of the elemental analysis, the analysis results of the amino acid composition are shown in Table 1-2 below.

Further, the ultraviolet absorption spectrum and infrared absorption spectrum of the present antibacterial substance A are shown in FIGS. 1 and 2, respectively.

To explain the ultraviolet absorption spectrum shown in FIG. 1, the above-mentioned antibacterial substance A has an extremely thick absorption characteristic of protein compounds derived from aromatic amino acids at a wavelength of 260 to 280 nm.

Further, to explain the infrared absorption spectrum shown in FIG. 2, the antibacterial substance A has a 0 wave number of 33006rn-
'Absorption of Niamide A, 0 wave number 2950CIrl'
:C-H stretching motion, O wave number 1650 crn-”
Niamide I(7) absorption, 0 wavenumber 1550C1rL-'
Absorption of Niamide ■, 0 wave number 1450 crn-1:C
Polypeptides and proteins, such as the bending vibrations of H2 and CH3, have unique absorption bands.

Further, the specific light absorption degree of the present antibacterial substance A is a value of 1.3 to 1.5.

In addition, the antibacterial properties of the antibacterial substance A are as shown in Table 2, as shown in Table 2.
S5 bacteria, PS-14 bacteria, Ingbri
tt) Shows strong antibacterial activity against bacteria and other oral streptococcus Streptococcus sanguis ATCC1
It also shows strong antibacterial activity against bacteria 0557, ATCC 10558, and Streptococcus salivarius.

The method for measuring the antibacterial activity of the present antibacterial substance A was as follows.

That is, after mixing indicator bacteria pre-cultured in Plain Bart Infusion liquid medium with 20 TL of sterilized Plain Bart Infusion Agar medium at a temperature that does not solidify the agar and do not kill the indicator bacteria, the mixture is placed in a sterile Petri dish with a diameter of 11α. Seven cylindrical cups with a diameter of 8 mm were placed on the plate, and the liquid was poured into the surrounding area of these cups.

After the agar has solidified, carefully remove each cup using tweezers.

Then, since seven circular depressions are formed by the cups in the solidified agar base in the petri dish, a certain amount of the aqueous solution of the antibacterial substance A is injected into each of these depressions, and incubated at 37°C for 24 hours. After that, the diameter of the growth inhibition zone formed around the depression was measured with a caliper, and this was taken as the antibacterial activity.

Furthermore, as shown in Figure 3, there is a linear relationship between the concentration of antibacterial substance A and the diameter of the inhibition zone, so the titer of antibacterial substance A can be measured by measuring the diameter of the inhibition zone. I can do it.

Furthermore, as shown in Table 3, the biochemical properties of this antibacterial substance A show that its antibacterial activity hardly decreases even after heat treatment at 100° C. for 10 minutes.

The activity is partially lost when the proteolytic enzymes trypsin and papain are added, but the activity is almost completely lost when the lipid-degrading enzyme lipase is added.

This suggests that lipids are included as active sites of antibacterial substances.

As mentioned above, the antibacterial substance A obtained by the present invention exhibits properties similar to those of previously reported bacteriocins, but its antibacterial spectrum and biochemical properties are different from those of conventional bacteriocins. Therefore, this antibacterial substance A was considered to be a new substance.

Furthermore, Table 4 shows the results of an experiment for inhibiting plaque formation of Streptococcus mutans using the antibacterial substance A produced by Streptococcus L-1.

This experimental method uses Streptococcus mutans 6 precultured in plain-vert infusion liquid medium.
A certain amount of 715 bacteria was inoculated into 5TfLl of plain infusion medium containing 5φ sucrose, and incubated at 37°C under anaerobic conditions.
After 6 hours of incubation, the medium was discarded, and the bacterial cells and the synthesized polysaccharide complex attached to the test tube wall were removed with 10-
It was redispersed in 511 L of 3M phosphate buffer and the absorption at 660 nm was optically measured.

This absorption at 660 nm is a measurement of so-called turbidity, and the amount of dispersed substance and absorbance show a proportional relationship.

As shown in Table 4, Streptococcus L.
When the antibacterial substance A produced by the -1 bacteria was prepared as a solution with a concentration of 10/- and added to the above reaction system, the amount of attached bacteria decreased as the antibacterial substance concentration increased.

Furthermore, when the amount of bacteria adhering to the synthetic polysaccharide involved in adhesion is taken as an index, the value is even less than half, indicating that it also has the effect of inhibiting the adhesion effect of polysaccharides.

In the present invention, the protein-based antibacterial substance A produced by Streptococcus L-1 bacteria is blended into the raw material for producing an oral hygiene composition to obtain a prescribed oral hygiene composition.

For example, when manufacturing toothpaste, the antibacterial substance A is blended in a predetermined ratio (for example, 20% by weight or less) with manufacturing raw materials such as calcium diphosphate, glycerin, a humectant, and a perfume.

Since the activity of this antibacterial substance A can be varied depending on the degree of purification, the amount to be added also varies depending on the degree of purification, and even in toothpaste, it varies depending on the type.

As described above, the present invention contains the protein-based antibacterial substance A produced by Streptococcus L-1 bacteria, so that it can be used to treat Gram-positive streptococci in the oral cavity, such as Streptococcus mutans, Streptococcus sanguis, or Streptococcus salivarius. It has a strong antibacterial effect against caries, and is particularly attracting attention as a caries pathogen.The pathogenic action is to demineralize teeth by producing large amounts of organic acids such as lactic acid from sugar, and to demineralize teeth from sucrose. It exhibits an antibacterial effect against Streptococcus mutans, which synthesizes polysaccharides and remains on the tooth surface for a long time, creating a foothold for plaque formation, and is thus the first step in the process of plaque formation. In other words, it can prevent the process by which Durham-positive cocci or bacilli form a foothold for plaque formation on the tooth surface, so it is more effective, easier, and more reliable than conventional disinfectants in preventing cavities and inhibiting plaque adhesion. It has advantages such as being able to prevent various diseases related to dental plaque and safely promoting oral hygiene.

[Example 1] A toothpaste having the following composition was manufactured.

Calcium diphosphate 50 weight Glycerin 2011 Sodium lauroyl sarcosinate 2 Carboxymethylcellulose 1 Saccharin
0.1//Fragrance 0.9 Streptococcus L-1 bacteria production 4//Antibacterial substance water Remaining weight 100 weight φ [Example 2] The following toothpaste was manufactured.

Calcium diphosphate glycerin sodium lauryl sulfate power lagenan saccharin sorbit Sodium monofluorophosphate fragrance Streptococcus L-1 bacteria production Antibacterial substance A water 50 weight Top 0 〃 2 〃 1 〃 Q,lu 10/1 0.05 /1 0.9〃 5 〃 remainder 100 weightφ [Example 3] A powdered toothpaste having the following composition was manufactured.

Calcium diphosphate calcium carbonate sorbit sodium lauryl sulfate saccharin fragrance Streptococcus L-1 bacteria production Antibacterial substance A sucrose monopalmitate water 50 weight rice cake 25 〃 10〃 1 〃 0.1 /1 0.9〃 5 〃 0.5〃 remainder 100 weight buns [Example 4] A liquid toothpaste having the following composition was manufactured.

Glycerin 35 Sodium polyacrylate by weight 5 Sodium lauryl sulfate 2 Saccharin
0.111 Fragrance 0.9 Ethanol 3 Streptococcus L-1 bacteria production 3 Antibacterial substance A lauroylgetanolamide 1 Water
Remaining weight of 100% All of the above examples were able to effectively prevent tooth decay and plaque formation.

[Brief explanation of drawings]

Figure 1 is an ultraviolet absorption spectrum of antibacterial substance A produced by Streptococcus L-1, Figure 2 is an infrared absorption spectrum of the same, and Figure 3 is a graph showing the relationship between the concentration of antibacterial substance A and the diameter of the inhibition circle. It is.

Claims (1)

[Claims] 1 Isolated from a culture of Streptococcus L-1 belonging to the Streptococcus mutans species obtained by mutating a bacterium belonging to the Streptococcus genus, it belongs to bacteriocins and has antibacterial effects against oral bacteria. An oral hygiene composition characterized by containing a protein-based antibacterial substance A having the following properties.