JPS5850718B2 - Method for producing protein-based antibacterial substances - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a protein-based antibacterial substance produced by Streptococcus I. Production of a novel bacteriocin-like protein-based antibacterial substance A that exhibits a strong antibacterial effect against bacteria in the oral cavity, especially cariogenic bacteria, by producing Stobococcus L-1 bacteria obtained by mutating bacteria. Regarding how to.

In recent years, much research has focused on the mycological properties of Streptococcus mutans, which has attracted attention as a bacterium that causes dental caries in humans. It has been reported that they produce bacteriocins that have antibacterial effects against.

For example, G.A.K.L. Strap, R.G.A.
Gibbons (J * KelstrupER, J, Gi
BB-ons) reports on bacteriocins produced by Streptococcus mutans and their activities.
an and Rod・ent S treptoco
cci;ArchsOralBiol,, 14.25
1-258 (1969)).

According to this report, the producing bacteria and their antibacterial activity are as shown in Table 1.Here, as shown below, 1 is a GS5 bacterium which is a common bacterium with Streptococcus mutans, which is used as an indicator bacterium by the present inventor. There is no antibacterial activity against both PK-1 and PK-1 bacteria, and the antibacterial spectrum is clearly different from the antibacterial spectrum of the antibacterial substance of the present invention.

It was.

"Bacteriocin-like activity of Streptococcus mutans" (Shigeyuki Hamada, Furu Oshima, Jun Mizuno, Journal of the Japanese Society of Basic Dental Medicine 15, 232, (1973)) describes BHT, FA-1, and GF7 of Streptococcus mutans.
Although it has been reported that one strain shows activity, as shown below, 6715 bacteria and O\
The 1Z-61 bacterium is described as lacking both bacteriocin production and indicator bacterium activity, and is different from the antibacterial substance A purified by the present inventor.

Despite the fact that the mycological properties of Streptococcus mutans have been studied,
Antibacterial substances, especially bacteriocins, produced by Streptococcus that act effectively against caries pathogens have never been isolated or purified.

The present inventor created a mutant strain of a bacterium of the genus Treptococcus,
While examining various properties, we discovered that this mutant strain produced a substance that exhibited antibacterial activity different from previously reported bacteriocins, and we isolated and purified this substance to determine its properties. As a result of the study, it was discovered that the mutant strain described above exhibits broad antibacterial activity against various bacteria of Streptococcus mutans and produces bacteriocin-like substances that act against Streptococcus mutans.As a result, the present invention has been made. This has led to the
The purpose of the present invention is to exhibit antibacterial activity against the cariogenic bacterium Streptococcus mutans, prevent oral infections, especially dental caries9, and use it as an active ingredient in oral hygiene to prevent dental plaque formation. Provides a method for producing a highly safe protein-based antibacterial substance A that can be used to promote oral hygiene, and in the current situation where the toxicity of synthetic bactericides and antibacterial substances to living organisms is a problem. It's about doing.

The present invention involves culturing Streptococcus ruu 1 bacteria obtained by mutating a bacterium belonging to the genus Streptococcus. This is a method for isolating and producing protein-based antibacterial substance A having the following properties.

The Streptococcus sulcus 1 strain used in the present invention is obtained by treating Streptococcus bacteria using a commonly used mutant strain production method (mutagenesis method).

Streptococcus slui 1, which is a mutant strain of the Streptococcus genus 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 sulcus 1 bacterium, as shown in Table 2, this Streptococcus sulcus 1 bacterium is non-motile, with a size of 1.0 to 1.2μ, and several to ten cocci. Streptococcus is a chain of several bacteria.

The L-1 bacteria is a non-spore-forming Durham-positive bacterium 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.

Table 2 (2) shows the growth status of the L-1 bacteria applied to each culture medium.

The growth media on which the L-1 bacterium grows relatively well are tributicase soy medium, BHI (presono1 infusion) medium, Myeteis salivarius medium, Todd Hevitt medium, and gum medium.

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

In addition, the physiological properties of the Streptococcus L-1 bacteria are as shown in Table 3, such as nitrate reduction, hydrogen sulfide production, indole production, starch hydrolysis, VP test,
Growth when ammonium dihydrogen phosphate is viewed as a mono-nitrogen, catalase, urease, and oxidase are denitrifying reactions,
The use of citric acid and the use of nitrate were each negative.
Both the MR test and the 0-F test are positive.

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

Regarding sugar fermentability, as shown in Table 4, inosit, starch, glycerin, and D-xylose were negative, and L-arabinose, D-glucose, D-mannose, and , D-galactose, maltose, sucrose, lactose, trehalose, D-fulvit, I) --r7nits, raffinose, and cellobiose are each positively fermented and produce functional products.

Also. As shown in Table 4, no gas production was observed for any of the above sugars.

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

0 Decomposes Aesculin and turns it blackish brown.

O shows no hippuric acid degrading activity.

0 Does not show arginine degrading activity.

0 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 quickly at 0°C to 40°C.

0 Growth on plain invert infusion agar medium supplemented with chouxcloth under normal anaerobic conditions (95 N2.5
Grows well in CO, ).

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

0 Hemolytic property against sheep blood: Hemolytic property is γ-hemolytic, not hemolytic.

The above microorganisms, namely Streptococcus L-i, are
Although it is a bacterium of the genus Streptococcus that is not described in Bacteriology, 7th edition (1957), it is classified according to RRFacklam's classification method (RRFacklam).

J @ of clinical Microbiol
ogy, Vol 5, 42.

Feb. (977)), it belongs to the Streptococcus mutans species because it utilizes D-mannite and lactose and does not decompose hippuric acid.

This L-1 bacterium was found to be new due to differences in antibacterial spectrum, etc., compared with the characteristics of other known bacterial strains, or to the indicator bacterium of the new antibacterial method obtained by culturing the L-1 bacterium as described below. It was thought that it was something.

The new antibacterial method according to the present invention can be obtained by culturing the above-mentioned Streptococcus L-1 bacterium 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.

1. Streptococcus L-1 bacteria is cultured in advance in a small amount of trypticase soy liquid medium under anaerobic conditions, and after inoculating this into another trypticase soy liquid medium, this is preferably cultured under anaerobic conditions. Incubate at 37°C and culture for 1-3 days.

Next, centrifugation is applied to this culture solution to obtain a culture supernatant.

Ammonium sulfate was added to the cultured supernatant to adjust the pH to 7.
After adjusting to the desired temperature, it is kept at a low temperature and left for one day.

As a result, a precipitate is formed, which is collected by centrifugation, redispersed in a small amount of water, and desalted by dialysis.

The crude antibacterial substance obtained by desalting in this way is 10-
After removal of anionic substances through DE-E-cellulose equilibrated with 3 molar phosphate buffer, then 10-3
It is adsorbed onto CM-cellulose equilibrated with a molar pH 7 phosphate buffer, thoroughly washed with the same phosphate buffer, and then salt is added to the solution in which this CM-cellulose is dispersed for antibacterial and other purposes. Elute the substance.

Furthermore, this solution is dialyzed and desalted, and the desalted product is frozen and freeze-dried to obtain a purified antibacterial substance.

In addition, in the manufacturing process of this purified antibacterial substance, 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 as substitutes. You can also do that.

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 employed.

To explain this point below, Streptococcus
Sangis ATCO10557 or ATCO105
58 bacteria were collected in advance by a normal culture method, and after heat sterilization, the Streptococcus sanguis cells were thoroughly washed with water, added to the desalted crude antibacterial substance, and thoroughly stirred. After that, centrifugation is performed to collect the bacterial cells, which are further suspended in saline to elute antibacterial and other substances, and centrifugal separation is performed again to obtain a supernatant.

The supernatant is then dialyzed and desalted, then frozen and dried to obtain purified antibacterial substance A.

The physicochemical properties of the antibacterial and other substances produced as described above are as shown in Table 5.

Based on the behavior of SDS acrylamide gel electrophoresis for proteins, which develops color by Buret color reaction,
It was confirmed that the protein had a molecular weight of about 5,000 to 15,000.

This antibacterial substance 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 above-mentioned antibacterial and other substances contain high molecular weight (5000 to 15
Since it is a protein-based substance (000), single-element analysis was difficult.

Therefore, instead of single element analysis, the analysis results of amino acid composition are shown in Table 5 below.

Further, the ultraviolet absorption spectrum and infrared absorption spectrum of the subject of this antibacterial method are shown in FIGS. 1 and 2, respectively.

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

Also, to explain the infrared absorption spectrum shown in FIG. 2, the above-mentioned antibacterial substance is included.

O wave number 3300 cIIL 'Absorption of Niamide A, 0 wave number 2950 cm': C-H (D stretching motion, O wave number 1650 (1771-1:7 mi)" I (7) absorption [, O
Wave number 1550 cIrL-1 Niamide ■ absorption, O wave number 1
450cIn": It has an absorption band peculiar to polypeptides and proteins, such as bending vibrations of CH2 and CH8.

Additionally, the specific light intensity of the present antibacterial method is 1.3 to 1.5.

In addition, as shown in Table 6, the antibacterial properties of the above-mentioned antibacterial substances include Streptococcus mutans JC2 bacteria, 6715 bacteria, OMZ-61 bacteria, PK-1 bacteria, which are cariogenic bacteria.
G35 bacteria, PS-14 bacteria.

In addition to exhibiting strong antibacterial activity against Ingbritt bacteria, it also exhibits strong antibacterial activity against other oral streptococci such as Streptococcus sanguis ATCO10557 and ATCO
It also shows strong antibacterial activity against bacteria 10558 and Streptococcus salivarius.

This antibacterial method The method for measuring the antibacterial activity of the subjects was as follows.

That is, after mixing indicator bacteria pre-cultured in Plain Bart Infusion liquid medium with sterilized Plain Bart Ink Knee John Agar medium at a temperature that does not solidify the agar and do not kill the indicator bacteria,
Seven tricylindrical cups with a diameter of 8 m were placed in a 1cIIL sterilized petri dish, and the mixture was poured into the enclosures 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 chassis, 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 substances and the diameter of the inhibition zone, so the potency of the antibacterial method can be measured by measuring the diameter of the inhibition zone. Can be done.

Furthermore, as shown in Table 7, the biochemical properties of the subjects of this antibacterial method show that their 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.

As mentioned above, the antibacterial substance obtained by the present invention exhibits strong antibacterial activity against human oral bacteria, especially Streptococcus mutans, which is attracting attention as a pathogen of dental caries. Since it also inhibits the formation of plaque produced by Streptococcus mutans, it sterilizes the dental caries bacteria Streptococcus mutans in a person's mouth, preventing oral infections such as preventing caries and plaque formation. Therefore, it can be used by adding it as an active ingredient to products related to oral hygiene.

Example 1 Streptococcus L-1 bacteria was cultured under anaerobic conditions in 10 ml of trypticase soy liquid medium in advance, and then inoculated into 10 t of trypticase soy liquid medium.

This was incubated at 37°C under anaerobic conditions, and
Cultured for ~3 days.

This culture solution was centrifuged at 100 rotations for 15 minutes to obtain a culture supernatant.

Next, this culture supernatant 1 was mixed with ammonium cisulfate 500
After adding t and adjusting the pH to around 7, the mixture was left at a low temperature for one day.

The precipitate formed from this left liquid was heated at 15,000 rpm for 3 minutes.
The samples were collected by centrifugation for 0 minutes, and then redispersed in a small amount of water and desalted by dialysis to obtain samples of crude antibacterial substances.

A sample of this crude antimicrobial material was first passed through DEAE-cellulose equilibrated with a 10-3 molar phosphate buffer.
After removing the anionic substances, it was adsorbed onto CM-cellulose equilibrated with a 10-3 molar pH 7 phosphate buffer, and then thoroughly washed with a 10-3 molar pH 7 phosphate buffer.

Antibacterial substance A was eluted by adding common salt to the solution in which CM-cellulose was dispersed to a final concentration of 1 mol.

Furthermore, this solution was desalted by dialysis against water,
The desalted product was frozen at 1°C and then freeze-dried to obtain white purified antibacterial substance A.

Purified antibacterial substance (the amount of people was 10 as described above/several tons per culture medium).

Example 2 Streptococcus sanguis AT C010557
Streptococcus sanguis cells were collected by a normal culture method and heat-sterilized at 100° C. for 15 minutes. After thoroughly washing with water, this was used as a sample of the desalted crude antibacterial substance obtained in Example 1 above. added to.

Next, after stirring thoroughly, the cells were centrifuged at 10,000 rpm for 20 minutes to collect the cells, and the cells were further suspended in 1 mol of saline to elute the antibacterial substances.

This was centrifuged at 10,000 rpm for 20 minutes to obtain a supernatant.

This supernatant was desalted by dialysis, then frozen at -20°C and freeze-dried to obtain purified antibacterial substance A.

The yield of purified antibacterial substance A per 10 tons of tributicase soy culture solution was several hundred mt.

In addition, ATCO10 was added to generation 9 of the above ATCO10557 bacterium.
Similar results were obtained when using 558 bacteria.

[Brief explanation of drawings]

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

Claims (1)

[Claims] 1. Streptococcus L-1 bacteria belonging to the Streptococcus mutans species obtained by mutating bacteria belonging to the Streptococcus genus were cultured, and this culture was found to belong to bacteriocins and have antibacterial activity against oral bacteria. 1. A method for producing a protein-based antibacterial substance, which comprises isolating a protein-based antibacterial substance A having the following properties.