JP2002000261A - Antibacterial agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a safe antimicrobial agent having a growth-inhibitory effect on a wide range of bacteria, especially harmful bacteria. SOLUTION: This antimicrobial agent uses a culture product of Streptomyces fulvissimus FERN P-16,347 or its variant or its gene recombinant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a safe bactericidal agent which acts on a wide variety of microorganisms, especially harmful bacteria, and inhibits the growth thereof.

[0002]

2. Description of the Related Art Many microorganisms are involved in spoilage and deterioration of foods and beverages and in human infectious diseases. To prevent such microorganisms, there is a method of killing these microorganisms or suppressing their growth. Used. Sterilization is carried out by heating, radiation / electron beam irradiation, addition of chemically synthesized substances having a bactericidal action, etc., but these methods for food and humans are not always suitable due to deterioration and safety issues. . In addition, many antibacterial substances that suppress the growth of microorganisms are known, such as chemically synthesized substances, substances and components extracted from plants, and substances and components produced by microorganisms. Has the advantage that it can be produced in large quantities and at low cost, and that it effectively acts on many microorganisms. However, it is not always suitable from the viewpoint of safety to the human body and the natural environment. On the other hand, it is known that many substances and components obtained from plants and microorganisms are safe except for special ones, and that they are degraded in the natural environment over time and have little effect on ecosystems. I have. For example, a well-known antibacterial substance produced by microorganisms is nisin, which is said to produce a special lactic acid bacterium isolated from cheese. This nisin is a peptide and is actually used as a food preservative in Europe. However, the effect is said to be antibacterial only for Gram-positive bacteria. Further, as one of the antibacterial substances produced by microorganisms, a cell wall lysing enzyme has attracted attention. Because cell wall lysing enzymes are also proteins, unlike chemical antibacterial agents and preservatives, they are not toxic by themselves and are very safe antibacterial agents. Has the advantage of not acting. However, disadvantages of cell wall lysing enzymes include:
Specificity is high because it is an enzyme, and specific microorganisms can be lysed well. However, even if they are closely related, they do not act on microorganisms having a cell wall of another structure. For example, many studies have been made on the antibacterial activity of foods with egg white lysozyme, but the target bacteria have been considerably limited due to the narrow lysis spectrum. Particularly, there is a report that the effect is small for gram-negative bacteria, and the effect is enhanced by using glycine in combination. In other words, many of the antibacterial substances produced by microorganisms that have been discovered so far have high safety for the human body and the environment, but are not sufficiently effective.

[0003]

In view of such circumstances, an object of the present invention is to provide a safe antibacterial agent which acts against a wide range of microorganisms, particularly harmful bacteria.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above-mentioned object, and as a result, by accident, the streptotype disclosed in Japanese Patent Application Laid-Open No. H11-5638 filed by the present applicant has been discovered. Streptomyces fulvi
ssimus) The culture containing the cell wall lytic enzyme produced by the TU-6 strain (FERM P-16347) has a growth inhibitory effect on a very wide range of microorganisms, is highly safe, and is the object of the present invention. They have found that they are extremely suitable as antibacterial agents, and have completed the present invention. That is, the present invention

[0005] Streptomyces flubisimus (Stre
ptmyces fulvissimus) TU-6 strain (FERM P-1)
6347) or a bactericidal agent characterized by comprising a culture of a mutant or a genetically modified product thereof as an active ingredient, in particular, an antibacterial agent useful as a preservative for foods, especially an antiseptic and an anti-caries agent of alcoholic beverages. A fungicide is provided. According to the present invention, it is possible to provide a safe antibacterial agent having a growth inhibitory effect against a very wide range of microorganisms, particularly harmful bacteria.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION A strain producing an active ingredient of the antibacterial agent of the present invention is Streptomyces fluvicimus TU.
-6 strains. This strain has been accredited by the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology on July 24, 1997, under the accession number FERM.
Deposited under P-16347. Hereinafter, this bacterium is abbreviated as TU-6 strain in this specification. This TU-6
The strain is a strain that produces the cell wall lytic enzyme (hereinafter abbreviated as TU-6 enzyme) described in JP-A-11-56348. In the present invention, if desired, not only this strain but also a mutant or gene obtained by subjecting the TU-6 strain to a mutation treatment and a gene recombination treatment to modify the productivity of the TU-6 enzyme by a method known per se. Recombinants can be used.

[0007] The active ingredient of the antibacterial agent of the present invention comprises the TU-
Six strains or mutants or recombinants thereof were transformed into a production medium, for example, 0.5% polypeptone, 0.1% chitin powder, 0.1% magnesium sulfate, 0.1% monopotassium phosphate, 0.1% PH 7.0 to 8.0 consisting of 3- (N-morpholino) propanesulfonic acid, preferably pH
A culture medium obtained by shaking culture or aeration and stirring culture using a liquid medium of 8.0 by a conventional method, and a culture itself such as a liquid from which cells have been removed, and a processed product thereof. As such a processed product, for example, from a culture solution,
The supernatant is separated by a known method such as centrifugation or filtration, and the supernatant is separated, for example, from an excluded molecular weight of 5,000 to 1
A powder obtained by concentrating by ultrafiltration using a 000 ultrafiltration membrane or ammonium sulfate, or by using a known drying method such as freeze-drying without concentrating, may be used. The concentrated solution may be dialyzed before drying, and may be used as a liquid without drying. The bactericidal agent of the present invention may be the active ingredient alone, or, if desired, combined with an appropriate additive or other lytic enzyme, an antibacterial agent and the like within a range not contrary to the object of the present invention, and a method known per se. Thus, an appropriate liquid or solid dosage form may be used.

As shown in the following examples, the antibacterial agent of the present invention contains TU-6 enzyme, and its effect is
This is probably due to the lytic activity of the -6 enzyme. That is, using the antibacterial agent of the present invention, for example, typical gram-positive bacteria such as lactobacilli and lactococci, in particular, cariogenic bacteria (Streptococcus mutans (Streptococcus mutans)
ccus mutans), spoilage of alcoholic beverages such as sake and beer, fire-killing bacteria and lactic acid bacteria that cause rotting, and Staphylococcus aureus (Staphylococcus that causes food poisoning) au
reus)) and Bacillus subtili
s) and Escherichia coli and Pseudomonas fluorescens, which are typical gram-negative bacteria, were oxidized under optimal conditions (40 ° C., pH 4.0 (40 ° C., pH 4.0) for the TU-6 enzyme. When the bacterium was lysed with a McIlvain buffer)), the above bacterium was well dissolved regardless of whether it was gram positive or negative. Further, when the bacteriolytic activity of the product of the present invention was examined under the condition that 15% of alcohol (ethanol) was present, the result was not inferior to the result measured under the condition where no alcohol was present. Therefore, it was found that the bacteriolytic activity of the antibacterial agent of the present invention has relatively high resistance to alcohol, and can be applied to alcoholic beverages such as sake and beer. Furthermore, when the bactericidal agent of the present invention was added to, for example, the above-mentioned bacterium growth medium, the growth degree of the bactericidal agent was compared with that of the bacterium not added. The growth of the fungus was inhibited. In particular, the growth of fire-killed bacteria and carious bacteria (Streptococcus mutans) was completely inhibited. In addition, when the results were compared with egg white lysozyme, which is a typical cell wall lysing enzyme and is already used as a food preservative, egg white lysozyme has almost no effect, and the antibacterial agent of the present invention is clearer. It was excellent in growth inhibitory activity.

On the other hand, the bactericide of the present invention is subjected to heat treatment (for 15 minutes in boiling water) to obtain TU-6 contained in the bactericide of the present invention.
After the enzyme was completely inactivated, the growth inhibitory effect was examined in the same manner. As a result, the growth inhibitory effect was reduced but not completely eliminated. From this, the growth inhibitory effect of the bactericidal agent of the present invention is not only due to the lytic activity of the cell wall lysing enzyme, but also due to a synergistic effect with an unknown substance contained in the culture solution of the TU-6 strain. Turned out to be something. Further, it can be said that the antibacterial agent of the present invention is stable against heat. In addition, Salmonella typhimurium (Salmonella typhimurium), which is a known method, is applied to the antibacterial agent of the present invention.
murium) When a mutagenicity test (Ames test) was performed using the TA98 strain, it was found that there was no mutagenicity at least at a concentration of 0.1%, indicating that the product of the present invention was safe. .

As described above, the use of the antibacterial agent of the present invention can effectively suppress the growth of a very wide range of harmful bacteria. In particular, it is useful for preventing burn-off, which is a problem in sake brewing, as an agent for preventing lactic acid bacteria contamination of alcoholic beverages such as beer, and as an agent for preventing tooth decay.

[0011]

EXAMPLES Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. “%” Is w / v% and the alcohol concentration is v / v%, except for the alcohol concentration. Example 1 Examination of the lysis spectrum of the bactericidal agent of the present invention (hereinafter, referred to as the present product) The lytic spectrum of the present product was examined by measuring the lytic activity against the target bacterium. The lytic activity was regarded as a decrease in turbidity of the cell suspension of the target bacterium, and the turbidity decrease rate was measured by the following method. Macilbain buffer (pH 4.0) so that the turbidity (OD ₆₆₀ ) of the target bacterial cell (logarithmic growth phase) suspension is 1.0 and the antibacterial agent of the present invention is finally 0.25%.
And the turbidity of the reaction solution after 2 hours of reaction was measured. As a control, turbidity of a sample similarly prepared except for the product of the present invention was measured in the same manner. The dissolution rate was determined by the following equation. Table 1 shows the results.

[0012]

(Equation 1)

[0013]

[Table 1]

As is apparent from Table 1, the microorganism of the present invention lysed the microorganisms studied by TU-6 enzyme which is a cell wall lysing enzyme contained therein. In addition, it was found that not only gram-positive bacteria (lactic acid bacteria, fire-killed bacteria, etc.) but also gram-negative bacteria (Escherichia coli, Pseudomonas sp., Etc.) lysed well and had a broad lysis spectrum. In particular, Saccharomyces oleus, which is said to be one of the causative bacteria of food poisoning, which is the causative fungus of burns such as sake,
In addition, Streptococcus mutans, which is a cariogenic bacterium, was well dissolved, and was shown to be useful for preventing burnout, preventing food poisoning, and preventing tooth decay.

Example 2 Examination of the bacteriolytic activity of the product of the present invention in the presence of alcohol In order to examine the lytic activity of the product of the present invention in the presence of alcohol, the reaction system of Example 1 was added to alcohol (ethanol).
The lysis rates of lactic acid bacteria and burnt bacteria when 15% was present and when they were not present were examined and compared. Table 2 shows the results.

[0016]

[Table 2]

As is evident from Table 2, the lytic activity of the product of the present invention was not inferior to the case where ethanol was present in a small amount, even in the presence of 15%. In other words, it was found that the bacteriolytic activity of the product of the present invention was sufficiently effective even when applied directly to alcoholic beverages such as sake or beer having an alcohol concentration of 15% or less.

Example 3 Growth Inhibition Test of Staphylococcus aureus Using the Product of the Invention The aqueous solution of the product of the present invention, which was sterilized by filtration, was used as an autoclaved Brain Heart Infusion Bouillon, a medium for general bacteria. "Nissui" (manufactured by Nissui Pharmaceutical Co., Ltd.) was added to a final concentration of 0.25%, and one obtained by adding sterilized distilled water in place of the aqueous solution of the product of the present invention was used. Staphylococcus aureus, the causative bacterium of food poisoning, was inoculated, and the changes over time in turbidity (OD ₆₆₀ ) when cultured with shaking at 37 ° C. for 24 hours were compared. For comparison, a case where egg white lysozyme (manufactured by Sigma) was similarly added to a final concentration of 0.25% was performed. The results are shown in FIG. For example, assuming that there is a bacteriostatic effect until the change in absorbance occurs, as apparent from FIG. 1, when egg white lysozyme is added to the medium at 0.25% as compared to the non-added group, A bacteriostatic effect was observed for about 2 hours. When the same amount of the product of the present invention was added, a bacteriostatic effect was observed for about 6 hours. Therefore, when the product of the present invention is applied to foods and the like, the growth of bacteria that cause food poisoning can be suppressed. The effect was much higher than that of lysozyme, which has been used as a preservative.

Example 4 Bacterial growth inhibition test using the product of the present invention An aqueous solution of the product of the present invention, which was sterilized by filtration, was mixed with an autoclaved SI medium (1% yeast extract, 0.5% polypeptone,
2.5% glucose, 1% sodium acetate, 0.01%
Magnesium sulfate, 0.00025% manganese sulfate,
0.00025% ferrous sulfate, 0.005% mevalonic acid, 10% ethanol, pH 5.1) to a final concentration of 0.2
5%, 0.125%, and 0.025% were added to the solution, and sterilized distilled water was added instead of the aqueous solution of the product of the present invention. The time-dependent changes in turbidity (OD ₆₆₀ ) when the cells were inoculated and cultured at 30 ° C. for 120 hours were compared. For comparison, a case where egg white lysozyme (manufactured by Sigma) was similarly added to a final concentration of 0.25% was performed. The results are shown in FIG. As is clear from FIG. 2, as compared with the non-added category,
In all the cases where the product of the present invention was added to the culture medium, no growth of H24 was observed, and there was a remarkable growth inhibitory effect on H12. On the other hand, egg white lysozyme had almost no growth inhibitory effect on Hiochi bacteria. In addition, the same examination was performed on other fire-killed bacteria, and the same result was obtained in each case. This study was performed in the presence of 10% ethanol, and it was found that the growth inhibitory effect of the product of the present invention was effective even in the presence of at least 10% ethanol. Therefore, by applying the product of the present invention, it is possible to prevent burnout of alcoholic beverages such as sake and beer and contamination of various bacteria by lactic acid bacteria.

Example 5 Growth Inhibition Test of Dental Caries Bacteria (Streptococcus mutans) Using the Product of the Present Invention An aqueous solution of the product of the present invention, which had been sterilized by filtration, was used as an autoclaved Brain Heart Infusion Bouillon, a medium for general bacteria. "Nissui" (manufactured by Nissui Pharmaceutical Co., Ltd.) added to final concentrations of 0.25%, 0.125% and 0.025%, and sterilized distilled water instead of the aqueous solution of the product of the present invention. To which decayed caries bacteria (Streptococcus mutans), which is the causative agent of the caries of one platinum loop, were inoculated and incubated for 24 hours at 37 ° C. for 24 hours, followed by turbidity (OD ₆₆₀ ). The changes were compared. For comparison, a case where egg white lysozyme (manufactured by Sigma) was similarly added to a final concentration of 0.25% was performed. The results are shown in FIG. As is clear from FIG. 3, the growth of the carious bacteria (Streptococcus mutans) was not observed at all in the case where the product of the present invention was added to the medium, and the growth was remarkable in any case as compared with the non-added case. It had a growth inhibitory effect on cariogenic bacteria. On the other hand, egg white lysozyme did not show any growth inhibitory effect on cariogenic bacteria. Therefore, by mixing the product of the present invention with toothpaste, powdered toothpaste, mouthwash, denture cleanser, etc., it suppresses the growth of dental caries, which is one of the causes of tooth decay, and is used as an anti-caries agent. I knew that I could do it.

Example 6 Examination of heat sensitivity of the product of the present invention In the same manner as in Examples 3, 4 and 5, when the final concentration of the product of the present invention was 0.25%, it was added to the medium. Before and after the heat treatment (in boiling water for 15 minutes), the effect of inhibiting the growth of the respective cells was examined and compared. The results are shown in FIGS. 4, 5 and 6. This embodiment is based on the TU that exists in the product of the present invention.
The bacteriolytic activity of the -6 enzyme is completely inactivated by heat, and the growth inhibitory effect of the microorganism of the present invention is due to the TU-6 enzyme alone or the growth inhibitory activity against a microorganism completely different from the TU-6 enzyme. The purpose of this study was to clarify whether or not the substance had the following. As is clear from FIG. 4, even when the enzyme was inactivated by heat treatment, there was a bacteriostatic effect for about 3 hours. Not only that, but other substances having a growth inhibitory effect were also found to be effective. In addition, in the case of the fire-killed bacteria (S24) in FIG. 5, regardless of the presence or absence of the heat treatment, the growth of the fire-killed bacteria was completely inhibited for at least 120 hours.
It was found that there were microorganism growth inhibitors other than the -6 enzyme. Furthermore, in the case of the carious bacteria of FIG.
Although the growth inhibition effect was reduced to some extent by heating, the result was obtained that the effect was not completely lost. From the above results, the growth inhibitory effect of the product of the present invention is slightly different depending on the target strain, but not only the lytic activity of the cell wall lytic enzyme works but is contained in the culture solution of the TU-6 strain. It turned out to be due to a synergistic effect with unknown substances. From the results, it can be said that the antibacterial agent of the present invention is stable against heat.

[0022]

According to the present invention, unlike a conventional antibacterial agent using egg white lysozyme, a single antibacterial agent can suppress the growth of fire-killed bacteria and can be used as a causative agent of food poisoning. Thus, it is possible to provide a safe and heat-stable antibacterial agent suitable for a wide range of uses, such as suppressing the growth of Staphylococcus aureus) and suppressing the growth of Streptococcus mutans, which is a cariogenic bacterium.

[Brief description of the drawings]

FIG. 1 is a view showing the results of examining the growth inhibitory effect of the product of the present invention on Staphylococcus aureus (Staphylococcus aureus).

FIG. 2 is a diagram showing the results of examining the growth inhibitory effect of the product of the present invention on fire-killed bacteria (S24).

FIG. 3 shows carious bacteria (Streptococcus and
FIG. 9 is a view showing the results of examining the growth inhibitory effect on C. mutans).

FIG. 4 is a diagram showing the results of examining the sensitivity of the product of the present invention to heat with respect to Staphylococcus aureus (Staphylococcus aureus).

FIG. 5 shows that the sensitivity of the product of the present invention to heat is determined by the fire-killed bacteria (S2
It is a figure showing the result of having examined about 4).

FIG. 6 is a graph showing the results of an investigation of the sensitivity of the product of the present invention to heat for carious bacteria (Streptococcus mutans).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 1/02 A61P 1/02 4C087 31/04 31/04 C12H 1/00 C12H 1/00 C12P 21/00 C12P 21/00 A // (C12N 1/20 (C12N 1/20 A C12R 1: 465) C12R 1: 465) (C12P 21/00 (C12P 21/00 A C12R 1: 465) C12R 1: 465) ( 72) Inventor Kenji Ozeki 4-9, Imazu, Iizazu-cho, Nishinomiya City, Hyogo Prefecture, Ozeki Research Institute (72) Inventor Masaaki Hamachi 4-9, Imazu, Iizazu, Iizazu-cho, Nishinomiya City, Hyogo Prefecture, Ozeki Co., Ltd. (72) Inventor Chieko Kumagai 4-9, Imazu, Iizazu-cho, Nishinomiya-shi, Hyogo F-term in the Ozeki Research Institute (reference) 4B021 MC01 MK06 MK07 MP01 MP02 4B028 AC10 AG04 AP29 AS1 8 4B064 AH19 CA04 CA19 CC24 DA01 DA10 4B065 AA50X AB01 AC14 BD14 CA27 CA41 CA42 CA44 4C083 AA031 AA032 CC41 EE32 FF01 4C087 BC23 ZA67 ZB35

Claims (4)

[Claims] [Claim 1] Streptomyces fulvicimus (St
reptmyces fulvissimus) TU-6 strain (FERM P-
16347) or a bactericidal agent comprising, as an active ingredient, a culture of a mutant or a recombinant thereof. 2. The antibacterial agent according to claim 1, which is a food preservative. 3. The antibacterial agent according to claim 1, which is an antiseptic for alcoholic beverages. 4. The antibacterial agent according to claim 1, which is a caries preventive agent.