JP7618281B2 - Biofilm remover - Google Patents

Description

The present invention relates to a biofilm remover and a biofilm removal method.

A biofilm is a structure formed by microorganisms, and it is known that the formation of this biofilm at a wound site on the surface of human skin can delay wound healing.
Therefore, methods for removing this biofilm have been studied, and for example, a gel-type biofilm remover has been proposed. When the biofilm remover is in gel form, it has the advantage that it flows into the wound and adheres to the wound bed, thereby providing an antibacterial effect to the entire wound and maintaining a moist environment in the entire wound.
As a gel-like biofilm remover, for example, a bactericidal composition suitable for use on skin and wounds, which contains an antibacterial agent source and an agent that destroys biofilms, has been proposed (see Patent Document 1).

However, the bactericidal composition described in Patent Document 1 contains an antibacterial agent source in addition to the biofilm-destroying agent. This bactericidal composition also contains a thickener to make the bactericidal composition into a gel. On the other hand, a biofilm remover is required not to cause undesirable side effects (e.g., cytotoxicity, hypersensitivity reactions, etc.) due to long-term use. Therefore, the bactericidal composition described in Patent Document 1 must be considered, including the side effects of each component.

JP 2013-209385 A

The present invention aims to provide a biofilm remover that is in gel form, has excellent biofilm removal performance, and places little strain on the human body, as well as a biofilm removal method using the same.

As a result of intensive research aimed at achieving the above object, the present inventors have discovered the following: That is, among the components capable of removing biofilms, there are some that can be used to make a biofilm remover into a gel by adjusting the concentration thereof, and have thus completed the present invention.
In other words, the biofilm remover of the present invention is a biofilm remover that is in a gel state at temperatures of 25°C and 40°C, contains water and sodium oleate, the amount of sodium oleate being 13% by mass or more and 30% by mass or less relative to the total amount of the biofilm remover, does not contain any antibacterial components other than the sodium oleate, and does not contain any thickening components other than the sodium oleate.

It is preferable that the biofilm removing agent related to the present invention does not contain any antibacterial component other than the potassium oleate or the sodium oleate.
A biofilm removal method related to the present invention is a biofilm removal method using the biofilm removing agent, characterized in that it comprises the steps of applying the biofilm removing agent and removing the applied biofilm removing agent with water.

The present invention provides a biofilm remover that is in gel form, has excellent biofilm removal performance, and imposes little burden on the human body, as well as a biofilm removal method using the same.

FIG. 2 is an explanatory diagram for explaining a biofilm removal method according to an embodiment of the present invention.

Hereinafter, embodiments of the biofilm removing agent and biofilm removing method of the present invention will be described.
[Biofilm remover]
First, the biofilm removing agent of the present embodiment will be described.
The biofilm removing agent of this embodiment is a gel-like biofilm removing agent that contains water and at least one selected from the group consisting of potassium oleate and sodium oleate, which will be described below. Because the biofilm removing agent of this embodiment is in a gel-like form, the biofilm removing agent flows into the wound and adheres to the wound bed, covering the entire wound to provide protection and maintain a moist environment for the entire wound.

The biofilm remover of this embodiment is preferably composed of water and at least one selected from the group consisting of potassium oleate and sodium oleate. The potassium oleate or sodium oleate can ensure biofilm removal performance. The potassium oleate or sodium oleate can also make the biofilm remover gel-like. In the biofilm remover of this embodiment, there is no need to use a biofilm removing component other than potassium oleate or sodium oleate, and there is no need to use a thickening component other than potassium oleate or sodium oleate. Therefore, it is preferable that the biofilm remover of this embodiment does not contain a biofilm removing component other than potassium oleate or sodium oleate. Furthermore, it is preferable that the biofilm remover of this embodiment does not contain a thickening component other than potassium oleate or sodium oleate.
Moreover, from the viewpoint of not using unnecessary components, the biofilm removing agent of this embodiment preferably does not contain any antibacterial components other than potassium oleate or sodium oleate.

The biofilm remover of this embodiment is preferably composed only of water and potassium oleate or sodium oleate, and does not substantially contain any other components. Potassium oleate is a potassium salt of oleic acid, a fatty acid that occurs in great abundance in nature, and has been confirmed to be a low burden on the human body. Sodium oleate is a sodium salt of oleic acid, a fatty acid that occurs in great abundance in nature, and has been confirmed to be a low burden on the human body. The biofilm remover of this embodiment contains substantially only potassium oleate or sodium oleate other than water, and therefore has a low burden on the human body.
The water used in the biofilm remover of this embodiment is preferably purified water. Examples of purified water include RO (Reverse Osmosis) water, deionized water, and distilled water. In addition, the purified water is more preferably purified water that conforms to the Japanese Pharmacopoeia or the quasi-drug raw material standard.
The potassium oleate used in the present embodiment may contain impurities such as potassium laurate, potassium myristate, potassium palmitate, potassium stearate, potassium palmitoleate, potassium linoleate, and potassium linolenate, etc. However, the content of impurities is preferably 30% by mass or less, more preferably 25% by mass or less, even more preferably 20% by mass or less, and particularly preferably 15% by mass or less, based on the total amount of potassium oleate.
The sodium oleate used in this embodiment may contain impurities such as sodium laurate, sodium myristate, sodium palmitate, sodium stearate, sodium palmitoleate, sodium linoleate, and sodium linolenate, etc. However, the content of impurities is preferably 30% by mass or less, more preferably 25% by mass or less, even more preferably 20% by mass or less, and particularly preferably 15% by mass or less.
The potassium oleate or sodium oleate used in this embodiment is obtained from oleic acid as a raw material and a potassium compound (potassium hydroxide, etc.) or a sodium compound (sodium hydroxide, etc.). Here, the raw material oleic acid usually contains impurities. Most of these impurities are linoleic acid, but other impurities include stearic acid, linolenic acid, palmitic acid, etc.

In this embodiment, the total amount of potassium oleate and sodium oleate must be 12% by mass or more and 32% by mass or less with respect to the total amount of the biofilm remover. If the amount of potassium oleate or sodium oleate is less than 12% by mass, the biofilm remover will be liquid at room temperature (25° C.). On the other hand, if the amount of potassium oleate or sodium oleate is more than 32% by mass, the gel will have too low fluidity and will be difficult to mix uniformly.
From the viewpoint of being able to maintain a gel state even in a high-temperature environment, the combined amount of potassium oleate and sodium oleate is preferably 17% by mass or more, more preferably 18% by mass or more, and particularly preferably 20% by mass or more, based on the total amount of the biofilm remover.
From the viewpoint of improving the fluidity of the gel, the total amount of potassium oleate and sodium oleate is preferably 30 mass% or less, more preferably 26 mass% or less, and particularly preferably 23 mass% or less, relative to the total amount of the biofilm remover.

The biofilm remover of the present embodiment may contain an additive within a range that does not impair the object of the present invention. The additive may be at least one selected from the group consisting of a pH adjuster and a chelating agent. More specifically, the additive may be at least one selected from the group consisting of citric acid, tartaric acid, lactic acid, malic acid, oxalic acid, malonic acid, fumaric acid, succinic acid, glycolic acid, phytic acid, or salts thereof.
In addition, when the above-mentioned additives are used, it is preferable that the biofilm removal agent of this embodiment consists of water, at least one selected from the group consisting of potassium oleate and sodium oleate, and the above-mentioned additives.

The biofilm remover of this embodiment can be produced by mixing water and at least one selected from the group consisting of potassium oleate and sodium oleate in the above-mentioned specified ratio and stirring the mixture.

[Biofilm removal method]
Next, the biofilm removal method of this embodiment will be described.
The biofilm removal method of the present embodiment is a biofilm removal method using the biofilm removing agent of the present embodiment, and is a method including an application step and a removal step, which will be described below.

In the application step, as shown in Figures 1(A) and 1(B), a biofilm removing agent 1 is applied to a wound site 2 on the skin surface and its surrounding area.
The application method may be any conventional method for applying a gel, such as (i) a method in which a biofilm remover in a tube is extruded from the tube and applied, or (ii) a method in which the biofilm remover is applied by hand.
From the viewpoint of ease of application of the gel, the temperature of the biofilm remover 1 is preferably 15°C or higher and 45°C or lower, and more preferably 20°C or higher and 35°C or lower.
The amount of the biofilm remover 1 to be applied is not particularly limited, and may be determined appropriately depending on the condition of the wound.

In this embodiment, after the application step, a step of covering the applied biofilm removing agent 1 with a coating material (coating step) may be provided.
Examples of the covering material include dressing materials such as gauze and film. By covering the wound site 2 with the covering material, the wound site 2 can be protected and evaporation of water in the biofilm removing agent 1 can be suppressed.
When this coating step is performed, a step of peeling off the coating material must be performed prior to the subsequent removal step.

In the removal step, the applied biofilm removing agent 1 is removed with water 3, as shown in FIG. 1(C).
Examples of the water 3 include purified water, physiological saline, and tap water.
From the viewpoint of being an appropriate temperature for humans, the temperature of the water 3 is preferably 15° C. or more and 40° C. or less, and more preferably 25° C. or more and 35° C. or less.

As described above, the biofilm removal method of this embodiment can remove biofilms using a gel-like biofilm remover. Furthermore, since the biofilm remover of this embodiment places less strain on the human body, the biofilm removal method of this embodiment can remove biofilms in a manner that places less strain on the human body.

Next, the present invention will be described in more detail with reference to examples and comparative examples, but the present invention is not limited to these examples.

[Example 1]
First, potassium oleate was prepared as follows.
That is, 78.3 g of purified water was added to the reaction tank, followed by 4.1 g of potassium hydroxide (100% by mass), and the reaction tank was placed in a warm bath at 70° C., and 17.6 g of oleic acid (purity: 87% by mass) was added while stirring with a stirrer to cause a reaction. The mixture was then cooled to obtain a biofilm remover (80% by mass of water, 20% by mass of potassium oleate).

[Examples 2 to 4 and Comparative Examples 1 to 3]
A biofilm remover was obtained in the same manner as in Example 1, except that the blending amounts of purified water and potassium oleate were changed as follows.
Example 2: 83% by weight of water, 17% by weight of potassium oleate
Example 3: 75% water, 25% potassium oleate
Example 4: 70% water, 30% potassium oleate
Comparative Example 1: 90% by weight of water, 10% by weight of potassium oleate
Comparative Example 2: 65% by weight of water, 35% by weight of potassium oleate
Comparative Example 3: 60% by weight of water, 40% by weight of potassium oleate

[Examples 5 to 9 and Comparative Examples 4 and 5]
A biofilm remover was obtained in the same manner as in Example 1, except that the blending amounts of purified water and sodium oleate were changed as follows.
Example 5: 87% water, 13% sodium oleate
Example 6: 84% water, 16% sodium oleate
Example 7: 83% water, 17% sodium oleate
Example 8: 80% water, 20% sodium oleate
Example 9: 70% water, 30% sodium oleate
Comparative Example 4: 90% by mass of water, 10% by mass of sodium oleate
Comparative Example 5: 65% by weight of water, 35% by weight of sodium oleate

[Example 10]
A biofilm remover was obtained in the same manner as in Example 1, except that the blending amounts of purified water, potassium oleate, and sodium oleate were changed as follows.
Example 10: 80% by weight of water, 10% by weight of potassium oleate, 10% by weight of sodium oleate

[Evaluation of biofilm remover (gel or not, applicability)]
The biofilm removers were evaluated (whether they were in a gel form, and their applicability) by the following method. The results are shown in Table 1. Table 1 also shows the amount of potassium oleate blended in each biofilm remover.
(1) Whether or not the biofilm remover was in a gel state The temperatures of the biofilm remover were set to 25° C. and 40° C., and the biofilm remover was observed to see whether or not it was in a gel state at each temperature. Then, the biofilm remover was evaluated according to the following criteria.
○: The biofilm remover is in gel form.
×: The biofilm remover is liquid.
(2) Coating Ability The biofilm removing agent was filled into a tube and the temperature was adjusted to 25° C. Then, the tube was pressed to extrude the biofilm removing agent and apply it. Then, the coating ability was evaluated according to the following criteria.
○: The biofilm remover was applied evenly.
×: The biofilm remover could not be applied evenly.

As shown in Table 1, the biofilm removers of Examples 1 to 10 were found to be in a gel state at a temperature of 25°C and to have good applicability. In contrast, when the combined amount of potassium oleate and sodium oleate was too small (Comparative Examples 1 and 4), the biofilm removers were found to be in a liquid state at a temperature of 25°C. Also, when the combined amount of potassium oleate and sodium oleate was too large (Comparative Examples 2, 3, and 5), the applicability was found to be poor.

[Evaluation of biofilm remover (biofilm removal)]
The biofilm remover was evaluated (biofilm removal ability) by the following method. Note that a gel biofilm remover can remove biofilms when the applied biofilm remover is removed with water. Therefore, biofilm removal ability can be evaluated by examining the biofilm removal rate of a biofilm remover diluted with water. Specifically, the following samples 1 to 7 were prepared, and the biofilm removal rate was measured by the following test method.
Sample 1: 1% by weight aqueous solution of potassium oleate (C18:1) Sample 2: 1% by weight aqueous solution of sodium oleate (C18:1) Sample 3: 1% by weight aqueous solution of potassium laurate (C12) Sample 4: 1% by weight aqueous solution of potassium linoleate (C18:2) Sample 5: 1% by weight aqueous solution of sodium laureth sulfate (LES) Sample 6: 1% by weight aqueous solution of sodium dodecyl sulfate (SDS) Sample 7: Softy EX-CARE Foam Hand Wash (Kao Corporation)

(Preparation of biofilm)
The passaged slant (OJ-1, ATCC No. BAA-2856) was pre-cultured in TSA (Trypticase soy agar) slant medium at 37 ° C. for 24 hours. Next, the bacteria were scraped off from the TSA slant medium and diluted with TSB (Tryptic Soybean Broth) so that the absorbance at a wavelength of 600 nm was 0.1 to obtain a bacterial solution. Next, the bacterial solution was dispensed in 1 mL portions into wells (Corning cell culture plate 12 well, for cultured cells). Next, the mixture was statically cultured at a temperature of 37 ° C. for 24 hours to form a biofilm. In addition, wells cultured without dispensing the bacterial solution were also prepared (used to calculate the removal rate).

(Biofilm Removal)
The supernatant of the biofilm was removed, and 1 mL of sterile saline was dispensed and removed once (hereinafter referred to as washing). Next, 1 mL of the sample that had been vortexed for a few seconds immediately before was dispensed into each well. Next, the wells were left to stand for 1 minute at room temperature. Next, the test substance was removed, and the wells were washed three times with 1 mL of sterile saline. The same procedure was performed for each sample, N=3. Instead of the sample, sterile saline was dispensed and measured, and used as a negative control (used to calculate the removal rate).

(Measurement of biofilm removal rate)
0.5 mL of 0.1% by mass crystal violet aqueous solution was dispensed and left to stand for 1 hour. Next, the supernatant was removed, and the wells were washed twice with 1 mL of sterile saline, and then 2 mL of 99.5% by volume ethanol was dispensed. Next, the insolubilized deposits at the bottom of the wells were scraped off with a plastic Corning rod, and the wells were left to stand for 1 hour to extract the crystal violet. Next, the wells were transferred to a new plate, and the absorbance at a wavelength of 570 nm was measured using a plate reader.
The absorbance of the well after contact with the sterile saline solution (Ac), the absorbance of the well cultured without the bacterial solution (Ab), and the absorbance of the well after contact with the sample (As) were measured. Then, the biofilm removal rate was calculated from these measured values based on the following formula (F1). The results are shown in Table 2.

As shown in Table 2, the biofilm removal rates of sample 1 (1% by mass aqueous solution of potassium oleate) and sample 2 (1% by mass aqueous solution of sodium oleate) were found to be higher than those of the other samples. This confirmed that biofilm removers containing potassium oleate have excellent biofilm removal performance.

The biofilm remover of the present invention is useful as a technique for healing wounds.

1... Biofilm remover 2... Wound area 3... Water

Claims (1)

A biofilm removing agent that is in a gel state at a temperature of 25° C. and a temperature of 40° C.,
Contains water, sodium oleate and its impurities ,
the impurities are at least one selected from the group consisting of sodium laurate, sodium myristate, sodium palmitate, sodium stearate, sodium palmitoleate, sodium linoleate, and sodium linolenate;
The content of the impurities is 15% by mass or less based on the total amount of the sodium oleate and its impurities,
The amount of sodium oleate and its impurities is 13% by mass or more and 20% by mass or less with respect to the total amount of the biofilm remover,
It does not contain any antibacterial components other than the sodium oleate and its impurities , and
Contains no thickening components other than sodium oleate and its impurities ;
A biofilm remover characterized by:

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