JP7668490B2 - Disinfectant - Google Patents

Description

The present invention relates to a disinfectant solution containing a disinfectant component derived from grapefruit seed extract.

Conventionally, liquid agents for sterilization and disinfection have been used that contain alcohols or chlorine-based sterilizing ingredients such as sodium hypochlorite. However, these ingredients require careful handling when used, as they can cause skin problems if they come into contact with the human body, and so the use of plant-derived sterilizing ingredients has been considered. One such ingredient known to have sterilizing properties is grapefruit seed extract (hereinafter, appropriately referred to as GSE).

For example, Patent Document 1 discloses a disinfectant solution prepared by dissolving GSE in water, adding phytic acid, and then adding brewer's alcohol, with the solution being 0.07-0.09% by mass of GSE, 0.04-0.06% by mass of phytic acid, 58-59.9% by mass of brewer's alcohol, and the remainder being water. This disinfectant solution exhibits a higher inactivation effect against feline calicivirus, a virus morphologically closely related to norovirus, than brewer's alcohol alone.

Patent Document 2 also discloses a virus inactivator in which a buffer is contained in an aqueous solution of GSE, and the pH is adjusted to 8 or higher. Sodium carbonate and sodium bicarbonate are used as the buffer, and it is described that the aqueous solution becomes a buffer solution and is stabilized, and that the virus inactivator can be stabilized over time in the weak alkaline range.

JP 2007-320924 A JP 2021-059512 A

In recent years, the demand for disinfectant solutions for general or medical use has been increasing, and there is a demand for disinfectant solutions with stronger disinfecting power and excellent safety and ease of handling. However, while the liquid preparations described in Patent Documents 1 and 2 have both been shown to be effective in inactivating viruses such as norovirus, their effectiveness against bacteria that form spores (hereinafter, appropriately referred to as spore-forming bacteria), for which a higher disinfecting power is required, has not been confirmed. In addition, the disinfectant solution in Patent Document 1 is a liquid preparation mainly composed of alcohol, which has problems with safety and ease of handling.

The present invention was made in light of this background, and aims to provide a disinfectant liquid that has high disinfecting power, is safe, and is easy to handle.

One aspect of the present invention is a mixture of carbonated water containing fine bubbles and an undiluted solution of grapefruit seed extract,
The carbonated water is high-concentration carbonated water having a carbon dioxide concentration of 300 mg/L or more.
The grapefruit seed extract concentrate contains 1 mg/100 g or more of polyphenols and 30 mg/100 g or more of citric acid,
The mixture has a pH of less than 7;
The blending ratio of the grapefruit seed extract concentrate in the mixed solution is 1 volume % or more to form a sterilization solution.

It has been found that a disinfectant liquid in which polyphenols derived from grapefruit seed extract are added as a disinfectant component to carbonated water not only has disinfectant and antiviral effects against general bacteria, but also has a high disinfecting power against spore-forming bacteria. The reason for this is not entirely clear, but it is thought that when the air bubbles contained in the carbonated water adhere to the surface of foreign bodies such as bacteria and viruses and burst, they have the effect of destroying the membrane that covers their surface, and further, the disinfectant component acts quickly on the bacteria and viruses whose membranes have been destroyed, progressing the disinfection. It is also presumed that the carbonated water has the effect of adjusting the pH of the disinfectant liquid, forming an environment suitable for disinfection, and that the air bubbles contained in the carbonated water are electrically charged and easily adsorb bacteria and viruses, contributing to the removal of various bacteria and viruses.

As described above, the above embodiment provides a disinfectant solution that has high disinfecting power, and is safe and easy to handle.

Hereinafter, an embodiment relating to the disinfectant solution will be described in detail.
In this embodiment, the disinfectant solution is an aqueous liquid agent containing carbonated water and polyphenols derived from grapefruit seed extract (hereinafter, abbreviated as grapefruit seed extract-derived). Preferably, the carbonated water contains fine bubbles. Also, preferably, the disinfectant solution is prepared so that the carbon dioxide concentration is 250 mg/L or more.

Preferably, the disinfectant liquid is prepared to have a pH of less than 7, and more preferably, to be a weakly acidic aqueous solution, for example, a pH in the range of 4.5 to 6. The carbonated water contained in the disinfectant liquid has a buffering effect that adjusts the disinfectant liquid to a weakly acidic region, and by containing finely bubbled carbon dioxide at a predetermined concentration, the disinfectant liquid is stably maintained in a predetermined weakly acidic region. The disinfectant liquid may further contain citric acid derived from grapefruit seed extract. Citric acid has the effect of adjusting the pH of the disinfectant liquid.

Grapefruit seed extract (GSE) is an extract of the seeds of grapefruit, a citrus fruit, and is extracted, for example, from crushed seeds using a solvent. GSE is known as a food additive whose main components are fatty acids and flavonoids (polyphenols), and in addition to these components, it also contains citric acid, etc. Citric acid is also recognized as a food additive. GSE (undiluted solution) that contains, for example, 1 mg/100 g or more of polyphenols and 30 mg/100 g or more of citric acid is preferably used.

Carbonated water is an aqueous liquid obtained by mixing carbon dioxide (CO ₂ ) gas with water, and preferably contains fine bubbles of the size of microbubbles or nanobubbles. Microbubbles are usually bubbles with a diameter of 1 μm or more and 100 μm or less, and nanobubbles are bubbles with a diameter of less than 1 μm. The particle size and number of fine bubbles can be measured, for example, for microbubbles, using an image analysis type particle size distribution measuring device, etc. Furthermore, nanobubbles can be measured by a dynamic light scattering method using scattering of laser light, a particle tracking method, a laser diffraction/scattering method, etc.

Such fine bubbles are stably dissolved in the disinfectant liquid, and can maintain the desired carbon dioxide concentration. In addition, the fine bubbles are negatively charged in the disinfectant liquid, for example, and are thought to have the function of easily adsorbing bacteria or viruses and destroying the coating on their surfaces, thereby assisting the disinfecting components in acting on the bacteria or viruses. Furthermore, the stable presence of fine bubbles in the disinfectant liquid stabilizes the pH of the disinfectant liquid, making it possible to maintain the effectiveness of the disinfectant liquid for a long period of time.

The disinfectant solution is preferably prepared so that the carbon dioxide concentration is 250 mg/L or more. The presence of more finely-dissolved carbon dioxide bubbles increases the probability of contact with bacteria or viruses, which is advantageous for disinfection or virus inactivation. Carbonated water with a carbon dioxide concentration of about 250 mg/L to 300 mg/L or more is generally considered to be a carbonated spring according to the Hot Springs Act, and is a higher concentration than that which exists naturally at room temperature.

Carbon dioxide can be dissolved in water at a predetermined concentration in advance to form carbonated water, which can be mixed with GSE. Any water can be used, such as ordinary tap water, natural water, or distilled water. The carbon dioxide concentration of the carbonated water is preferably 300 mg/L or more, and more preferably 500 mg/L or more, making it possible to adjust the carbon dioxide concentration of the sterilizing liquid to the desired concentration.

There are no particular limitations on the method for preparing carbonated water, and any method can be used as long as carbon dioxide is mixed and dissolved in water so that fine bubbles are contained at the desired concentration. In this case, the amount of carbon dioxide that can be dissolved in carbonated water is limited by pressure, and at atmospheric pressure, for example, the upper limit is about 1000 mg/L. Therefore, it is preferable to increase the amount of carbon dioxide that can be dissolved by making the pressure during production of carbonated water higher than atmospheric pressure and mixing under pressure. Usually, it is preferable to adjust the carbon dioxide concentration of the carbonated water to be in the range of 1000 mg/L or more and 1300 mg/L or less.

When mixing carbonated water and GSE to make a disinfectant solution, it is desirable to use a concentrate of GSE and set the mixing ratio at 0.1% by volume or more in order to effectively exert the disinfecting action. There is no particular upper limit to the mixing ratio of the concentrate of GSE, but a range of 25% by volume or less will provide sufficient practical effects. Preferably, the mixing ratio of GSE can be appropriately set in the range of 1% by volume or more and 10% by volume or less so that the desired disinfecting effect can be obtained.

The disinfectant solution obtained in this way is an aqueous liquid agent using only naturally derived ingredients, and is highly safe. It is also easy to handle and can be used directly, but it can also be diluted with water, such as tap water, if necessary or depending on the application. In that case, the disinfectant solution can be placed in a spray bottle or the like and water added to achieve the desired dilution ratio, or the disinfectant solution can be added to water in a humidifier tank or the like and sprayed in the form of a mist.

As described above, the disinfectant liquid of this embodiment is made by adding a disinfectant component derived from grapefruit seed extract to carbonated water in which fine carbon dioxide bubbles are dissolved, and it not only has disinfectant and antiviral effects against general bacteria, but also has high disinfecting power against spore-forming bacteria. It is also safe and easy to handle, making it useful as a disinfectant liquid.

The reason why such an effect is obtained is not entirely clear, but it is believed that a major contributing factor is that the fine bubbles contained in the disinfectant liquid are electrically charged and therefore easily adsorb bacteria and viruses, and when they adhere to the surface of foreign bacteria or viruses, they burst and destroy their coating. This makes it easier for the disinfectant components to act on the bacteria and viruses whose coatings have been destroyed, and disinfection proceeds quickly. It is also believed that the fine bubbles are stable in the disinfectant liquid, have a buffering effect that keeps the pH almost constant in the weak acidic range, and form an environment suitable for disinfection, which contributes to making it possible to remove various bacteria and viruses.

The present invention will be explained in more detail below by showing examples of the disinfectant solution, as well as antibacterial tests or virus inactivation tests conducted on various bacteria or viruses in comparative examples or reference examples, and the results thereof. However, the present invention is not limited to the following examples.

(Examples 1 to 3)
A disinfecting solution consisting of a mixture of grapefruit seed extract stock solution (hereinafter, GSE stock solution) and high-concentration carbonated water containing fine bubbles was prepared as follows, and a test was conducted to confirm the effect of the obtained disinfecting solution. As the GSE stock solution, a commercially available product having the component composition shown in Tables 1 and 2 was used. As shown in Table 1, the GSE stock solution contains polyphenols as disinfecting components, lipids (fatty acids), as well as citric acid, fructose, and proteins. Table 2 shows the fatty acid composition constituting the lipids in Table 1.

The carbonated water containing fine bubbles was high-concentration carbonated water prepared by mixing tap water and carbon dioxide gas under a pressure of 0.35 MPa to give a carbon dioxide concentration of 1000 mg/L. It was confirmed by a particle size measuring device using scattering of laser light that the carbonated water prepared in this manner contained nanobubbles with particle sizes of less than 1 μm at 1.9 to 2.0 x ¹⁰⁷ pieces/mL.

It was also confirmed that the carbonated water prepared in this way maintains a pH of about 5, regardless of the pH of tap water (usually in the range of 5.8 to 8.6). It was also confirmed that even when carbon dioxide is mixed with alkaline water of pH 10, the pH remains around 6 due to the buffering effect of the carbonated water.

500 ml of the high-concentration carbonated water obtained as described above was mixed with the GSE stock solution to obtain a content ratio of 2% by volume to obtain a disinfectant solution for testing. This disinfectant solution for testing was used to conduct an antibacterial test by pour plate culture method to confirm the disinfectant effect against spore-forming bacteria. The test bacteria solution to be added to the disinfectant solution was prepared using a Bacillus subtilis solution obtained in advance by culturing Bacillus subtilis, and 1 ml of the bacteria solution was inoculated into 9 ml of the disinfectant solution for testing. As shown in Table 3, after the addition of the bacteria solution and allowing it to act for a specified time at room temperature, the test solution was pour-cultured on a standard agar medium and the number of bacteria was counted. The results are shown in Table 3 as Examples 1 to 3.

In addition, the same operation as in Example 1 was performed using physiological saline as a comparative control. The results are shown in the control solution column in Table 3. As shown in the results in Table 3, the control solution of Example 1 not containing a disinfectant solution had a bacterial count of 1.7 x ¹⁰⁵ (/ml) immediately after addition. In contrast, in Example 1, the bacterial count was reduced to below the lower limit of quantification immediately after addition, just like the control solution, and similar results were obtained after 1 hour and 2 hours in Examples 2 and 3. In this way, it was confirmed that the disinfectant solution obtained by mixing GSE containing polyphenols as a disinfectant component and carbonated water containing fine bubbles of carbon dioxide exhibits a high disinfecting effect against spore-forming bacteria.

(Comparative Examples 1 to 3)
A comparative test solution was prepared by mixing 500 ml of high-concentration carbonated water obtained in the same manner as in Example 1 with no GSE stock solution. 1 ml of the bacterial solution prepared in advance in the same manner as in Example 1 was inoculated into 9 ml of this test solution, and an antibacterial test was carried out to confirm the sterilization effect against spore-forming bacteria. As shown in Table 3, after the bacterial solution was allowed to act for a predetermined time after addition, the test solution was pour cultured on a standard agar medium and the number of bacteria was counted. The results are shown in Table 3 as Comparative Examples 1 to 3.

As a control, physiological saline was used and the same operation as in Comparative Example 1 was carried out. The results are shown in the control solution column in Table 3. As shown in the results in Table 3, in all of Comparative Examples 1 to 3, the bacterial counts were on the order of ¹⁰⁵ , which was almost the same as the control solution, and it was found that high-concentration carbonated water alone was not effective in eliminating spore-forming bacteria.

(Comparative Examples 4 to 7)
As a comparative test solution, a GSE stock solution without high-concentration carbonated water was used, and an antibacterial test against spore-forming bacteria was performed in the same manner as in Example 1. 0.1 ml of a test bacteria liquid prepared in advance was added to 30 ml of the GSE stock solution as a test solution, and mixed. After contact at room temperature for 10 minutes, 30 minutes, and 1 hour, 1 ml of the liquid was taken and diluted with sterile physiological saline. This diluted liquid was pour-cultured on a standard agar medium, and the number of live bacteria was measured after a predetermined time. For the test bacteria liquid, spore-forming bacteria in which vegetative bacteria were killed by heating after culturing Bacillus subtilis to form spores were used. The results are shown in Table 3 as Comparative Examples 4 to 7.

As a control, sterilized purified water was used, and the same operations as in Comparative Examples 4 and 7 were carried out. The results are also shown in the control solution column in Table 3. As shown in the results in Table 3, in the case of using only the GSE undiluted solution in Comparative Examples 4 to 7, the bacterial count was on the order of ¹⁰⁵ , which was equivalent to the control solution immediately after addition or one hour later, and it was found that no sterilization effect against spore-forming bacteria was observed.

(Reference Examples 1 to 3)
For reference, test solutions with different concentrations of the GSE stock solution used in Example 1 were prepared and antibacterial tests were conducted against three types of food poisoning bacteria to confirm the presence or absence of an effect and the concentration at which the effect was observed. As shown in Table 4, the test solutions were prepared by diluting the GSE stock solution with sterilized water to 0.1%, 0.01%, and 0.001% aqueous solutions (volume %), and 1 ml of the test bacteria solution for addition was added to each of 9 ml of these test solutions and stirred. The test bacteria solutions were prepared by culturing three types of bacteria, Escherichia coli, Salmonella spp, and Staphylococcus aureus, using test strains in advance and preparing them to have a predetermined bacterial count level.

The number of bacteria (colonies) that appeared in the test solution immediately after addition and stirring was counted by pour plate culture method using standard agar medium (cultured at 35±1°C for 48 hours). As shown in Table 4, the number of bacteria was on the order of ¹⁰ for all bacteria in the physiological saline solution (control sample 1) used as a comparison control. In contrast, in Reference Example 1, which was added with 0.1% GSE stock solution, Escherichia coli (E. coli) was reduced by about one order of magnitude to ¹⁰ , Salmonella spp. was reduced by about two orders of magnitude, and Staphylococcus aureus was reduced by about one-fifth. No reduction in the number of bacteria was confirmed in Reference Examples 2 and 3, which were added with 0.01% and 0.001% GSE stock solutions.

Based on the results in Table 4, a test solution containing 2.0% (volume %) GSE stock solution was prepared, and a similar antibacterial test was carried out for Reference Examples 4 to 6 in which the reaction time was changed. 1 ml of the test bacteria solution for adding the three types of bacteria was added to 9 ml of the test solution and stirred, and the number of bacteria was measured using the pour plate culture method immediately after addition, after 1 hour of reaction (room temperature), and after 2 hours of reaction (room temperature). The results are shown in Table 4, along with the results of control samples 2 to 4 for comparison.

As shown in Table 4, in Control Samples 2 to 4, the bacterial counts for all bacteria were on the order of ¹⁰⁵ , and no change was observed with time of action. In contrast, in Reference Examples 4 to 6, in which 2.0% GSE stock solution was added, the bacterial counts for Escherichia coli (E. coli) and Salmonella spp. were 10 or less immediately after addition. For Staphylococcus aureus, the bacterial count was 40 (/ml) immediately after addition, but after 1 and 2 hours of action, it was 10 or less, and the bacteria were no longer detectable.

(Reference Examples 7 to 9)
For reference, the GSE stock solution used in Example 1 was subjected to an antibacterial test against enterohemorrhagic Escherichia coli O157 (hereinafter, O157). As the test solution, 1 ml of a test bacterial solution for addition, which had been prepared in advance using a preserved bacterial strain, was inoculated into 9 ml of the GSE stock solution, and the solution was allowed to act for a predetermined time from the addition of the bacterial solution, as shown in Table 5. The test solution was then pour cultured on a standard agar medium (35°C, 24±2 hours), and the number of bacteria was counted. The results are shown in Table 5 as Reference Examples 7 to 9, together with the results of a control solution using physiological saline.

As shown in the results in Table 5, even the test solution containing only the GSE concentrate showed a sterilizing effect against O157 immediately after addition, reducing it below the lower limit of quantification.

(Reference examples 10 to 12)
For reference, the GSE stock solution used in Example 1 was subjected to an antibacterial test against MRSA (methicillin-resistant Staphylococcus aureus). 0.1 ml of a test bacteria solution prepared in advance was added to 10 ml of the GSE stock solution as a test solution, and the test solution was reacted for a predetermined time shown in Table 5, and then diluted 100 times with LP dilution solution to stop the reaction. Next, 1 ml of the test solution was taken out and mixed with 9 ml of dilution saline to dilute 10 times. This diluted solution was mixed and cultured on a standard agar medium (at 37°C for 48 hours), and the number of colonies was counted to determine the viable cell count. The test bacteria solution was prepared by culturing a stock bacteria in advance to remove medium components and then adjusting the bacteria count to a predetermined range with purified water. The results are shown in Table 6 as Reference Examples 10 to 12.

The results of a similar test performed on a control solution using sterilized purified water are also shown in Table 6. For the control solution, the results are shown for the same specified times (1 minute, 5 minutes, and 10 minutes) as in Reference Examples 10 to 12, as well as the number of bacteria immediately after inoculation as control sample 5.

As shown in the results in Table 6, in the case of control sample 2, the number of bacteria did not change significantly immediately after inoculation, whereas in the case of the test solutions containing GSE concentrate, as in Reference Examples 10 to 12, a clear sterilization effect against MRSA was observed in a reaction of 1 minute, and the number of viable bacteria decreased to less than 100 (/ml) in a reaction of 10 minutes.

(Reference examples 13 to 20)
For reference, a virus inactivation test against influenza A virus and feline calicivirus was performed on the GSE stock solution used in Example 1. The GSE stock solution was used as the test solution, and the virus solution was added and mixed to the working solution, and the virus infectivity was measured after a predetermined time (room temperature). As shown in Table 7, the influenza virus was measured for the working solution immediately after the start of the action, 10 minutes later, 1 hour later, and 3 hours later. The feline calicivirus was measured for the working solution immediately after the start of the action, 15 seconds later, 1 minute later, and 10 minutes later. These results are shown in Table 7 as Reference Examples 13 to 16 and Reference Examples 17 to 20, together with the results of the control solution (purified water).

Feline calicivirus is an alternative virus to norovirus. The virus solution was prepared by centrifuging a virus culture solution of a cell culture and diluting the supernatant 10-fold with purified water. 0.1 ml of this virus solution was added to 1 ml of test solution to prepare an action solution, which was allowed to act at room temperature for a predetermined time, and then diluted 1000-fold with cell maintenance medium. The virus infectivity (log TCID ₅₀ /ml) is the 50% tissue culture infectious dose measured based on the TCID ₅₀ (median tissue culture infectious dose) method.

As shown in Table 7, in Reference Example 14, the viral infectivity of influenza virus was less than 4.5 (log TCID ₅₀ /ml) after 10 minutes of exposure, and in Reference Example 18, the viral infectivity of feline calicivirus was less than 3.5 (log TCID ₅₀ /ml) after 15 seconds, and the virus was no longer detectable.

(Reference Example 21)
For reference, the GSE stock solution used in Example 1 was subjected to an acute oral toxicity test (limit test) in mice in accordance with the OECD Guidelines for Toxicity Testing of Chemical Substances (1987). The GSE stock solution was diluted 1000 times with purified water, and the test solution was orally administered to male and female mice in a single dose of 20 ml/kg. No abnormalities were observed during the observation period after the test, and no difference was observed in weight gain compared to the control group. The control group of male and female mice was administered 0.7 ml or 0.6 ml of purified water instead of the test solution, and the observation period was 14 days.

For reference, the GSE concentrate used in Example 1 was tested according to "Testing Methods for Pesticides, Feed Additives, or Veterinary Drug Ingredients Residual in Food" (Notification No. 0124001 from the Food Safety Bureau dated January 24, 2005), and the following were detected in all test items:

The above results show that GSE concentrate does not pose any acute oral toxicity to animals, and no residual pesticides have been detected. The same is likely true for the disinfectant solution obtained by mixing GSE concentrate with carbonated water, so by using such a disinfectant solution in place of conventional disinfectants, it will be possible to achieve both disinfecting performance and safety.

This type of disinfectant solution contains only GSE, a naturally derived ingredient, and carbonated water, and is a weakly acidic aqueous solution that has little effect on the human body, making it not only safe but also easy to handle. Furthermore, by combining GSE, which contains disinfectant ingredients, with carbonated water in which fine carbon dioxide bubbles are dissolved, it exhibits a high disinfecting effect against various bacteria and viruses, and can also maintain stable quality for a long period of time.

The present invention is not limited to the above-described embodiments and examples, and can be applied to various embodiments without departing from the spirit of the present invention.

Claims (7)

It is made of a mixture of carbonated water containing fine bubbles and a concentrate of grapefruit seed extract.
The carbonated water is high-concentration carbonated water having a carbon dioxide concentration of 300 mg/L or more.
The grapefruit seed extract concentrate contains 1 mg/100 g or more of polyphenols and 30 mg/100 g or more of citric acid,
The mixture has a pH of less than 7;
A disinfecting solution, in which the blending ratio of the grapefruit seed extract concentrate in the mixed solution is 1 volume % or more . The disinfectant solution according to claim 1, wherein the carbonated water is high-concentration carbonated water having a carbon dioxide concentration of 1000 mg/L or more and 1300 mg/L or less . The disinfectant solution according to claim 2 , wherein the fine bubbles include nanobubbles having a particle diameter of less than 1 μm . The disinfectant solution according to any one of claims 1 to 3 , wherein the blending ratio of grapefruit seed extract in the mixed solution is 2 volume% or more . The disinfectant solution according to any one of claims 1 to 4 , wherein the mixed solution has a pH of 4.5 or more and 6 or less . The disinfecting solution according to any one of claims 1 to 5, wherein the grapefruit seed extract concentrate further contains a fatty acid . The disinfectant solution according to any one of claims 1 to 6 , which is intended for the removal of spore-forming bacteria, which are spore-forming bacteria .