Sterilization liquid

Abstract

To provide a disinfectant solution having high sterilizing effect for various bacteria and viruses, excellent in safety and handleability.SOLUTION: The disinfectant solution contains a carbonic water and a polyphenol derived from grapefruit seed extract. Preferably, the carbonic water contains fine bubbles. The disinfectant solution with the polyphenol derived from grapefruit seed extract added as sterilizing component exhibits high sterilizing action and antiviral action.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a disinfectant solution containing a disinfectant component derived from grapefruit seed extract. [Background technology]

[0002] Traditionally, disinfectants containing alcohol or chlorine-based disinfectant components such as sodium hypochlorite have been used as disinfectants and sanitizers. However, these components can cause skin problems if they come into contact with the human body, requiring careful handling during use. Therefore, the use of plant-derived disinfectant components is being considered. Grapefruit seed extract (hereinafter referred to as GSE as appropriate) is known as such a component, possessing disinfectant properties.

[0003] For example, Patent Document 1 discloses a disinfectant solution prepared by dissolving GSE in water, adding phytic acid, and then adding brewing alcohol, such that the solution contains 0.07-0.09% by mass of GSE, 0.04-0.06% by mass of phytic acid, 58-59.9% by mass of brewing alcohol, and the remainder being water. This disinfectant solution shows a higher inactivation effect against feline calicivirus, a virus closely related to norovirus in terms of morphological characteristics, than when brewing alcohol is used alone.

[0004] Furthermore, Patent Document 2 discloses a virus inactivator in which a buffering agent is contained in an aqueous solution of GSE and adjusted to a pH of 8 or higher. It is stated that sodium carbonate and sodium bicarbonate are used as buffering agents, and the aqueous solution stabilizes as a buffer, thereby obtaining the long-term stability of the virus inactivator in the weakly alkaline range. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2007-320924 [Patent Document 2] Japanese Patent Publication No. 2021-059512 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] In recent years, the demand for disinfectants for general or medical use has increased, and there is a need for disinfectants that are more effective at killing germs, and are also safer and easier to handle. However, although the liquids described in Patent Documents 1 and 2 have shown effectiveness in inactivating viruses such as norovirus, their effectiveness against bacteria that form spores (hereinafter referred to as spore-forming bacteria) has not been confirmed, which requires a higher level of disinfection power. Furthermore, the disinfectant described in Patent Document 1 is an alcohol-based liquid, which has drawbacks in terms of safety and ease of handling.

[0007] This invention was made in view of the above background, and aims to provide a disinfectant solution that has high disinfecting power and is excellent in terms of safety and ease of handling. [Means for solving the problem]

[0008] One aspect of the present invention is a disinfectant solution comprising carbonated water and polyphenols derived from grapefruit seed extract. [Effects of the Invention]

[0009] A disinfectant solution containing polyphenols derived from grapefruit seed extract as a disinfecting component in carbonated water has been found to exhibit not only disinfectant and antiviral effects against common bacteria, but also high disinfecting power against spore-forming bacteria. The reason for this is not entirely clear, but it is thought that the bubbles in the carbonated water adhere to the surface of foreign substances such as bacteria and viruses and, upon bursting, destroy the protective film covering their surfaces. Furthermore, the disinfecting component rapidly acts on the bacteria and viruses whose protective films have been destroyed, leading to disinfection. In addition, it is presumed that the carbonated water adjusts the pH of the disinfectant solution, creating a favorable environment for disinfection, and that the electrically charged bubbles in the carbonated water make it easier to adsorb bacteria and viruses, thus enabling the removal of various bacteria and viruses.

[0010] As described above, according to the above embodiment, it is possible to provide a disinfectant solution that has high disinfecting power and is excellent in terms of safety and ease of handling. [Modes for carrying out the invention]

[0011] The embodiments of the disinfectant solution will be described in detail below. In this embodiment, the disinfectant solution is an aqueous solution containing carbonated water and polyphenols derived from grapefruit seed extract (hereinafter abbreviated as "grapefruit seed extract-derived"). Preferably, the carbonated water contains microbubbles. Preferably, the disinfectant solution is prepared so that the carbon dioxide concentration is 250 mg / L or higher.

[0012] Preferably, the disinfectant solution is prepared so that its pH is less than 7, and more preferably, it is prepared so that it is a weakly acidic aqueous solution, for example, in the range of pH 4.5 to 6. The carbonated water contained in the disinfectant solution has a buffering effect that adjusts the disinfectant solution to a weakly acidic range, and by containing carbon dioxide in the form of fine bubbles at a predetermined concentration, it stably maintains the disinfectant solution in the predetermined weakly acidic range. The disinfectant solution may also further contain citric acid derived from grapefruit seed extract. Citric acid has the effect of adjusting the pH of the disinfectant solution.

[0013] Grapefruit seed extract (GSE) is an extract of the seeds of the 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 approved as a food additive. GSE (undiluted solution) containing, for example, 1 mg / 100g or more of polyphenols and 30 mg / 100g or more of citric acid is preferably used.

[0014] Carbonated water is an aqueous solution obtained by mixing carbon dioxide (CO2) gas with water, and preferably contains microbubbles or nanobubbles. Microbubbles are typically bubbles with a diameter of 1 μm to 100 μm, while nanobubbles are bubbles with a diameter of less than 1 μm. The particle size and number of microbubbles can be measured, for example, by an image analysis type particle size distribution analyzer for microbubbles. Nanobubbles can be measured by dynamic light scattering using laser light scattering, particle tracking, laser diffraction and scattering, etc.

[0015] Such microbubbles can stably dissolve in the disinfectant solution and maintain the desired carbon dioxide concentration. Furthermore, it is believed that the microbubbles, for example, become negatively charged in the disinfectant solution, making them more likely to adsorb bacteria or viruses, and also have the function of destroying the surface film of these bacteria or viruses, thereby assisting the disinfectant components in acting on them. In addition, the stable presence of microbubbles in the disinfectant solution stabilizes the pH of the disinfectant solution, making it possible to maintain the effectiveness of the disinfectant solution for a long period of time.

[0016] The disinfectant solution is preferably prepared to have a carbon dioxide concentration of 250 mg / L or higher. The greater the amount of finer, bubble-like carbon dioxide dissolved in it, the higher the probability of contact with bacteria or viruses, thus advantageously acting for disinfection or virus inactivation. Carbonated water with a carbon dioxide concentration of approximately 250 mg / L to 300 mg / L or higher is generally considered to be the concentration of a carbonated spring under the Hot Spring Law, and is higher than the concentration naturally present at room temperature.

[0017] Carbon dioxide can be mixed with GSE as carbonated water, which is pre-dissolved in water at a predetermined concentration. Any type of 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 higher, preferably 500 mg / L or higher, which allows the carbon dioxide concentration of the disinfectant solution to be adjusted to the desired concentration.

[0018] The method for preparing carbonated water is not particularly limited, and any method may be used as long as carbon dioxide is mixed with water and dissolved so that fine bubbles are contained at a desired concentration. At that time, the amount of carbon dioxide that can be dissolved in carbonated water is limited by pressure. For example, at atmospheric pressure, about 1000 mg / L is the upper limit. Therefore, preferably, the pressure during the production of carbonated water is made higher than atmospheric pressure, and by performing mixing under pressure, the amount of carbon dioxide that can be dissolved can be increased. Usually, it is preferable to adjust so that the carbon dioxide concentration of the carbonated water is in the range of 1000 mg / L or more and 1300 mg / L or less.

[0019] When mixing carbonated water and GSE to obtain a disinfectant solution, from the viewpoint of effectively exerting its disinfecting action, it is desirable to use the undiluted solution of GSE and set its mixing ratio to 0.1% by volume or more. Also, the upper limit of the mixing ratio of the undiluted solution of GSE is not particularly limited, but if it is in the range of 25% by volume or less, a practically sufficient effect can be obtained. Preferably, the mixing ratio of GSE can be appropriately set within the range of 1% by volume or more and 10% by volume or less so as to obtain a desired disinfecting effect.

[0020] The disinfectant solution thus obtained is an aqueous solution using only natural-derived components and has high safety. Also, it has excellent handling properties and can be used directly, but if necessary or depending on the application, it can also be diluted with water such as tap water and used. In that case, the disinfectant solution can be put into a spray bottle or the like and water can be added to obtain a desired dilution ratio, or the disinfectant solution can be added to the water in a humidifier tank or the like and sprayed in a mist form for use.

[0021] As described above, the disinfectant solution of this embodiment is obtained by adding a disinfecting component derived from grapefruit seed extract to carbonated water in which fine bubble-like carbon dioxide is dissolved, and not only has a disinfecting action and an antiviral action against general bacteria, but also exhibits high disinfecting power against spore-forming bacteria. Also, it is excellent in safety and handling properties and is useful as a disinfectant solution.

[0022] While the exact reason for these effects is not entirely clear, it is believed that the microbubbles contained in the disinfectant solution become electrically charged, making them more likely to adsorb bacteria and viruses. When they adhere to the surface of these foreign substances, they burst, destroying their protective coatings. This allows the disinfectant components to act more effectively on the damaged coatings, leading to rapid disinfection. Furthermore, the stable presence of the microbubbles in the disinfectant solution, along with its buffering effect that maintains a nearly constant pH in the weakly acidic range, creates a favorable environment for disinfection, which is thought to contribute to the removal of various bacteria and viruses. [Examples]

[0023] The present invention will be described in more detail below with reference to examples of disinfectant solutions and the antibacterial or viral inactivation tests conducted against various bacteria or viruses in comparative examples or reference examples, along with their results. However, the present invention is not limited to the following examples.

[0024] (Examples 1-3) A disinfectant solution was prepared as follows, consisting of a mixture of grapefruit seed extract concentrate (hereinafter referred to as GSE concentrate) and high-concentration carbonated water containing microbubbles, and tests were conducted to confirm the effectiveness of the obtained disinfectant solution. A commercially available GSE concentrate with the component composition shown in Tables 1 and 2 was used. As shown in Table 1, the GSE concentrate contains polyphenols and lipids (fatty acids) as disinfectant components, as well as citric acid, fructose, and protein. Table 2 shows the fatty acid composition that makes up the lipids in Table 1.

[0025] The carbonated water containing microbubbles was prepared by mixing tap water and carbon dioxide gas under a pressure of 0.35 MPa to achieve a carbon dioxide concentration of 1000 mg / L. The carbonated water thus prepared was then analyzed using a particle size analyzer employing laser light scattering to determine that it contained nanobubbles with a particle size of less than 1 μm, resulting in a density of 1.9 to 2.0 × 10⁻⁶. 7 It was confirmed that it contained particles / ml.

[0026] Furthermore, it was confirmed that the pH of the carbonated water prepared in this way remains at around 5, regardless of the pH of tap water (typically in the range of 5.8 to 8.6). It was also confirmed that even when carbon dioxide is mixed with alkaline water with a pH of 10, the buffering effect of the high-concentration carbonated water keeps the pH at around 6.

[0027] To obtain a test disinfectant solution, 500 ml of high-concentration carbonated water obtained as described above was mixed with GSE stock solution to a concentration of 2% by volume. Using this test disinfectant solution, an antibacterial test was conducted to confirm the disinfectant effect against spore-forming bacteria using the pour plate culture method. The test bacterial suspension added to the disinfectant solution was prepared using a Bacillus subtilis suspension obtained in advance by culturing Bacillus subtilis, and 1 ml of the bacterial suspension was inoculated into 9 ml of the test disinfectant solution. As shown in Table 3, after allowing the solution to react at room temperature for a predetermined time after adding the bacterial suspension, the test solution was pour plate cultured on standard agar medium and the number of bacteria was measured. The results are listed in Table 3 as Examples 1 to 3.

[0028] [Table 1]

[0029] [Table 2]

[0030] [Table 3]

[0031] Furthermore, the same procedure as in Example 1 was performed using physiological saline as a control. The results are listed in the control solution column in Table 3. As shown in the results in Table 3, the bacterial count immediately after addition of the control solution in Example 1, which did not contain the disinfectant solution, was 1.7 × 10⁶. 5The result was ( / ml). In contrast, in Example 1, the bacterial count was reduced to below the limit of quantification immediately after addition, the same as the control solution, and similar results were obtained in Examples 2 and 3 after 1 hour and 2 hours. Thus, it was confirmed that the disinfectant solution, which is a mixture of GSE containing polyphenols as a disinfectant component and carbonated water containing carbon dioxide microbubbles, shows a high disinfectant effect against spore-forming bacteria.

[0032] (Comparative Examples 1-3) 500 ml of high-concentration carbonated water, obtained in the same manner as in Example 1, was prepared as a comparative test solution without mixing it with the GSE stock solution. 1 ml of a pre-prepared bacterial suspension, similar to that in Example 1, was inoculated into 9 ml of this test solution, and an antibacterial test was conducted to confirm the bactericidal effect against spore-forming bacteria. As shown in Table 3, after allowing the bacterial suspension to act for a predetermined time, the test solution was cultured on standard agar plates, and the bacterial count was measured. The results are listed in Table 3 as Comparative Examples 1-3.

[0033] Furthermore, the same procedure as in Comparative Example 1 was performed using physiological saline as a control. The results are listed in the control solution column in Table 3. As shown in the results in Table 3, the bacterial count in Comparative Examples 1 to 3 was 10. 5 The results were almost identical to the control solution, indicating that high-concentration carbonated water alone does not provide a disinfecting effect against spore-forming bacteria.

[0034] (Comparative Examples 4-7) For comparative testing, an antimicrobial test against spore-forming bacteria was performed using a stock GSE solution without mixing with high-concentration carbonated water, similar to the test in Example 1. 30 ml of stock GSE solution was mixed with 0.1 ml of a pre-prepared test bacterial solution. After contact at room temperature for 10 minutes, 30 minutes, and 1 hour, 1 ml of the mixture was taken and diluted with sterile physiological saline. This diluted solution was cultured on standard agar plates, and the number of viable bacteria was measured after a predetermined time. The test bacterial solution used was spore-forming bacteria obtained by culturing Bacillus subtilis to form spores, and then killing the vegetative bacteria by heating. The results are listed in Table 3 as Comparative Examples 4-7.

[0035] Furthermore, the same procedure as in Comparative Examples 4 and 7 was performed using sterile purified water as a control. The results are listed in the control solution column in Table 3. As shown in the results in Table 3, in the case of GSE stock solution alone in Comparative Examples 4-7, the bacterial count was 10 in all cases. 5 The results were found to be equivalent to those of the control solution immediately after addition or one hour later, indicating no bactericidal effect against spore-forming bacteria.

[0036] (Reference examples 1~3) For reference, the GSE stock solution used in Example 1 was used to prepare test solutions with varying concentrations, and antimicrobial tests were conducted against three types of foodborne pathogens to confirm the presence or absence of effectiveness and the concentration at which effectiveness was observed. As shown in Table 4, the GSE stock solution was diluted with sterile water to obtain 0.1%, 0.01%, and 0.001% aqueous solutions (volume %), and 9 ml of each test solution was mixed with 1 ml of the test bacterial suspension for addition. The test bacterial suspensions were prepared by pre-cultivating the test strains of three species: Escherichia coli, Salmonella spp., and Staphylococcus aureus, and adjusting them to the specified bacterial count levels.

[0037] Immediately after adding and stirring the test solution, the number of bacteria (colony count) was measured using the pour plate method with standard agar medium (35±1°C, 48 hours of incubation). As shown in Table 4, for the physiological saline (control sample 1) used as a comparison, the number of bacteria for all types of bacteria was 10. 5 This was the order. In contrast, Reference Example 1, in which 0.1% GSE stock solution was added, showed that E. coli (E. coli) was 10 4 The bacterial counts showed a decrease of approximately one order of magnitude, with Salmonella showing a decrease of approximately two orders of magnitude and Staphylococcus aureus showing a decrease of approximately one-fifth. In reference examples 2 and 3, to which 0.01% and 0.001% GSE stock solutions were added, no decrease in bacterial count was observed.

[0038] [Table 4]

[0039] Based on the results in Table 4, test solutions containing 2.0% (vol.%) of GSE stock solution were prepared, and similar antibacterial tests were performed on reference examples 4-6 with varying reaction times. 1 ml of each of the three bacterial cultures was added to 9 ml of the test solution and stirred. The bacterial count was measured immediately after addition, after 1 hour (room temperature), and after 2 hours (room temperature) using the pour plate method. The results, along with the results for control samples 2-4, are shown in Table 4.

[0040] As shown in Table 4, the number of bacteria in each of the control samples 2-4 was 10. 5 No changes were observed in the order of order based on the duration of action. In contrast, in reference examples 4-6 to which 2.0% GSE stock solution was added, the bacterial counts of Escherichia coli (E. coli) and Salmonella decreased to 10 or less immediately after addition. Although the bacterial count of Staphylococcus aureus was 40 ( / ml) immediately after addition, it decreased to 10 or less after 1 hour and 2 hours of action, and the bacteria became undetectable.

[0041] (Reference examples 7~9) For reference, an antimicrobial test against enterohemorrhagic Escherichia coli O157 (hereinafter referred to as O157) was performed on the GSE stock solution used in Example 1. As the test solution, 9 ml of GSE stock solution was inoculated with 1 ml of a test bacterial suspension prepared in advance using a preserved bacterial strain, and allowed to react for a predetermined time after the addition of the bacterial suspension, as shown in Table 5. Subsequently, the test solution was cultured on standard agar plate (35°C, 24 ± 2 hours), and the number of bacteria was measured. The results are listed in Table 5 as Reference Examples 7-9, along with the results for a control solution using physiological saline.

[0042] [Table 5]

[0043] As shown in the results in Table 5, even the test solution containing only the GSE stock solution showed a bactericidal effect against O157 immediately after addition, and the levels decreased to below the limit of quantification.

[0044] (Reference examples 10~12) For reference, an antimicrobial test against MRSA (methicillin-resistant Staphylococcus aureus) was performed on the GSE stock solution used in Example 1. As the test solution, 0.1 ml of a pre-prepared test bacterial suspension was added to 10 ml of the GSE stock solution and allowed to react for the specified time shown in Table 5. The reaction was then stopped by diluting it 100-fold with LP diluent. Next, 1 ml of this solution was taken and mixed with 9 ml of physiological saline for dilution, resulting in a 10-fold dilution. This diluted solution was cultured on standard agar plate (at 37°C for 48 hours), and the number of colonies was measured to determine the number of viable bacteria. The test bacterial suspension used was prepared by culturing stored bacteria in advance, removing the culture medium components, and adjusting it to the specified bacterial count range with purified water. The results are listed in Table 6 as Reference Examples 10-12.

[0045] [Table 6]

[0046] Furthermore, the results of the same test conducted with a control solution using sterile purified water are also shown in Table 6. For the control solution, the bacterial count immediately after inoculation is shown as control sample 5, along with the results after reacting for the same predetermined time (1 minute, 5 minutes, 10 minutes) as in Reference Examples 10-12.

[0047] As shown in the results in Table 6, while the bacterial count in control sample 2 did not change significantly immediately after inoculation, the test solution containing the GSE stock solution showed a clear antibacterial effect against MRSA after a 1-minute reaction, as in Reference Examples 10-12, and the number of viable bacteria decreased to less than 100 ( / ml) after a 10-minute reaction.

[0048] (Reference examples 13~20) For reference, a virus inactivation test was conducted on the GSE stock solution used in Example 1 against Influenza A virus and Feline calicivirus. As the test solution, the GSE stock solution was used, and for the working solution obtained by adding and mixing the virus solution, the virus infectivity titer after a predetermined time (room temperature) was measured. As shown in Table 7, for Influenza A virus, measurements were made on the working solutions immediately after the start of the action, after 10 minutes, after 1 hour, and after 3 hours. Also, for Feline calicivirus, measurements were made on the working solutions immediately after the start of the action, after 15 seconds, after 1 minute, and after 10 minutes. These results are listed together with the results of the control solution (purified water) in Table 7 as Reference Examples 13 - 16 and Reference Examples 17 - 20.

[0049] Note that Feline calicivirus is an alternative virus to norovirus. The virus solution is the supernatant obtained by centrifuging the virus culture solution in the cell culture medium and diluted 10 - fold with purified water. For the working solution obtained by adding 0.1 ml of this virus solution to 1 ml of the test solution and allowing it to act at room temperature for a predetermined time, it was then diluted 1000 - fold with the cell maintenance medium. The virus infectivity titer (log TCID 50 / ml) is the 50% tissue culture infectious dose measured based on the TCID 50 (median tissue culture infectious dose) method.

[0050]

Table 7

[0051] As shown in Table 7, for Influenza A virus, in Reference Example 14 after 10 minutes of action, the virus infectivity titer was less than 4.5 (log TCID 50 / ml), and for Feline calicivirus, in Reference Example 18 after 15 seconds of action, the virus infectivity titer was less than 3.5 (log TCID 50 / ml), and the virus was no longer detected.

[0052] (Reference Example 二十一) 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). A test solution prepared by diluting the GSE stock solution 1000 times with purified water was administered orally as a single dose of 20 ml / kg to male and female mice in the test group. No abnormalities were observed during the observation period after the test, and there was no difference in weight gain compared to the control group. Male and female mice in the control group were administered 0.7 ml or 0.6 ml of purified water instead of the test solution, and the observation period was 14 days.

[0053] Furthermore, for reference, the GSE stock solution used in Example 1 was subjected to testing based on the "Test Methods for Substances Constituting Pesticides, Feed Additives, or Veterinary Drugs Residing in Food" (Food Safety Notification No. 0124001, dated January 24, 2005), and the following detections were observed for all test items.

[0054] Based on these results, no acute oral toxicity to animals was observed with the GSE concentrate, and no residual pesticides were detected. The same is likely to be true for the disinfectant solution obtained by mixing the GSE concentrate with carbonated water. Therefore, by using such a disinfectant solution in place of conventional disinfectants, it is possible to achieve both disinfecting performance and safety.

[0055] This type of disinfectant solution contains only naturally derived GSE and carbonated water, and is a weakly acidic aqueous solution with minimal impact on the human body. Therefore, it is not only safe but also easy to handle. Furthermore, by combining GSE containing disinfectant components with carbonated water containing dissolved carbon dioxide in the form of fine bubbles, it exhibits high disinfecting efficacy against various bacteria and viruses, while maintaining stable quality over a long period.

[0056] The present invention is not limited to the embodiments and examples described above, and can be applied to various embodiments without departing from the spirit of the invention.

Claims

1. A disinfectant solution containing carbonated water and polyphenols derived from grapefruit seed extract.

2. The disinfectant solution according to claim 1 , wherein the carbonated water contains fine bubbles.

3. The disinfectant solution according to claim 2 , wherein the fine bubbles include microbubbles or nanobubbles.

4. The disinfectant solution according to any one of claims 1 to 3, wherein the carbon dioxide concentration is 250 mg / L or more.

5. The disinfectant solution according to any one of claims 1 to 4, having a pH of less than 7.

6. The disinfectant solution according to any one of claims 1 to 5, further comprising citric acid derived from the grapefruit seed extract.

7. The disinfectant solution according to any one of claims 1 to 6, which is a mixed solution of the carbonated water and the grapefruit seed extract.

8. The disinfectant solution according to claim 7, wherein the carbon dioxide concentration of the carbonated water is 300 mg / L or more.

9. The disinfectant solution according to any one of claims 1 to 8, wherein the blending ratio of the grapefruit seed extract is 0.1% by volume or more.

10. The disinfectant solution according to any one of claims 1 to 9, wherein the blending ratio of the grapefruit seed extract is 1% by volume or more.