KR102152292B1 - Composition for the bacterial inactivation comprising spring water and fruit extract - Google Patents

Abstract

The present invention relates to an antibacterial and antifungal effect of hot spring water and lemon extract, as well as a synergistic effect that appears in a mixture of two components.
As a result of the absorbance test of the bacterial culture solution for the experimental group in which distilled water, hot water and lemon extract was added to each experimental group, antibacterial and antifungal effects were confirmed in the mixed composition of the hot spring water and lemon extract. It was confirmed that it was not due to pH change due to. The anti-fungal effect of the hot spring water and lemon extract was confirmed to decrease the growth of the strain by measuring the inhibition of mold growth in the solid culture solution. As a result of cytotoxicity tests in animal cells, the hot spring water and lemon extract exhibiting such antimicrobial effects show that the cell culture solution made with the hot spring water, lemon extract and a mixture of the two components compared to the cell culture solution containing 0.02% benzoic acid, was insignificant. Toxicity was observed. Through this, a composition using hot spring water and lemon extract can function as a natural preservative that is harmless to the human body while exhibiting an antimicrobial effect.

Description

Composition for killing microorganisms comprising spring water and fruit extract {Composition for the bacterial inactivation comprising spring water and fruit extract}

The present invention relates to a composition for killing microorganisms comprising hot spring water and fruit extract.

Microorganisms such as bacteria and fungi grow inside over time in foods and household goods, and toxins from these microorganisms have a harmful effect on the human body, causing diseases.[1, 2] However, chemically stable preservatives are used. When added, the proliferation of these microorganisms can be suppressed. For example, paraben-type preservatives induce the release of the contents of the cell and apoptosis by destroying the phospholipid bilayer by binding a specific functional group to the cell membrane.[3] Parabens, benzoic acid, and dihydroacetic acid are commonly used preservatives. [4] Among them, benzoic acid is the most commonly used preservative for household products such as body lotions and wet tissues.[5] However, direct exposure to benzoic acid can cause harmful diseases such as asthma and urticaria. [6, 7] As such, preservatives such as benzoic acid can cause serious side effects to the human body. There are rules that limit it to less than [5]. In recent years, as consumers' health-related knowledge and inclination to pursue it have increased, the use of products with artificial chemical preservatives such as benzoic acid has been gradually avoided.[8] Research is underway to find natural preservatives that are harmless. Examples of natural substances that can be used as preservatives include plant-derived antibacterial substances, animal-derived antibacterial substances, and microorganism-derived antimicrobial substances.[9] Many of these are due to the difficulty of accessing the ingredients or the purification process of ingredients. There may be restrictions on use in the actual industry. Therefore, it is very important to develop a material that has good accessibility and does not require purification of ingredients or is simple.

Infection caused by pathogenic fungi is the cause of some psoriasis and athlete's foot disease among diseases that occur in the skin.[10] Hot spring water has been known to be effective in curing these diseases.[11] This can be thought to be due to the anti-fungal effect of the hot spring water. In this way, the hot spring water can exhibit antimicrobial effects such as preservatives by the ingredients of the water itself without the addition of special substances. It has been reported that this may be the action of the sulfur component as the antimicrobial effect appears differently depending on the presence or absence of sulfur among the components contained in the hot spring water.[11-13]

Lemon is a substance that anyone can easily access, and because of its antibacterial effect, it has been used for storage of drinking water and for removing harmful bacteria from food.[14] In addition, essential oil extracted from lemon also exhibits such antibacterial effects.[15] The cause of this characteristic of lemon is that citral, a type of monoterpene present in lemon, is effective against Salmonella and Staphylococcus aureus fungi. This can be known through related research.[16]

In this study, we investigated the antibacterial and antifungal performance of each lemon-derived natural substance and the hot spring water itself, and furthermore, through the synergistic effect that appears when these two components are mixed, the possibility of applying the mixture composition to replace preservatives I want to find out.

An object of the present invention is to provide a mixture of fruit extract and hot spring water capable of functioning as a natural preservative that is harmless to the human body while exhibiting an antimicrobial effect.

The present invention is a hot spring water containing a sulfur component; And

It provides a composition for killing microorganisms comprising an extract of one or more fruits selected from the group consisting of lemon, calamansi and lime.

In addition, the present invention provides a sterilization method comprising treating the above-described composition for killing microorganisms on a subject for killing.

In addition, the present invention provides a food composition comprising the composition for killing the aforementioned organisms.

The present invention provides an increased antibacterial and antifungal effect of a mixture of hot spring water and fruit extract (lemon extract).

As a result of the absorbance test of the bacterial culture solution in the embodiment of the present invention, it was possible to confirm the antibacterial and antifungal effects in the mixture of hot spring water and lemon extract, and it was proved that this effect was not due to the pH change caused by each component itself. In addition, the anti-fungal effect of the mixture of hot spring water and lemon extract was confirmed to decrease strain growth through the test for inhibiting mold growth in the solid culture solution. In addition, as a result of the cytotoxicity test in animal cells of the mixed solution, insignificant cytotoxicity was observed in the culture solution of the mixed solution as compared to the cell culture solution containing 0.02% of benzoic acid.

Accordingly, it can be seen that the composition using the hot spring water and fruit extract according to the present invention exhibits antimicrobial and antifungal effects and functions as a natural preservative that is harmless to the human body.

1 is a graph showing the results of a cytotoxicity change test according to the content of a mixture of hot spring water and lemon extract.
2 is a graph showing the antibacterial effect of a mixture of hot spring water and lemon extract.
3 is a graph showing the anti-fungal effect of a mixture of hot spring water and lemon extract.

The present invention is a hot spring water containing a sulfur component; And

It provides a composition for killing microorganisms comprising an extract of one or more fruits selected from the group consisting of lemon, calamansi and lime.

In the present invention, the term “microbial killing” means killing or inhibiting the growth of microorganisms including viruses, fungi (fungi), protozoa and bacteria (bacteria). In addition, in the present invention, the term "composition for killing microorganisms" refers to a composition containing a substance having an effect of killing microorganisms. Accordingly, the composition for killing microorganisms has antibacterial activity, antifungal activity, antiviral activity, and the like.

In addition, the composition for killing microorganisms in the present invention may be expressed as a fruit extract and a hot spring water mixture or a hot spring water containing a fruit extract.

Hot spring water containing a sulfur component according to the present invention; And

A composition comprising an extract of one or more fruits selected from the group consisting of lemon, calamansi and lime has a killing effect against a wide range of microorganisms, and can be used in foods, cosmetics and pharmaceuticals.

In the present invention, the hot spring water contains a sulfur component, and Kim's paper (Kim N, J Beloor, et al., 2014. Quantitative Assessment of Antibacterial, Antioxidant and Cytoprotective Properties of Sulfurous Spring Water. International Journal of Scientific Research, 3(2) ).), you can use the hot spring water of Ildong Jeil Onsen located in Gyeonggi-do.

Since the hot spring water of Ildong Jeil Onsen contains sulfur, it exhibits an excellent effect on killing microorganisms.

The hot spring water may be hot spring water containing 0.1 to 0.5 mg/L of sulfate ions. In addition, in one embodiment, the hot spring water is sulfate ion 0.1 to 0.5 mg/L, sodium 70 to 100 mg/L, potassium 0.1 to 1.5 mg/L, fluorine 8 to 15 mg/L, zinc 0.002 to 0.08 mg/L, Chlorine ion 16.5 to 29.5 mg/L, iron 0.005 to 0.07 mg/L, and manganese 0.001 to 0.01 mg/L.

In the present invention, the extract of one or more fruits selected from the group consisting of lemon, calamansi and lime (hereinafter, fruit extract) is a fruit, that is, after obtaining juice through a juicer from the fruit, that is, the fruit part of lemon, calamansi and lime, filtered Means one filtrate.

In the present invention, the concentration in the composition of the fruit extract may be 0.01 to 4 vol%, 0.01 to 1.5 vol%, 0.5 to 1.2 vol%, or 0.8 to 1.0 vol%. It has an excellent microbial killing effect in the concentration range. When the concentration exceeds 4 vol%, there is a concern that cytotoxicity may be exhibited upon contact through the skin and respiratory system, so it is recommended to adjust to the above concentration range.

The pH of the composition for killing microorganisms according to the present invention may be 2.0 to 7.0 or 5.0 or 7.0. The microbial killing effect of the composition according to the present invention is not due to pH, but due to the components of the composition itself, which can be confirmed from showing excellent microbial killing effect even at pH 7.0.

The composition for killing microorganisms according to the present invention may exhibit a killing effect on harmful microorganisms among microorganisms. In one embodiment, the type of the microorganism is not particularly limited, and Fusarium Oxysporum, Escherichiac coli O157:H7, Shigella spp., Salmonella Typhimurium, Pseudomonas Bacillus (Pseudomonas aeruginosa), Listeria monocytogenes, Aspergillus oryzae and Vibrio sepsis (Vivrio vulnificus), Staphylococcus aureas, and Pseudomonas aeruginosa.

In the following examples, it was confirmed that the bacteria BL21 and the fungus fusarium Oxysporum showed an excellent antimicrobial effect by deriving a synergistic effect of the hot spring water and fruit extract contained in the composition. Accordingly, the composition for killing microorganisms according to the present invention includes hot spring water and fruit extracts, but exhibits a significant microbial killing effect, and thus can be widely applied to foods, cosmetics, or pharmaceuticals.

In addition, the present invention provides a method for killing microorganisms comprising treating the aforementioned composition for killing microorganisms on a subject for killing.

In the present invention, due to the synergistic effect of hot spring water and fruit extract, it is possible to derive an excellent microbial killing effect even when natural substances are used. Treatment of the composition may vary depending on the target for killing microorganisms, but may be, for example, 1 to 30 minutes or 1 to 1 minute. In the present invention, even if it is treated for a short time compared to the prior art, it can have an excellent microbial killing effect due to synergistic effect.

In this case, the object may be food, cosmetics, or pharmaceuticals, and may be widely used to achieve purposes such as antibacterial, sterilization, disinfection, and preservation.

One embodiment of the present invention provides a food composition comprising the composition for killing microorganisms described above.

When the composition of the present invention is prepared as a food composition, the composition may be used as a substitute for food preservatives because it prevents the food from being deteriorated by microorganisms. The composition may be included in the food composition in an amount acceptable for food.

In addition to the composition according to the present invention, the food composition may include ingredients that are commonly added during food production, and may include, for example, proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents. Examples of the carbohydrate include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. When the food composition of the present invention is prepared as a drink, it may further include citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, headworm extract, jujube extract, licorice extract, and the like in addition to the sterilizing composition.

In addition, an embodiment of the present invention provides a cosmetic composition comprising a composition for killing microorganisms.

When the composition is prepared as a cosmetic composition, the composition may be used as a substitute for cosmetic preservatives because it prevents the cosmetic composition from being deteriorated by microorganisms. The composition may be included in the cosmetic composition in an amount acceptable for cosmetics.

In addition to the composition, the cosmetic composition may include ingredients commonly added during the manufacture of cosmetics, for example, a solubilizing agent, a thickening agent, a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, Fragrances, surfactants, water, ionic emulsifiers, nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic activators, lipophilic activators or lipids It may contain adjuvants commonly used in cosmetic fields such as vesicles.

Cosmetics can be prepared in the form of a general emulsified or solubilized formulation. For example, a lotion such as a flexible lotion or a nutritional lotion; Emulsions such as facial lotion or body lotion; Creams such as nourishing cream, moisture cream, or eye cream; essence; Cosmetic ointment; spray; Gel; pack; Sunscreen; Makeup base; Foundations such as liquid type, solid type or spray type; powder; Makeup removers such as cleansing cream, cleansing lotion, and cleansing oil; It may have a formulation such as a cleaning agent such as a cleansing foam, soap, and body wash.

In addition, an embodiment of the present invention provides a pharmaceutical composition comprising a composition for killing microorganisms.

The composition may be included in the pharmaceutical composition in an amount acceptable for pharmaceuticals.

In addition to the composition, the pharmaceutical composition may include ingredients that are commonly added during the manufacture of pharmaceuticals, and may include, for example, a pharmaceutical and acceptable carrier. Such a carrier may contain various components such as a buffer solution, sterile water for injection, normal saline or phosphate buffered saline, sucrose, histidine, or polysorbate.

In addition, hot spring water containing a sulfur component; And

It provides a food composition for killing microorganisms comprising lemon extract.

The composition may prevent food from being deteriorated by microorganisms, and may be used as a substitute for food preservatives.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example .

Example 1. Manufacture of lemon extract and hot spring water mixture

(1) Experimental material

The sulfur-containing hot spring water used in the present invention was collected at Ildong Jeil Hot Spring in Gyeonggi-do, referring to Kim's paper [13], and the collected hot spring water was filtered using a 0.22 μm membrane filter (Millipore, Bilerica, MA).

For lemon extract, cut a commercially available lemon in half, extract the juice from the flesh part excluding the skin through a juicer, and filter the filtrate at 4℃ using a 0.22μm millipore membrane filter (Millipore, Bilerica, MA). Used while storing.

The fungal strain was Fusarium Oxysporum [17] and was used after being distributed from the Center for Fungal Genetics Resources. The fungal strain was collected from the solid medium in which the strain was activated on the solid medium that was first distributed using McGinnis' method [18], and then transferred to a 14 mL Round-Bottom Tube filled with sterilized distilled water and sealed. It was stored in an environment at 25 ° C.

In addition, BL21 was used for bacterial species by referring to Kim's paper [13].

(2) Manufacture of lemon extract and hot spring water mixture

A mixture (spring+Lemon or spring/Lemon) was prepared by adding lemon extract to the hot spring water. The concentration of lemon extract in the mixture was 1%, 2% and 4% (vol%).

Comparative example 1. Manufacture of lemon extract and distilled water mixture

A mixed solution (DW+Lemon, DW/Lemon) was prepared by adding lemon extract to distilled water. The concentration of lemon extract in the mixture was 1%, 2% and 4% (vol%).

Experimental example .

Statistical processing

The experiments conducted in the present invention were all carried out in three repeated experiments, and the experimental values were expressed as mean ± standard deviation. Significant difference analysis for the experimental results was performed through Student's T test, and a p value of less than 0.05 was treated as having statistical significance.

Experimental example 1. Biotoxicity of lemon extract and hot spring water mixture

(1) method

After adding Dulbecco's Modified Eagle Medium powder (Thermo Scientific, Waltham, MA) and 3.7 g/L NaHCO ₃ to each composition solution including lemon extract in distilled water and hot spring water to make a cell culture solution for each composition solution for use in the experiment. Cell culture solutions were prepared by adding 10% FBS and 1% penicillin-streptomycin. Then, adjust the pH to 7.0 using 1M HCl and 1M NaOH solution so that the pH of all cell culture samples is the same, and finally filter through a 0.45μm syringe filter (Millipore, Bilerica, CA) and treat the cells. Used after.

The cytotoxicity test of each composition was carried out by measuring cytotoxicity using a CCK-8 assay kit (Dojindo Laboratories, Kumanoto, Japan) 24 hours after treatment with the culture solution, and then the result analysis was carried out according to the manufacturer's instructions. .

(2) result

In the present invention, Figure 1 is a graph showing the results of the cytotoxicity change experiment according to the content of a lemon extract and hot spring water mixture. In FIG. 1, DW represents distilled water, BA represents a mixture of 0.02% benzoic acid and distilled water, DW+Lemon represents a mixture of lemon extract and hot spring water, and Spring+Lemon represents a mixture of lemon extract and distilled water.

First, a cytotoxicity verification experiment was conducted to confirm whether hot spring water and distilled water containing lemon extract are toxic to skin cells and lung epidermal cells. To compare toxicity according to the content of lemon extract, cytotoxicity was confirmed in human dermal fibroblast, an animal eukaryotic cell, and MLE12, a lung epidermal cell line of mice. In general, when lemon extract is added, the pH of the composition is lowered, which may affect cell growth compared to distilled water and hot spring water under normal conditions, so the pH of the cell culture solution was equally limited in all experimental groups. Therefore, the results shown in the experiment are independent of the pH of the composition of each experimental group, and the cytotoxicity shown in the experiment results can be considered to have occurred due to the components of lemon extract, hot spring water and benzoic acid (BA) added to each cell culture solution. Although the amount of benzoic acid added to distilled water is very small, it shows antimicrobial effect and is set to 0.02%, which is also the amount added to the actual product.[22]

As a result of comparing cytotoxicity by adding 1%, 2% and 4% lemon extract to distilled water, human dermal fibroblast and MLE12 showed little toxicity in cell culture solution made with distilled water containing 1% lemon extract. When% and 4% of lemon extract were contained, cytotoxicity was observed at 30% and 53%, respectively. (Fig. 1A) Similarly, by adding 1%, 2% and 4% lemon extract to hot spring water, cytotoxicity was reduced. As a result of comparison, human dermal fibroblast and MLE12 showed little toxicity in the cell culture solution made with hot spring water containing 1% lemon extract, but when 2% and 4% lemon extract were contained, 27% and 51% were respectively. It showed cytotoxicity. (Fig. 1B)

This is compared with the fact that the cell viability decreased by 9.3% on average when compared with the cell culture solution made with distilled water or hot spring water containing 0.02% benzoic acid, compared with the fact that more than 2% lemon extract contained cytotoxicity upon contact through the skin and respiratory system. Show that it can represent

Experimental example 2. Antibacterial effect of natural substances derived from hot spring water and lemon

(1) method

In the present invention, the antibacterial effect of the mixed solution was measured by measuring the absorbance of the bacterial culture solution.

BL21 was used as the bacterial species, and the growth rate of bacteria in the culture medium of each composition was measured by referring to Sezonov's method [19]. One Luria-Bertani culture solution (Becton, Dickinson and Co.) made based on the water of each experimental group was filtered through a 0.45 μm syringe filter (Millipore, Bilerica, MA), then sterilized and used for bacterial culture. In BL21 culture extract with an OD600 value of 1.0, dilute 1000 times in each experimental group and inject the culture solution, and culture in a shaking incubator at 37°C and 190RPM every 2 hours, SpectraMax M2 spectrophotometer (Molecular devices, Sunnyvale, CA) was used to measure the OD600 value of each experimental group.

(2) result

The antibacterial effect of hot spring water containing 1% lemon extract was investigated based on the results of previous experiments that showed no toxicity to skin and respiratory cells when 1% lemon extract was added to distilled water and hot spring water.

Absorbance measurement using a bacterial culture solution was confirmed by measuring the absorbance measurement value (OD600) at a wavelength of 600 nm after bacterial culture in the culture solution inoculated with BL21 every 2 hours after inoculation.

Distilled water (DW), distilled water containing 0.02% benzoic acid (BA Ehsms DW/BA), hot spring water (Spring), distilled water containing 1% lemon extract (DW) to find out the antibacterial effect of hot spring water containing lemon extract. /Lemon) and 1% of lemon extract-containing hot spring water (Spring/Lemon) were used to prepare a bacterial culture solution, and then the same amount of BL21 was inoculated and the OD600 value was measured every 2 hours.

The experimental results are shown in FIG. 2.

2 is a graph showing the antibacterial effect of hot spring water and lemon extract. As shown in FIG. 2, after 8 hours of BL21 inoculation, the absorbance value decreased by 18% similar to the experimental result using Kim's hot spring water. 13] In distilled water and hot spring water containing 1% lemon extract, it was possible to observe a decrease in absorbance of 23% and 26%, respectively (Fig. 2A, left graph).

In addition, as a result of comparing the area under the curve of the absorbance curve of each composition, 18% in hot spring water, 22% in distilled water containing lemon extract, and 33% in hot spring water containing lemon extract compared to the area under the curve shown in the bacterial culture solution made using distilled water. Area reduction could be confirmed. (Fig. 2A, right graph) When it is seen that the area reduction of up to 50% was confirmed in distilled water containing benzoic acid, it was confirmed that a considerable antibacterial effect appeared even in hot spring water containing lemon extract, and the antibacterial effect of hot spring water was improved by adding lemon extract. I could see that it was.

In order to prove that these experimental results were not caused by the pH change of the composition solution due to the addition of lemon extract, the degree of bacterial culture after BL21 inoculation was observed in a pH 4.0 environment similar to when 1% lemon extract was added to distilled water and hot spring water. As a result of the experiment, it was confirmed that there was little difference in absorbance over time in the bacterial culture solution made with distilled water and hot spring water and the bacterial culture solution made after adjusting the pH of distilled water and hot spring water to 4.0, while distilled water and hot spring water containing lemon extract showed a decrease in absorbance. .(Figure 2B)

Through the results of this experiment, it was found that the antibacterial effect of the addition of lemon extract was not caused by pH change, but by the lemon extract itself.

Experimental Example 3. Anti-fungal effect of natural substances derived from hot spring water and lemon

(3) method

① Measurement of mold growth inhibition in solid culture solution

Potato Dextrose Agar (BD, Franklin Lakes, NJ) was used to prepare a solid medium for fungal culture. For activation of the activated fungus strain, a certain amount of the supernatant was collected from the stock, and a 100-fold diluted 5000 spores/mL solution was inoculated into a solid medium by 50 μL and incubated at 25° C. for 3 days. After that, referring to Islam's experimental method [20], the activated strain fungus strain portion was isolated, and the portion was transferred to a new solid medium and cultured at 25°C for 3 days. After that, the areas located at the same distance from the center of the strain mold strain grown sufficiently radially in the PDA solid medium were separated and transferred to the PDA solid medium made from each composition to be tested, and the average of the fungal strains in each experimental group at 24 hour intervals. The diameter was measured.

② Measurement of dry mass of mold culture solution

The test for measuring the dry mass of the mold culture was carried out by referring to Alharbi's experiment [21]. To make a liquid culture solution, the Potato Dextrose Broth culture solution was filtered through a 0.45 μm syringe filter (Millipore, Bilerica, CA), and then 10 mL of the solution of each experimental group was divided into 50 mL tubes. After collecting a certain amount of the supernatant from the Fusarium Oxysporum strain stock, a 100-fold diluted 5000 spores/mL solution was inoculated into the PDB culture medium of each composition by 200 μL, and cultured in a 140RPM shaking incubator at 37°C for 4 days. To measure the dry mass of each experimental group at 24 hour intervals, first dry a 0.22μm membrane filter (Millipore, Bilerica, CA) to be used as a filter paper for liquid culture solution for one day at 65℃, and then weigh the membrane filter itself before filtering. Measured. After filtering each experimental group, incubate again for one day at 65℃ to remove all excess moisture remaining in the membrane filter, and before filtering with the weight of the membrane filter after filtering after the membrane filter is dried. The dry mass of the mold was measured by subtracting the weight of the membrane filter of.

(2) result

After observing the antibacterial effect of the above lemon extract-containing hot spring water, the following experiment was conducted to confirm whether the composition of the lemon extract and the hot spring water also exhibited antifungal effect. The solid medium that grows mold can inhibit the radial growth of mold strains on the solid medium, depending on the composition of the composition. This can be confirmed by measuring the diameter of the strain.[23] Through this, the antifungal performance of each of the hot spring water and lemon extract anf and the synergistic effect of the two components were confirmed by measuring the diameter of the strain every 24 hours after inoculation of the strain.

The experimental results are shown in FIG. 3.

Figure 3 is a graph showing the anti-fungal effect of hot spring water and lemon extract, as shown in Figure 3, after 4 days of inoculation of the strain, in the medium made of hot spring water, a 14% reduction in diameter value appears, as well as 1% of lemon In distilled water and hot spring water containing the extract, strain diameter reductions of 15% and 28% were observed, respectively.

When it was confirmed that the diameter value decreased up to 35% in the medium made of benzoic acid-containing distilled water (DW/BA), it was confirmed that a considerable anti-fungal effect was observed in the hot spring water containing lemon extract. It was found that the mold effect was improved. (Figure 3A)

In order to prove that these experimental results are not caused by the pH change of the composition solution due to the addition of lemon extract, a culture medium was prepared in a pH 4.0 environment similar to that when 1% lemon extract was added to distilled water and hot spring water, and then the strain was inoculated to The degree of culture was observed. As a result of the experiment, there is little difference in the diameter of the strain in the culture medium made of distilled water and hot spring water and 4 days after inoculation of the strain in the culture medium made after adjusting the pH of the distilled water and hot spring water to 4.0, whereas the strain in distilled water and hot spring water containing lemon extract It was confirmed that a decrease in diameter was observed. (FIG. 3B) Through the results of this experiment, it was found that the antifungal effect according to the addition of the lemon extract was not caused by the change in pH, but by the lemon extract itself.

In addition, the effect of inhibiting growth according to the composition of the culture medium of the mold can be observed through the liquid medium. After inoculation of the same mass of mold strains into each composition, the mold was collected every 24 hours and the dry mass of the mold was measured. In the liquid culture medium, the dry mass increased from the 2nd day, whereas in the culture medium made with hot spring water, a significant change in the experimental value began to appear from the 3rd day. On the other hand, in the other experimental groups, there was no noticeable change in the dry mass until the 4th day (Fig. 3C, left figure). This is because the cell culture solution made with hot spring water suppresses the growth of mold than the culture solution made with distilled water, so the dry mass increased. You might think that it took longer to do it. In addition, in the remaining three other experimental groups, there was no significant change in the dry mass of the mold until the fourth day. In order to prove that the anti-fungal effect of the lemon extract observed in this way is not the effect of the pH itself, the result of an experiment that measured the dry mass of the mold after adjusting all of the pH of the experimental group to 4.0, is a cell culture solution made of distilled water. The increase in the dry mass of the mold in the cell culture solution made with a solution of pH lowered to 4.0 in distilled water and distilled water was almost the same, whereas in the remaining experimental groups with lemon extract added, the dry mass of the mold did not change significantly for 4 days. Became. (Figure 3C, right figure)

The antimicrobial effect of hot spring water and lemon extract confirmed through the above experiments is consistent with the results of previous research papers using a mixture containing a sulfur component and essential oil of lemon. Although the exact mechanism of action of the mixture containing elemental sulfur has not yet been determined, the antibacterial effect is significantly different between Gram-positive bacteria and Gram-negative bacteria, and it has been reported that the effect is particularly strong in Gram-positive bacteria.[24] This is not due to the weak basicity of pH 8.5, which is indicated by the hot spring water itself, but is consistent with the results of previous studies known to show different antibacterial efficacy in the state of containing and removing sulfur from the spring water.[13] Essential oil extracted from lemon also exhibits antibacterial effect, so there was a previous study that reported that it showed very strong fungal effect in Gram-positive bacteria, but the effect was reduced in Gram-negative bacteria.[25] In the aspect of anti-fungal, the exact mechanism of action has not been identified, but the results of studies showing that salts containing sulfur reduce spoilage caused by fungal infections [26, 27][27] and studies on the anti-fungal effect of essential oil extracted from lemon Results have been published [26] In addition, it has been reported that when two components with antibacterial effect are present together, the two components exhibit a synergistic effect and a stronger antibacterial effect.[28] These results seem to be contrary to the results that hot water and 1% lemon extract are harmless to cells in the cytotoxicity test, but this can be considered due to the structural aspects of the cells used in the experiment. Bacteria are prokaryotic cells, which have a cell wall made of peptidoglycans as structures.[29] In addition, although not prokaryotic cells, fungi are eukaryotic cells that have a cell wall made of chitin, glucan, glycoprotein, etc. [30]. In both microorganisms, cell walls are very important for survival, and cell wall synthesis is essential during differentiation. 30, 31] On the other hand, human dermal fibroblast or MLE12 cells are animal eukaryotic cells without cell walls [32], and do not require cell wall synthesis during differentiation. In other words, it can be thought that both the hot spring water and the lemon extract are involved in the cell wall synthesis process and inhibit cell proliferation, which means that the cell wall of both the hot spring and lemon extract is directly exposed to the outside, and the composition of the cell wall is larger than that of the cell membrane. It can be thought that this is reflected through the fact that positive bacteria have a stronger effect than Gram negative bacteria. These results can be considered that the proliferation of bacteria was inhibited due to the low pH caused by the addition of lemon extract, which is strongly acidic. Many types of bacteria have strategies to survive in the extreme pH environment [33]. In the experiment where the pH of the lemon extract as much as 1% was added to the total volume of distilled water was equally limited to 4.0, it was found that the antibacterial effect of the lemon extract was not due to a change in pH, but because of its own components (Figure 1C). On the other hand, in the case of benzoic acid, the result of inhibiting the reproduction of cells in all experiments regardless of the presence of the cell wall was found.As reported in the previous paper, the action of benzoic acid to induce apoptosis through inhibition of intracellular pH balance maintenance. This is because the mechanism works directly inside the cell. [34] Although the amount of benzoic acid was very small in the above experiments, according to the Korean regulations on the use of preservatives, the maximum allowable amount of benzoic acid in most household products was limited to 0.5%.[5] In fact, benzoic acid in many household products Since the content of is much larger than 0.02%, it can be considered that the cell damage to the preservatives we encounter in real life will be greater than the results confirmed in this study. Based on the experiments in this study, the antimicrobial effect of each of the hot spring water and lemon extract, and the stronger antimicrobial effect due to the synergistic effect of the mixed composition, are most widely distributed on the market, but they are harmful when direct contact with the human body. It is possible to think of the potential for natural preservatives that can replace the benzoic acid indicated. However, the exact mechanism of action of the hot spring water and lemon extract has not yet been identified, and additional research is needed on the development of a natural-derived preservative that is powerful and harmless to the human body through a clear identification of the ingredients that exhibit antimicrobial effects and the purification process of the ingredients. I think it will do.

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Claims (6)

Hot spring water containing sulfur; And
It comprises an extract of one or more fruits selected from the group consisting of lemon, calamansi and lime,
The concentration of the extract of the fruit is 0.01 to 4 vol%,
The hot spring water is sulfate ion 0.1 to 0.5 mg/L, sodium 70 to 100 mg/L, potassium 0.1 to 1.5 mg/L, fluorine 8 to 15 mg/L, zinc 0.002 to 0.08 mg/L, chloride ion 16.5 to 29.5 mg A composition for killing microorganisms comprising /L, 0.005 to 0.07 mg/L of iron and 0.001 to 0.01 mg/L of manganese.
delete delete The method of claim 1,
The pH of the composition is a composition for killing microorganisms of 2.0 to 7.0.
The method of claim 1,
The microorganism is a composition for killing microorganisms selected from the group consisting of bacteria and fungi.
A food composition comprising the composition according to claim 1.

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