KR102450693B1 - A lactobacillus paracasei uos1 strain and antibacterial composition comprising the same - Google Patents

Abstract

The present invention relates to a lactobacillus paracasei UOS1 strain having antibacterial activity, and also provides an antibacterial composition containing a culture, lysate, extract or purified product of the strain as an active ingredient. An objective of the present invention is to provide a natural antibacterial agent which is derived from a new strain with excellent biosafety and antibacterial activity and can replace conventional synthetic preservatives.

Description

Lactobacillus paracasei UOS1 strain, and antimicrobial composition comprising the same {A LACTOBACILLUS PARACASEI UOS1 STRAIN AND ANTIBACTERIAL COMPOSITION COMPRISING THE SAME}

The present invention relates to a Lactobacillus paracasei UOS1 strain having antibacterial activity and uses thereof.

Various microorganisms exist on human skin, most of which are not harmful to humans, but bacteria such as Listeria monochitogenes, Salmonella genus, Staphylococcus aureus and some pathogenic E. can threaten

Recently, natural ingredients have been widely used in cosmetics, and contamination by microorganisms may occur easily, resulting in quality deterioration such as discoloration, discoloration, and changes in viscosity and pH.

Cosmetics consist of a mixture of various substances with high molecular weight lipids or water as the main component. Among these substances, glycerin and sorbitol, which are carbon sources for microorganisms, are contained, and amino acid derivatives and proteins are mixed as nitrogen sources. Bacteria and mold using carbon and nitrogen sources as mediators can easily propagate.

As such, cosmetics are vulnerable to microbial contamination due to their use, so preservatives have to be used to protect cosmetics for a long period of time, and since cosmetics are repeatedly used on the skin, it is necessary to develop a preservative with excellent biosafety and less skin irritation.

Therefore, in various products including cosmetics, an appropriate antiseptic system against microorganisms occupies an important part in product development.

Parabens, which are preservatives that have been widely used in cosmetics, drugs, and processed foods, have been used for a long time because of their broad and effective spectrum of antibacterial activity against gram-positive and gram-negative bacteria.

However, parabens cause an imbalance in hormones in the body and have recently been restricted and controversial due to problems related to carcinogenesis such as breast cancer.

In general, synthetic raw materials have excellent antibacterial activity, but they can cause dermatitis, allergies and hormone secretion abnormalities, and there is a risk that they do not biodegrade and remain on the skin. There is a need to discover and commercialize materials.

Natural preservatives to replace synthetic preservatives include plant extracts such as safflower extract, grapefruit extract, hinokitol, and magnolol, but these are difficult to use publicly due to their high production cost and low preservative efficacy, allowing microorganisms to reproduce There are risks that can be

Therefore, in recent years, interest in microorganisms that produce natural antibacterial substances that are non-toxic and do not harm humans is increasing. We are strengthening our market entry strategy.

In the case of microorganisms widely present in nature, they secrete metabolites for survival and dominance in the habitat, and these metabolites have the effect of inhibiting the growth or killing of other microorganisms.

Metabolites with antibacterial activity produced by microorganisms include biosurfactant and bacteriocin (Jiang et al., 2012; Sharma and Saharan, 2014). In addition, various enzymes and siderophores, which are antibacterial and antifungal substances produced by microorganisms, are also available. reported (Nagarajkumar et al., 2004).

Because these microorganism-derived metabolites have a structure and efficacy similar to those of chemically synthesized components, they are spotlighted as substitutes for chemically synthesized components that are presently present with various risks as natural products. In addition, since it is easy to operate and can be cultured in large quantities, it has advantages in terms of economy and utility.

As a result of culturing and studying microorganisms isolated from cabbage kimchi aged for more than 3 months, the present inventors can produce metabolites that inhibit the growth of microorganisms, have antibacterial activity against various types of microorganisms, and are stable even in a wide range of pH It was confirmed that it can exhibit antibacterial activity, and the present invention was completed.

(Patent Document 1) Republic of Korea Patent No. 10-1990820
(Patent Document 2) Republic of Korea Patent No. 10-1467990

The present invention is to solve the problems of the prior art, and an object of the present invention is to provide a natural antibacterial agent derived from a new strain having excellent biosafety and antibacterial activity that can replace conventional synthetic preservatives.

According to one aspect of the present invention, there is provided a Lactobacillus paracasei UOS1 strain deposited with accession number KACC 81213BP.

In one embodiment, the strain has a citric acid (Citric acid), lactic acid (Lactic acid), and acetic acid (acetic acid) producing ability, the citric acid, lactic acid, and acetic acid 35 to 40: 15 to 20: 44 to It can be produced in a weight ratio of 46.

According to another aspect of the present invention, there is provided an antibacterial composition comprising the strain, its culture, lysate, extract or purified product as an active ingredient.

In one embodiment, the antimicrobial composition is Escherichia coli , Staphylococcus aureus , Pseudomonas aeruginosa ), Candida albicans ) And Aspergillus Brazil Reensis ( Aspergillus brasiliensis ) It may have antibacterial activity against one or more microorganisms selected from the group consisting of.

In one embodiment, the antibacterial composition may have antibacterial activity in a pH range of 4.0 to 9.0.

According to another aspect of the present invention, there is provided a cosmetic composition comprising the antibacterial composition.

According to another aspect of the present invention, there is provided a composition for external application to the skin comprising the antibacterial composition.

According to another aspect of the present invention, (a) pre-culturing the Lactobacillus paracasei UOS1 strain deposited with accession number KACC 81213BP; (b) inoculating the strain into a culture medium and fermenting; And (c) removing the strain and recovering the supernatant; is provided a method for producing an antimicrobial composition comprising a.

According to the present invention, the metabolites of the strain can exhibit antibacterial activity against various types of microorganisms, and thus can be added to pharmaceuticals, food and cosmetics to be used as a preservative to increase the shelf life of the product.

Since the culture medium containing the metabolites of the strain stably exhibits antibacterial activity even at pH in a wide range, it can be widely applied to various types of products.

The antibacterial composition has low toxicity to the human body and has excellent antibacterial effect, so it can replace conventional synthetic preservatives.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a 16s rDNA nucleotide sequence result for Lactobacillus paracasei USO1 strain according to an embodiment of the present invention
2 is a component analysis result of the Lactobacillus paracasei UOS1 strain culture solution according to an embodiment of the present invention.
3 and 4 are results of evaluating the antimicrobial activity of the Lactobacillus paracasei UOS1 strain culture solution according to an embodiment of the present invention.

The terms used in this specification have been selected as currently widely used general terms as possible while considering the functions in the present invention, which may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like.

In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Numerical ranges are inclusive of the values defined in that range. Every maximum numerical limitation given throughout this specification includes all lower numerical limitations as if the lower numerical limitation were expressly written. Every minimum numerical limitation given throughout this specification includes all higher numerical limitations as if the higher numerical limitation were expressly written. All numerical limitations given throughout this specification will include all numerical ranges that are better within the broader numerical limits, as if the narrower numerical limitations were expressly written.

Hereinafter, examples of the present invention will be described in detail, but it is obvious that the present invention is not limited by the following examples.

According to one aspect of the present invention, there is provided a Lactobacillus paracasei UOS1 strain deposited with accession number KACC 81213BP.

The "Lactobacillus paracasei UOS1 strain" is a novel lactic acid bacterium having 98.11% of 16s rDNA sequence homology with the Lactobacillus paracasei NRIC strain 1938.

The present inventors isolated the above strains from cabbage kimchi aged for a long time, and among them, Lactobacillus paracasei UOS1 strain that has antibacterial activity against various types of microorganisms and can stably exhibit antibacterial activity even at a wide pH range is isolated did.

The strain effectively inhibits the growth of microorganisms for cosmetics or food by producing organic acids such as citric acid, lactic acid, and acetic acid as metabolites, and in addition to various microorganism growth inhibitory substances Through this, it is possible to suppress the growth of contamination-causing bacteria, pathogenic bacteria, and putrefactive bacteria.

In particular, the Lactobacillus paracasei UOS1 strain can maximize antibacterial activity by producing citric acid, lactic acid, and acetic acid as metabolites in an optimal ratio.

Specifically, the Lactobacillus paracasei UOS1 strain can produce the citric acid, lactic acid, and acetic acid in a weight ratio of 35 to 40: 15 to 20: 44 to 46, and optimal antibacterial activity can be implemented in the above range. .

The present inventors cultured the strain under optimal conditions through medium optimization technology, analyzed metabolites according to the culture, and confirmed that the culture medium can effectively inhibit the growth of various types of microorganisms.

According to another aspect of the present invention, there is provided an antibacterial composition comprising the strain, its culture, lysate, extract or purified product as an active ingredient.

Metabolites of the strain may exhibit antibacterial activity against various types of microorganisms, and the strain or a culture solution thereof may be usefully used as a natural antibacterial agent.

The antibacterial composition includes the strain or its culture, lysate, extract or purified product, and can exhibit excellent antibacterial activity because it contains microbial inhibitory metabolites such as organic acids.

The culture may be the culture itself obtained by culturing the strain, or a culture supernatant obtained by removing the strain therefrom, or a concentrate or lyophilizate of the culture supernatant.

In one embodiment, the culture comprises the steps of (a) pre-culturing the Lactobacillus paracasei UOS1 strain deposited with accession number KACC 81213BP; (b) inoculating the strain into a culture medium and fermenting; And (c) removing the strain and recovering the supernatant; can be obtained through.

The culture comprises at least one carbon source selected from the group consisting of glucose, lactose, fructose, mannose, galactose, xylose, ribose, maltose, sucrose, dextrin, glycerin, and inulin; And it can be obtained by culturing the strain in a medium containing at least one nitrogen source selected from the group consisting of soy protein hydrolyzate, skim milk powder, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium oxalate, and diammonium phosphate inulin.

The carbon source or nitrogen source used in the culture medium is, for example, about 0.1 to 30 w / v%, 1 to 25 w / v%, 1 to 20 w / v%, 1 to 10 w / v%, 1 to 5 w / v %, or 1 to 1.5 w/v%.

The culture medium may include commonly used salts, and the salts may include, for example, one or more salts selected from the group consisting of potassium nitrate, potassium sulfate, magnesium carbonate, magnesium sulfate, and magnesium nitrate, The salt may be included, for example, at about 0.001 to 10 w/v%, 0.1 to 7 w/v%, or 0.5 to 5 w/v%.

The culture medium may include about 0.01 to 10 w/v%, 0.05 to 5 w/v%, or 0.1 to 2 w/v% of yeast autolysate, the culture medium having a pH of about 2.0 to 7.0. may be, but is not limited thereto.

The culture cultured the strain, for example, about 24 to 144 hours, 48 to 120 hours, or 15 to 96 hours under a temperature condition of about 20 to 42 ° C, about 25 to 35 ° C, or about 27 to 30 ° C. It may be obtained by

The culture conditions of the strain may include the step of fermenting by adding the strain to the medium, and the fermentation may use a commonly used fermentation method, for example, the method may use fed batch fermentation. can

The extract may be obtained by extracting the culture of the isolated strain with various organic solvents.

The lysate is obtained by isolating the strain from the culture of the strain, disrupting the cell wall or membrane through mechanical methods or nonmechanical methods, and releasing intracellular products. it could be

The purified product may be obtained by culturing the strain and extracting and purifying the active ingredient from the liquid medium or the fermentation process is completed.

The antimicrobial composition may exhibit antimicrobial activity against various types of microorganisms, for example, Escherichia coli , Staphylococcus aureus , Pseudomonas aeruginosa , Candida albicans ( Candida albicans ) and Aspergillus brasiliensis ( Aspergillus brasiliensis ) It may have antimicrobial activity against one or more microorganisms selected from the group consisting of, but is not limited thereto.

The antibacterial composition may have antibacterial activity in a pH range of 4.0 to 9.0.

Since the antibacterial composition can be stably maintained in antibacterial activity despite a wide range of pH changes, it is easy to apply to various products such as food and external preparations for skin as well as cosmetic products formulated in various forms.

According to another aspect of the present invention, there is provided a cosmetic composition or an external preparation for skin comprising the antibacterial composition.

The skin external preparation or cosmetic composition may be formulated by a conventional method. In the formulation of external skin preparations, reference may be made to the contents disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, and in the formulation of cosmetic compositions, International cosmetic ingredient dictionary, 6th ed., The cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1995 may be referred to.

Specifically, the cosmetic composition may be prepared in the form of a general emulsified formulation and a solubilized formulation. For example, lotion, such as a softening lotion or a nourishing lotion; emulsions such as facial lotions and body lotions; creams such as nourishing creams, moisturizing creams, and eye creams; essence; makeup ointment; spray; gel; pack; sunscreen; makeup base; foundations such as liquid type, solid type or spray type; powder; makeup removers such as cleansing creams, cleansing lotions, and cleansing oils; Or it may be formulated as a cleaning agent such as a cleansing foam, soap, body wash, but is not limited thereto. In addition, the external preparation for skin may be formulated as an ointment, patch, gel, cream or spray, but is not limited thereto.

In the skin external preparation or cosmetic composition, in each formulation, other ingredients may be appropriately blended within the range that does not impair the purpose according to the present invention, depending on the type of formulation or the purpose of use, in addition to the essential ingredients.

The external preparation for skin or cosmetic composition may include a conventionally acceptable carrier, for example, oil, water, surfactant, humectant, lower alcohol, thickener, chelating agent, pigment, preservative, fragrance, etc. may be appropriately blended, but this It is not limited.

The acceptable carrier may vary depending on the formulation. For example, when formulated into an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or their Mixtures may be used.

When the skin external preparation or cosmetic composition is formulated as a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or mixtures thereof may be used as a carrier component, and in the case of a spray, chloro It may further comprise a propellant such as fluorohydrocarbon, propane, butane or dimethyl ether.

When the skin external application or cosmetic composition is formulated as a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, Propylene glycol, 1,3-butylglycol oil may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, fatty acid esters of polyethylene glycol or sorbitan may be used. can

When the skin external preparation or cosmetic composition is formulated as a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, such as Suspending agents, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like can be used.

When the cosmetic composition is formulated as a soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolyzates, isethionate, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugars, etc. can be used

The skin external application or cosmetic composition is a fatty substance, an organic solvent, a solubilizer, a thickener, a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, and a foaming agent commonly used in the industry according to the quality or function of the final product. ), fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, common in cosmetics It may additionally contain adjuvants commonly used in the field of cosmetics or dermatology, such as any other ingredients used as

However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.

However, the following Examples and Experimental Examples only illustrate the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example: Isolation and identification of lactic acid bacteria from kimchi

Take 1 mL of a sample obtained by grinding 10 g of cabbage kimchi aged for more than 3 months, and then dilute with sterile distilled water by decimal dilution method using sterile distilled water. Then, 100 μl of MRS solid medium (1% peptone, 1% beef) extract, 0.4% yeast extract, 2% glucose, 0.5% sodium acetate hydrate, 0.1% tween 80, 0.2% potassium phosphate monobasic, 0.2% triammonium citric acid, 0.02% magnesium sulfate, 0.005% manganese sulfate monohydrate, 1.5% agar) was plated and incubated at 30°C for 3 days.

A single colony formed after culturing was taken, separated and inoculated in the same medium by a stroke plate method, and then cultured at 30° C. for 3 days to obtain a pure single species.

The isolated single strain was inoculated into 3 mL of MRS liquid medium and cultured with shaking at 30 °C for 2 days. Stored, re-inoculated as needed, and used for later experiments.

Example: Identification of the isolated strain

For taxonomic identification of the isolated strain, 16s rDNA sequencing was performed as follows.

Amplification of the 16s rDNA gene of the isolated strain

A single colony of the above strain purely cultured from Chinese cabbage kimchi was inoculated into MRS liquid medium (3 mL) and cultured with shaking at 30 °C for 3 days.

After that, the culture solution was incubated at 13,000 rpm and 4° C. for 3 minutes. Cells were recovered by centrifugation.

Chromosomal DNA (gDNA) is extracted from the strain cells according to the DNA isolation kit (Intron lifetechnology) and the manufacturer's protocol, and the extracted DNA is used as a template and 16s rDNA base sequence using primers having the nucleotide sequences shown in Table 1 below. was amplified.

division sequence (5'-3') SEQ ID NO: 1 agagtttgatcmtggctcag SEQ ID NO: 2 tacggytaccttgttacgactt

Specifically, 1 μL of extracted DNA solution, 1 μL of primer mixture (10 pmole / μL) and 18 μL of sterile distilled water were added to each pfu polymerase PCR mix (Bioneer), and then T-100 Thermal cycler DNA amplifier (Bio -rad) was used for PCR.

PCR was performed by repeating the reaction process at 95°C for 5 minutes, 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 3 minutes, 25 times.

The obtained PCR product was confirmed by applying a voltage of 120V on a 1% agarose gel to electrophoresis for 20 minutes, and cutting out a DNA amplification product having a length of about 1.5 kb and purified using a DNA fragment purification kit (Intron lifetechnology).

16s rDNA sequencing

In order to determine the nucleotide sequence of the obtained 16s rDNA gene, the 16s rDNA nucleotide sequence (SEQ ID NO: 3) was requested to Bionics Co., Ltd. and read NCBI BLAST program ( http://blast.ncbi.nlm.nih.gov/Blast ) .cgi ) was used for analysis.

As a result, the strain isolated from cabbage kimchi was found to have 98.11% homology with the Lactobacillus paracasei NRIC 1938 strain (FIG. 1).

Accordingly, as a novel lactic acid bacteria derived from plants, the isolated strain was named Lactobacillus paracasei UOS1 and deposited with the National Academy of Agricultural Sciences Microbial Bank (KACC) as accession number 81213BP on May 10, 2022.

[SEQ ID NO: 3]

tagagttatgatcatggctcaggatgaacgctggcggcgtgcctaatacatgcaagtcgaacgagttctcgttgatgatcggtgcttgcaccgagattgaacatggaacgagtggcggacgggtgagtaacacgtgggtaacctgcccttaagtgggggataacatttggaaacagatgctaataccgcatagatccaagaaccgcatggttcttggctgaaagatggcgtaagctatcgcaaaaggatggacccgcggcgtattagctagggggtgaggtaatggctcaccaaggcgatgatacgtagccgaactgagaggttgatcggaaacattgggactgagacacggcccaaactcctacgggaggcagcagtagggaatcttccacaatggacgcaagtctgatggagcaacgccgcgtgagtgaagaaggctttcgggtcgtaaaactctgttgttggagaagaatggtcggcagagtaactgttgtcggcgtgacggtatccaaccagaaagccacggctaactacgtgccagcagccgcgggtaatacgtaggtgccaagcgttatccggatttattgggcgtaaagcgagcgcaggcggtttcccaagtctgatgtgaaagccctcggcttaaccgaggaagcgcatcggaaactgggaaacttgagtgcagaagaggacagtggaactccatgtgtagcggtgaaatgcgtagatatatggaagaacaccagtggcgaaggcggctgtctggtctgtaactgacgctgaggctcgaaagcatgggtagcgaacaggattagataccctggtagtccatgccgtaaacgatgaatgctaggtgttggagggtttccgcccttcagtgccgcagctaacgcattaagcattccgcctggggagtacgagggcaaggttgaaactcagaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaa ccttaccaggtcttgacatcaaaagatcacctgagagatcaggtttccccttcgggggcaaaatgacaggtggtgccaggttgtcgtcagctcgtgtcgtgagatgatgggttaagtcccgcaacgagcgcaacccttatgactagttgccagcatttagttgggcactctagtttgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggatggtacaacgagttgcgagagggcgaggtcaagctaatctcttaaagccattctcagttcggactgtaggctgcaactcgcctacacgaagtcggaatcgctagtaatcgcggatcagcacgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgagagtttgtaacacccgaagccggtggcgtaacccttttagggagcgagccgtctaaggtgggacaaatgattagggtgaagtcgtaacaaggtagccgta

Example: Preparation of strain culture and fermentation purified product

Culture Preparation

Since the identified microorganism produces a microbial growth inhibitory material such as an organic acid through fermentation and secretes it out of the cell, the microorganism was cultured and a culture solution was obtained therefrom.

The appropriate culture medium conditions for the identified microorganism are glucose or lactose 1 to 1.5 w / v%, skim milk or soy protein hydrolyzate is 1 to 7 w / v%, yeast autolysate yeast extract 0.2 to 0.4 w / v v%, 0.001 to 0.4 w/v% of malt extract, and 0.001 to 1.5 w/v% of each inorganic salt were used, sterilized at 121 °C for 15 minutes.

After culturing for 48 to 96 hours at 200 rpm at 25 to 35 ° C with the prepared culture solution, ultrasonic extraction was performed for 1 to 3 hours to obtain an extract, and the supernatant separated at 12,000 rpm for 15 to 30 minutes was treated with a 0.22 μm membrane filter. It was recovered after sterilization.

Preparation of cultured products

The recovered and filtered culture solution was purified using an impurity remover.

Examples of impurities removers include activated carbon, ion exchange resin, clay, magnesium silicate, diatomaceous earth, orange peel and lemon peel.

The amount of the impurity remover used was collected after sterilization using a 0.22 μm membrane filter after adding 0.1% to 10% of the recovered culture solution and stirring at 25 to 35° C. for 20 to 90 minutes.

Comparative Example 1: Lactobacillus paracasei ( Lactobacillus paracasei ) manufacture of fermented products

A single strain of Lactobacillus paracasei obtained from the market was inoculated into MRS liquid medium sterilized at 121 ° C. for 15 minutes, incubated at 25 to 35 ° C. at 200 rpm for 48 hours to 96 hours, and then in a centrifuge at 12,000 rpm for 15 minutes to 30 minutes. The separated supernatant was collected after sterilization using a 0.22 μm membrane filter.

The recovered culture solution was recovered after proceeding in the same manner as in Example using an impurity removing agent.

Comparative Example 2: Lactobacillus Sake ( Lactobacillus sakei ) manufacture of fermented products

It was cultured and recovered in the same manner as in Comparative Example 1 using a commercially available Lactobacillus sacai single strain.

Comparative Example 3: Lactobacillus plantarum ( Lactobacillus plantarum ) manufacture of fermented products

It was cultured and recovered in the same manner as in Comparative Example 1 using a commercially available Lactobacillus plantarum single strain.

Experimental Example 1: Analysis of strain culture components

Components included in the culture obtained in Examples were analyzed as follows.

A sample solution for analysis was prepared using a 0.2 μm membrane filter after adding and mixing purified water in a 1:1 ratio to the culture.

Components included in the mixed solution were analyzed using an HPLC (Waters Alliance HPLC 2695, USA) system. As a column, MetaCarb 87H (250 × 4.6 mm, Agilent) was used. A PDA (Photodiode Array Detector) was used as the detector.

10 μL of the sample was injected into the column, and the flow rate was detected at 0.5 mL/min.

It was analyzed with a wavelength of 220 nm (Wavelength), and the temperature of the sample and the oven was maintained at 25 °C.

Referring to Figure 2, except for the extraction solvent peak, the highest concentration of the substance was detected at the three peak retention times, and this was achieved through the selective ion monitoring mode and the library of compounds included in the Agilent MassHunter workstation software. As a result of the analysis, the detected peaks were identified as citric acid, lactic acid, and acetic acid.

Citric acid, lactic acid and acetic acid were prepared by preparing a solution of a certain concentration for each material, and after analysis, the contents of citric acid, lactic acid and acetic acid in the sample were confirmed by comparative analysis with the sample analysis results (Table 2).

division Citric acid Lactic acid Acetic acid Example 0.7 0.3 0.8 Comparative Example 1 0.1 1.0 0.5 Comparative Example 2 0.05 1.1 0.3 Comparative Example 3 0.2 0.6 0.6

Referring to Table 2, as a result of the component analysis for Examples, citric acid was analyzed as 0.7%, lactic acid as 0.3%, and acetic acid as 0.8%.

On the other hand, in the case of the comparative group, although there were slight differences between components, citric acid was analyzed at a level of 0.05 to 0.2%, lactic acid at a level of 0.6 to 1.5%, and acetic acid at a level of 0.3 to 0.6%, and the organic acid ratio was different.

Experimental Example 2: Evaluation of antibacterial activity

The minimum inhibitory concentration (MIC) and minimum killing concentration (MBC) of the strain culture obtained in Examples were measured.

The method used in Experimental Example 2 was carried out based on the M07-A9 and M27 methods of the Clinical Laboratory Standards Institute (CLSI).

The strain culture obtained in Example was diluted with a 2-fold dilution method using sterile water to a volume of 100 ul per well using a 96 well plate.

E. coli, S. aureus, and P. aeruginosa strains suspended at 1.0 to 9.0 × 10 ⁵ CFU/mL in the diluted culture were inoculated into each well with the treated culture at 100 μL each.

C. albicans and A. brasiliensis were inoculated at a concentration of 1.0 to 9.0 x 10 ⁴ CFU / mL and inoculated at 100 μL each into a culture well.

Each strain used in Experimental Example 2 was cultured in an optimal growth medium and an optimal growth temperature and used in the experiment.

E. coli, S. aureus, and P. aeruginosa were sterilized with commercially available TSB medium (Tryptic Soy Broth) of difco at 121°C for 15 minutes, and the culture obtained by culturing in a 37°C incubator with shaking for 24 hours was used. .

C. albicans was obtained by culturing a culture sterilized by adding 4% glucose and 1% peptone to sterile distilled water in a 30°C incubator for 48 hours with shaking.

A. brasiliensis was used as a culture obtained by sterilizing the commercially available PDB medium (Potato Dextrose Broth) of difco, and culturing with shaking in an incubator at 30° C. for 48 hours.

The suspension method was prepared by diluting with the optimal medium used for each strain.

After inoculation of the 96-well plate is completed, check the turbidity after stationary culture for 24 or 48 hours at the optimal growth temperature for each strain, and set the culture concentration in the well where turbidity is not visible to the MIC, which is the minimum concentration that inhibits the growth of bacteria. decided.

After determining the MIC concentration, 1.5% of agar was added to the optimal growth medium for each strain, and 20 μL of each was smeared on a plate medium hardened in a petri dish. The minimum concentration was determined as MBC.

The results of measuring the MIC and MBC for the samples obtained in Examples for 5 types of microorganisms are shown in Table 3 and FIGS. 3 and 4 .

division MIC MBC Example E. coli ≤1.0% ≤1.0% S. aureus ≤1.0% ≤1.0% P. aeruginosa ≤1.0% ≤1.0% C. albicans ≤1.0% ≤1.0% A. brasiliensis 2.0% 2.0% Comparative Example 1 E. coli 15% 15% S. aureus 25% 25% P. aeruginosa 15% 15% C. albicans 30% 40% A. brasiliensis 50% >50% Comparative Example 2 E. coli 15% 15% S. aureus 20% 25% P. aeruginosa 15% 15% C. albicans 40% 40% A. brasiliensis 40% 50% Comparative Example 3 E. coli 20% 15% S. aureus 20% 25% P. aeruginosa 20% 20% C. albicans 40% 50% A. brasiliensis >50% >50%

Referring to Table 3, the samples of Examples exhibited strong antibacterial activity of less than 1.0% in both MIC and MBC results of harmful bacteria E. coli, S. aureus, P. aeruginosa and C. albicans , and A. brasiliensis was about 2.0 % concentration effectively inhibited the growth of microorganisms.

On the other hand, the control group Lactobacillus paracasei ( Lactobacillus paracasei ), Lactobacillus sakei ( Lactobacillus sakei ) and Lactobacillus plantarum ( Lactobaciilus plantarum ) In the case of strain cultures , MIC and All MBC exhibited antibacterial activity at a level of 15.0% to 20.0%, and C. albicans and A. brasiliensis showed a significant difference in antimicrobial activity as the growth of microorganisms was inhibited at a level of about 30.0% to 50.0% or higher.

When comparing Comparative Examples 1 to 3 and Examples, it exhibited a difference in antibacterial activity of at least 15 times to maximum 30 times or more compared to Examples against harmful bacteria E. coli, S. aureus, and P. aeruginosa , and C. albicans, A. brasiliensis exhibited antibacterial activity at a concentration of about 40 to 50%, and a difference in antibacterial activity of at least 25 times and at most 33 times compared to the example was confirmed.

The above results suggest that the sample of Example can exhibit a higher level of antibacterial activity compared to three types of comparative groups of bacteria of the same species, and can effectively inhibit the growth of microorganisms at a predetermined concentration.

Experimental Example 3: Evaluation of stability over time

After the samples of Examples and Comparative Examples were stored in glass bottles under the same conditions, stability over time was evaluated in a chamber that can be shielded from light.

The color change was visually observed while stored at 25° C. for 48 weeks.

<Evaluation Criteria>

No abnormal color change: O, slight browning: △, browning: X

division 1 week
lapse 4 weeks
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lapse 36 weeks
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lapse Example O O O O O O O O O Comparative Example 1 O O O △ △ △ △ X X Comparative Example 2 O O △ △ △ X X X X Comparative Example 3 O O △ △ △ △ X X X

Referring to Table 4, while no color change was observed in the samples of Examples, the formulation stability was excellent, whereas in Comparative Examples 1 to 3, browning gradually occurred over time, and thus stability over time was evaluated as relatively poor.

The above results suggest that the samples of Examples not only have high antibacterial activity but also have excellent stability over time, so that they can be usefully used as raw materials for cosmetics.

The foregoing description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and likewise components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

Name of deposit institution: National Academy of Agricultural Sciences

Accession number: KACC81213BP

Deposit date: 20220523

Claims (8)

Lactobacillus paracasei UOS1 strain deposited with accession number KACC 81213BP. The method of claim 1,
The strain has citric acid, lactic acid, and acetic acid production ability,
The citric acid, lactic acid, and acetic acid 35 to 40: 15 to 20: Lactobacillus paracasei UOS1 strain producing a weight ratio of 44 to 46. Lactobacillus paracasei UOS1 strain deposited with accession number KACC 81213BP, its culture, lysate, extract or purified;
From the group consisting of Escherichia coli , Staphylococcus aureus , Pseudomonas aeruginosa , Candida albicans and Aspergillus brasiliensis from the group consisting of An antimicrobial composition having antimicrobial activity against one or more selected microorganisms. delete 4. The method of claim 3,
An antibacterial composition having antibacterial activity in the range of pH 4.0 to 9.0. A cosmetic composition comprising the antibacterial composition of claim 3 or 5. A composition for external application to the skin comprising the antibacterial composition of claim 3 or 5. (a) pre-culturing the Lactobacillus paracasei UOS1 strain deposited with accession number KACC 81213BP;
(b) inoculating the strain into a culture medium and fermenting; and
(C) removing the strain and recovering the supernatant; Antimicrobial composition manufacturing method comprising a.

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