Oat-derived lactobacillus rhamnosus ICL-46 strain and application thereof in resisting inflammation, resisting allergy, enhancing skin immunity, improving atopic dermatitis, soothing skin and regulating skin microorganisms

Abstract

Relates to a novel lactobacillus rhamnosus ICL-46 strain and application thereof in resisting inflammation, resisting allergy, relieving skin and regulating skin microorganisms. The strain according to one embodiment has the effects of resisting inflammation, resisting allergy, enhancing skin immunity, improving atopic dermatitis, soothing skin, and regulating skin microorganisms, and thus can be applied to cosmetics, medicines, foods, and the like in various ways.

Description

Technical Field

[0001] Relates to a novel microorganism derived from oats and its uses in anti - inflammation, anti - allergy, enhancing skin immunity, improving atopic dermatitis, soothing the skin, and regulating skin microbiota.

[0002] Background Art

[0003] Probiotics refer to live microorganisms that provide health benefits. Representative strains of probiotics include Lactobacillus and Bifidobacterium strains. In recent years, research on manufacturing functional cosmetics by applying probiotic microorganisms to cosmetics has been actively carried out.

[0004]

[0005]

[0006] On the other hand, oats (Avena sativa) contain ingredients useful for the skin and are thus widely used in the cosmetic field in the form of oat extracts, oat oil, oat peptides, oat kernel powder, etc.

[0007]

[0008] However, research on microorganisms derived from oats and their skin - beautifying effects is not yet sufficient. Summary of the Invention

[0010] One embodiment provides a novel strain of Lacticaseibacillus rhamnosus.

[0011]

[0012] Another embodiment provides a lysate, culture broth, extract of the culture broth, fermentation broth of the strain, or any combination thereof.

[0013]

[0014] Another embodiment provides a method for preparing the lysate of the strain.

[0015] Still another embodiment provides a composition comprising the strain of Lacticaseibacillus rhamnosus, its lysate, culture broth, extract of the culture broth, fermentation broth, or any combination thereof.

[0016]

[0017] Yet another embodiment provides the uses of the composition in anti - inflammation, anti - allergy, enhancing skin immunity, soothing the skin, and regulating skin microbiota.

[0018]

[0019] Another embodiment provides a method for preventing, improving or treating the skin condition of an individual, which comprises the step of administering an effective amount of the composition to an individual in need.

[0020] One embodiment provides a Lacticaseibacillus rhamnosus strain ICL-46.

[0021] The strain may be isolated. The term "isolated" in this specification means that it does not exist in nature but can be artificially isolated and utilized. The strain may be isolated from oats (Oat or Avena sativa). The strain may be isolated from the kernel of oats, the bran of oats, or a mixture thereof.

[0022] The strain may be a strain isolated by inoculating oats into MRS (De Man, Rogosa, Sharpe) medium and performing pure isolation and cultivation of the cultured colonies. In this specification, "Lacticaseibacillus rhamnosus isolated from oats" may be used interchangeably with "Lacticaseibacillus rhamnosus of oat origin".

[0023] The strain may be a strain belonging to the genus Lacticaseibacillus sp.

[0024] The strain may contain 16S rRNA having a sequence identity of about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or about 99.9% or more with SEQ ID NO:1. The strain may contain the 16S rRNA of SEQ ID NO:1.

[0025] The term "sequence identity" used in this specification refers to the degree of identity of amino acid residues or bases between sequences after aligning two sequences to be as consistent as possible in a specific comparison region. The percentage of sequence identity can be determined using known sequence comparison programs. As an example, BLASTN (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc), etc. can be cited.

[0026] The Lacticaseibacillus rhamnosus strain may be a strain deposited under the accession number KCTC15702BP.

[0027] The strain may have the effects of anti - inflammation, anti - allergy, enhancing skin immunity, soothing the skin, or regulating skin microbiota.

[0028] Another embodiment provides a disrupted product of the strain, a culture solution, an extract of the culture solution, a fermentation broth, or any combination thereof.

[0029] The specific content of the strain is as described above.

[0030] The term "disrupted product" in this specification may refer to the intracellular biological components contained in microorganisms, such as components like cell walls or membranes, as well as disrupted products containing useful components, such as cultures containing metabolites released extracellularly during the cultivation of microorganisms. The disrupted product can be prepared by pressure or ultrasonic disruption, excluding methods that induce modification of microbial metabolites, such as irradiation or heat inactivation. In a specific example, the disrupted product of the strain can be prepared by a Microfluidizer (M.F.). The useful components in the microorganism are prone to deterioration due to excessive temperature and energy. Therefore, the disrupted product can be prepared by using a Microfluidizer, which can most stably obtain the useful components in the microorganism in the existing production process.

[0031] The disrupted product may include the disrupted product itself, its concentrate, or its freeze - dried product.

[0032] The term "culture solution" in this specification can be used interchangeably with "culture supernatant", "conditioned culture solution", or "conditioned medium". It can refer to the entire medium obtained by culturing the strain in a medium that can provide nutritional components for the Lactobacillus rhamnosus strain to grow and survive in vitro for a specified time, containing the strain, its metabolites, additional nutritional components, etc. And the culture solution can also refer to the culture solution obtained by removing the cells from the cell culture solution obtained by culturing the strain. On the other hand, the liquid obtained by removing the cells from the culture solution is also called "supernatant", which can be obtained by allowing the culture solution to stand for a specified time, removing the sedimented part at the bottom layer, and only taking the upper liquid, or by filtering to remove the cells, or by centrifuging the culture solution to remove the precipitate at the bottom and only taking the upper liquid. The "cells" refer to the strain of the present invention itself, including the strain itself isolated and screened from samples, etc., or the strain cultured and isolated from the culture solution. The cells can be obtained by centrifuging the culture solution and taking the sedimented part at the bottom layer, or since they settle to the bottom layer of the culture solution due to gravity, they can be obtained by removing the upper liquid after allowing it to stand for a specified time.

[0033] The culture solution may include the culture solution itself obtained by culturing the strain, its concentrate, or its freeze - dried product, or the culture supernatant obtained by removing the strain from the culture solution, its concentrate, or its freeze - dried product.

[0034] The culture solution of the strain can be separated by centrifugation into cells and supernatant. In this specification, the "crush product" can be a mixture of the culture solution of the strain and the cell crush product.

[0035] Those of ordinary skill in the art to which the present invention pertains can utilize it by appropriately selecting and modifying the culture medium and culture conditions for culturing the Lactobacillus rhamnosus strain. For example, the medium can be a medium made from one or a mixture selected from glucose, yeast extract, malt extract, peptone, glutamic acid, K2HPO4 (dipotassium hydrogen phosphate), KH2PO4 (potassium dihydrogen phosphate), NaCl (sodium chloride), MnSO4 (manganese sulfate), and MgSO4 (magnesium sulfate). And the culture conditions can be culturing at a temperature of 30°C to 40°C for 12 hours to 72 hours, but are not limited thereto.

[0036] The term "culture solution extract" in this specification refers to an extract obtained from the culture solution or its concentrate, and can include an extract solution, a dilution or concentrate of the extract solution, a dry product obtained by drying the extract solution, or a crude purification or purification product thereof, or a fraction obtained by fractionating them.

[0037] Another embodiment provides a method for preparing a crush product of the strain.

[0038] The method may include: a step of inoculating a Lactobacillus rhamnosus strain according to an embodiment into a medium and then culturing to obtain a strain culture solution;

[0039] a step of centrifuging the culture solution to separate cells and supernatant; and

[0040] a step of mixing the crush product obtained by crushing the cells and the supernatant to obtain a strain crush product.

[0041] Before the step of obtaining the strain culture solution, a step of sterilizing the medium may further be included. The sterilization can be carried out according to conventional methods in the art. For example, it can be carried out at 110-130°C for 1 minute to 1 hour.

[0042] The type of the culture medium is not limited as long as it can culture Lactobacillus rhamnosus strains. Those of ordinary skill in the technical field to which the present invention pertains can utilize it by appropriately selecting and modifying the culture medium and culture conditions for culturing the Lactobacillus rhamnosus strains. For example, the culture medium can be a culture medium made from one or a mixture of glucose, yeast extract, malt extract, peptone, glutamic acid, K2HPO4, KH2PO4, NaCl, MnSO4, and MgSO4. And the culture conditions can be culturing at a temperature of 30°C to 40°C for 12 hours to 72 hours, but are not limited thereto. Through the above culture, a strain culture solution can be obtained.

[0043] The step of centrifuging the culture solution to separate the bacterial cells and the supernatant can further include a step of filtering after centrifugation. The filtering can be carried out according to conventional methods in the art.

[0044] In the step of obtaining the strain lysate, a microfluidizer can be used to break the bacterial cells, but is not limited thereto.

[0045] Another embodiment provides a use of the Lactobacillus rhamnosus strain, its lysate, culture solution, extract of the culture solution, fermentation broth, or any combination thereof. Specifically, a composition containing the Lactobacillus rhamnosus strain, its lysate, culture solution, extract of the culture solution, fermentation broth, or any combination thereof is provided.

[0046] In the above use or composition, the Lactobacillus rhamnosus strain can be the Lactobacillus rhamnosus ICL-46 strain according to the above embodiment.

[0047] The uses of the strain can include anti-inflammatory, anti-allergic, enhancing skin immunity, soothing the skin, regulating skin microorganisms, preventing or treating skin diseases.

[0048] The strain can inhibit or reduce the expression of inflammation-related genes (such as COX-2 (cyclooxygenase-2), iNOS (inducible nitric oxide synthase)); or inhibit or reduce the release of the major factor β-hexosaminidase related to allergy or immunity; inhibit or reduce skin harmful bacteria (such as P. aeruginosa (Pseudomonas aeruginosa), S. aureus (Staphylococcus aureus)) without affecting skin beneficial bacteria (such as S. epidermidis (Staphylococcus epidermidis)).

[0049] The term "anti-inflammatory" can be used interchangeably with "inflammation improvement" and "inflammation inhibition", and can refer to any effect of alleviating the immune response and inhibiting the production of COX-2 or iNOS.

[0050] The term "anti-allergy" can be used interchangeably with "allergy improvement" and "allergy inhibition", and can refer to any action that inhibits or improves the body's allergic reactions.

[0051] The term "enhancing skin immunity" can refer to all actions that enhance skin immunity. For example, it can be achieved by inhibiting allergenic substances (such as: β-hexosaminidase) or harmful skin bacteria, but is not limited thereto.

[0052] The term "skin disease" can be an inflammatory skin disease, an allergic skin disease, or an immune skin disease. The term "prevention" includes inhibiting the occurrence of a disease. The term "treatment" includes inhibiting, alleviating, or eliminating the development of a disease.

[0053] The inflammatory skin disease can be one selected from the group consisting of skin trauma, dermatitis, atopic dermatitis, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, drug eruption, and acne.

[0054] The allergic skin disease can be one selected from the group consisting of atopic dermatitis, urticaria, drug eruption, and pruritus.

[0055] The immune skin disease is a general term for skin diseases related to immunity and can include, for example, atopic dermatitis, seborrheic dermatitis, psoriasis, etc.

[0056] The term "soothing the skin" refers to all actions that soothe and stabilize the heat sensation, pain, etc. of the irritated skin. The soothing of the skin can include soothing the skin that has become sensitive due to inflammation, allergy, or decreased immunity.

[0057] The term "regulating skin microbiota" can refer to increasing beneficial skin bacteria and reducing harmful skin bacteria; or only inhibiting or reducing harmful skin bacteria without affecting beneficial skin bacteria. Specifically, "regulating skin microbiota" can be inhibiting or reducing the growth of harmful skin bacteria without affecting the growth of beneficial skin bacteria. Beneficial and harmful skin bacteria can include all known strains. In a specific example, the beneficial skin bacteria can be Staphylococcus epidermidis, but is not limited thereto. The harmful skin bacteria can be Pseudomonas aeruginosa or Staphylococcus aureus, but is not limited thereto.

[0058] According to an embodiment, the composition can inhibit or reduce the expression of one or more of COX-2 and iNOS by including the Lactobacillus rhamnosus strain, its disrupted product, culture solution, extract of the culture solution, fermentation broth, or any combination thereof. Therefore, the composition can exhibit an anti-inflammatory effect and a preventive or therapeutic effect on inflammatory skin diseases.

[0059] The composition according to one embodiment can inhibit or reduce the major allergy factor β-hexosaminidase by including the Lactobacillus rhamnosus strain, its lysate, culture broth, extract of the culture broth, fermentation broth, or any combination thereof. Accordingly, the composition can exhibit anti-allergy effects, enhance skin immunity, and have preventive or therapeutic effects on allergic skin diseases and immune skin diseases.

[0060] The composition according to one embodiment can inhibit or reduce the growth of skin harmful bacteria (P. aeruginosa, S. aureus) by including the Lactobacillus rhamnosus strain, its lysate, culture broth, extract of the culture broth, fermentation broth, or any combination thereof, without affecting the growth of skin beneficial bacteria (S. epidermidis). Accordingly, the composition can exhibit the effect of regulating skin microflora.

[0061] The composition according to one embodiment can exhibit a skin soothing effect through one or more of the above-described anti-inflammatory, anti-allergy, skin immunity-enhancing, and skin microflora-regulating effects.

[0062] In another specific example, the strain can exhibit a synergistic effect when used together with other strains having an effect of improving skin condition.

[0063] The composition can include 0.001 wt% to 80 wt%, for example, 0.01 wt% to 60 wt%, 0.01 wt% to 40 wt%, 0.01 wt% to 30 wt%, 0.01 wt% to 20 wt%, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% to 10 wt%, or 0.1 wt% to 5 wt% of the strain, its lysate, culture broth, extract of the culture broth, fermentation broth, or any combination thereof, based on the total weight of the composition.

[0064] The term "comprising as an active ingredient" in this specification means adding the strains, their disrupted products, culture broths, extracts of culture broths, fermentation broths or any combination thereof in this specification to such an extent that the effects can be exhibited, which means adding various components as auxiliary components and formulating them into various forms for transportation and stabilization of drugs, etc.

[0065] The composition may be in a liquid state or a dry state. In a specific example, the composition may be in the form of dry powder.

[0066] The drying method for preparing the composition in a dry state may use the methods commonly used in the art, and there is no particular limitation thereto. Non-limiting examples of the drying method include air drying method, natural drying method, spray drying method, freeze drying method, etc. These methods may be used alone, or at least two methods may be used together.

[0067] The composition may contain an effective amount of an additive sufficient to reduce the deterioration of the strains, their disrupted products, culture broths, extracts of culture broths, fermentation broths or any combination thereof. The additive may be, for example, a binder, but is not limited thereto.

[0068] The composition may further contain a cosmetically, pharmaceutically, or foodologically acceptable carrier. The composition may be formulated with the carrier and provided as a cosmetic, a drug, a food additive, etc.

[0069] The composition may be a cosmetic composition.

[0070] In addition to the active ingredients disclosed in this specification, the cosmetic composition may further contain various components commonly used in cosmetic compositions, functional additives, etc. For example, it may contain conventional auxiliaries such as antioxidants, stabilizers, solubilizers, surfactants, dispersants, preservatives, vitamins, pigments, fragrances, etc., as well as carriers.

[0071] The cosmetic composition is not particularly limited to a specific dosage form and may be appropriately selected according to the purpose. The cosmetic composition may have, for example, a soluble dosage form, an emulsified dosage form or a dispersed dosage form. The cosmetic composition may have, for example, a lotion, a nourishing lotion, a massage cream, a nourishing cream, a serum, a mask, a gel, an ampoule or a skin-adhering cosmetic dosage form.

[0072] The composition may be a composition for external use on the skin.

[0073] In this specification, the topical skin preparation may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal delivery patch, a medicated bandage, a lotion, or a combination thereof. The topical skin preparation may be appropriately formulated with components commonly used in topical skin preparations such as cosmetics and pharmaceuticals, for example, aqueous components, oily components, powder components, alcohols, humectants, thickeners, ultraviolet absorbers, skin lightening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof.

[0074] The composition may be a pharmaceutical composition.

[0075] The pharmaceutical composition may further comprise a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, mannitol, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxypropyl cellulose, or a combination thereof. The disintegrant may be calcium carboxymethyl cellulose, sodium starch glycolate, anhydrous monobasic calcium phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, or a combination thereof. The lubricant may be magnesium stearate, silica, talc, or a combination thereof.

[0076] The pharmaceutical composition may be formulated into oral or parenteral dosage forms. Oral dosage forms include granules, powders, solutions, tablets, capsules, dry syrups, etc. Parenteral dosage forms may be injections, ointments, etc.

[0077] The composition may be a food composition. In this case, it may be formulated into conventional health functional food dosage forms known in the art. Thus, the food composition may be a health functional food composition.

[0078] The food composition may use the strain, its lysate, culture solution, extract of the culture solution, fermentation broth, or any combination thereof alone, or may be used together with other foods or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use (prevention, health care or therapeutic treatment). There is no particular limitation on the type of the health functional food. Among the types of health functional foods, the beverage composition may contain various flavoring agents or natural carbohydrates as additional ingredients, such as conventional beverages. The natural carbohydrates are monosaccharides, such as glucose and fructose; disaccharides, such as maltose and sucrose; polysaccharides, such as dextrin and cyclodextrin; and sugar alcohols, such as xylitol, sorbitol and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used. The food composition may also contain nutritional agents, vitamins, electrolytes, flavors, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerol, alcohol, carbonating agents used in carbonated beverages, or combinations thereof. The food composition may also contain the pulp for preparing natural fruit juices, fruit juice beverages, vegetable beverages, or combinations thereof.

[0079] Another embodiment provides a method for preventing, improving or treating the skin condition of an individual, which comprises the step of administering an effective amount of the composition to an individual in need.

[0080] The skin condition of the individual may be a condition related to inflammation, allergy or immunity.

[0081] The terms "administer", "introduce" and "implant" in this specification are used interchangeably and may refer to arranging a composition according to a specific example into an individual by a method or via a route that causes the composition according to a specific example to be at least partially localized at a desired site.

[0082] Administration can be carried out by methods known in the art. Administration can be directly administered to the individual by any means, such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. The administration can be systemic or local administration.

[0083] The individual may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat or cat. The individual may be an individual in need of improving the skin condition, such as an individual in need of anti-inflammatory, anti-allergic, enhancing skin immunity, soothing the skin or regulating the skin microbiota effects.

[0084] The administration may be administering 0.1 mg to 1000 mg, such as 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg of the composition according to a specific example to each individual per day. However, the dose can be prescribed in various ways according to factors such as the formulation method, administration method, age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity of the patient, and those skilled in the art can consider these factors to appropriately adjust the dose. The number of administrations can be once a day, or more than twice within the range of clinically acceptable side effects. For the administration site, it can be administered at one or more than two sites. The total number of days of administration for each treatment can be 1 day to 30 days, and it can be once a day or at intervals of 2 days to 5 days. If necessary, the same treatment can be repeated after an appropriate time. For animals other than humans, the dose per kg can be the same as that for humans, or for example, the above dose can be converted and administered according to the volume ratio (e.g., average value) of organs (such as the heart) between the target animal and humans.

[0085] A novel Lactobacillus rhamnosus strain according to an embodiment, which has the effects of anti - inflammation, anti - allergy, enhancing skin immunity, improving atopic dermatitis, soothing the skin, or regulating skin microorganisms, and thus can be applied to cosmetics, pharmaceuticals, foods, etc. in various ways. Description of the Drawings

[0086] Figure 1 To show the results of COX - 2 mRNA expression levels according to the types of lactic acid bacteria strains sourced from oats.

[0087] Figure 2 To show the results of iNOS mRNA expression levels according to the types of lactic acid bacteria strains sourced from oats.

[0088] Figure 3 To show the results of β - hexosaminidase inhibition effects according to the types of lactic acid bacteria strains sourced from oats.

[0089] Figure 4 To show the comparison results of the growth degree according to the treatment with the disrupted product of Lactobacillus rhamnosus ICL - 46 when co - culturing the skin - beneficial bacterium S. epidermidis (Staphylococcus epidermidis) and the harmful bacterium P. aeruginosa (Pseudomonas aeruginosa).

[0090] Figure 5 When co-culturing the skin-beneficial bacterium S. epidermidis (Staphylococcus epidermidis) and the harmful bacterium S. aureus (Staphylococcus aureus), it is based on the comparison results of the growth degree treated with the broken product of Lactobacillus rhamnosus ICL-46. Detailed implementation mode

[0091] Hereinafter, the present invention will be described in more detail through examples. However, these examples are only used to exemplarily describe the present invention, and the scope of the present invention is not limited to these examples.

[0092] Example 1. Isolation and identification of oat-derived strains

[0093] Isolate and identify microorganisms from oats.

[0094] Specifically, use a sterilized grinder to crush undried oat kernels and oat bran. Add 10 g of the crushed product to 90 mL of MRS (de Man, Rogosa and Sharpe) liquid medium and culture at 37 °C for about 48 hours.

[0095] Dilute the cultured liquid medium 10-fold with physiological saline, spread it on an MRS plate, and culture for more than 48 hours under the same culture conditions. Then randomly isolate strains from the resulting colonies.

[0096] Subculture the randomly isolated strains more than 3 times, drop the isolated pure colonies on a BCP (Bromocresol Purple) agar plate (manufacturer: eiken chemical, product name: BCP plate count agar), and confirm the acid-producing ability. Screen strains with milky white colonies and yellow surroundings. Analyze the base sequence of the 16S rRNA gene of the selected colonies for identification and classification. Conduct a similarity search for the analyzed 16S rRNA base sequence in NCBI (National Center for Biotechnology Information, USA), and the result shows 99.67% homology with Lactobacillus rhamnosus.

[0097] The newly isolated microorganism Lactiplantibacillus rhamnosus strain (named "ICL-46") from oats was deposited at the Korea Research Institute of Bioscience and Biotechnology (KCTC) on November 20, 2023, with the deposit number KCTC15702BP. The ICL-46 strain has a 16S rRNA sequence of SEQ ID NO:1 (complementary DNA).

[0098] Example 2. Preparation of the disrupted product of Lactiplantibacillus rhamnosus ICL-46

[0099] The culture medium was sterilized at 121 °C for 1 minute. The culture medium can be appropriately selected according to the type of microorganism inoculated. For example, a culture medium made of one or a mixture selected from glucose, yeast extract, malt extract, peptone, glutamic acid, K2HPO4, KH2PO4, NaCl, MnSO4, and MgSO4 can be used.

[0100] The Lactiplantibacillus rhamnosus ICL-46 strain of Example 1 was inoculated into the sterilized culture medium and then cultured at 37 °C for 24 hours.

[0101] After the culture was completed, the cells and the supernatant were separated by centrifugation. The cells were disrupted by passing them through a microfluidizer twice at 800 bar. The disrupted product of the Lactiplantibacillus rhamnosus ICL-46 strain was obtained by mixing the disrupted cells and the supernatant.

[0102] Comparative Examples 1 to 4. Isolation and preparation of disrupted products of lactic acid bacteria from oats

[0103] Lactic acid bacteria were isolated from oats in the same manner as in Example 1. Through the base sequence analysis of the 16S rRNA gene, Lactiplantibacillus plantarum strain, Lactococcus lactis strain, Latilactobacillus sakei strain, and Lactiplantibacillus rhamnosus strain were identified. The Lactiplantibacillus rhamnosus strain belongs to the same species as the ICL-46 strain of Example 1, but has a different 16S rRNA sequence.

[0104] The disrupted products of the lactic acid bacteria from oats were prepared in the same manner as in Example 2. The disrupted product of the Lactiplantibacillus plantarum strain was used as Comparative Example 1, the disrupted product of the Lactococcus lactis strain was used as Comparative Example 2, the disrupted product of the Latilactobacillus sakei strain was used as Comparative Example 3, and the disrupted product of the Lactiplantibacillus rhamnosus strain was used as Comparative Example 4.

[0105] Experimental Example 1. Confirmation of anti-inflammatory efficacy - Expression analysis of COX-2 and iNOS

[0106] To confirm the anti-inflammatory effect of the lysate of Lactobacillus rhamnosus ICL-46 strain, the gene expressions of COX-2 (cyclooxygenase-2) and iNOS (Nitric oxide synthases) were analyzed by real-time PCR (Real-Time Fluorescent Quantitative PCR).

[0107] Specifically, after treating mouse macrophages Raw 264.7 with LPS (liposaccharides), 0.5% (v / v) of the lysates of the strains of Example 2 and Comparative Examples 1 to 4 were added, and the expression levels of COX-2 and iNOS genes were confirmed. The group untreated with any substance (MO: media only) was used as the negative control group, and the group treated only with LPS was used as the positive control group.

[0108] Figure 1 To show the results of the COX-2 mRNA expression levels according to the types of lactic acid bacteria strains derived from oats.

[0109] Figure 2 To show the results of the iNOS mRNA expression levels according to the types of lactic acid bacteria strains derived from oats.

[0110] As a result, as Figure 1 and Figure 2 shown, it was confirmed that the lysate of Lactobacillus rhamnosus ICL-46 strain had the best effect in inhibiting the expressions of COX-2 and iNOS genes derived from oats compared with other lactic acid bacteria strains.

[0111] From the above results, it was confirmed that the lysate of Lactobacillus rhamnosus ICL-46 strain exhibited skin soothing efficacy through a significantly excellent anti-inflammatory effect.

[0112] Experimental Example 2. Confirmation of anti-allergic efficacy - Inhibition of the release of β-hexosaminidase

[0113] To confirm the anti-allergic effect of the lysate of Lactobacillus rhamnosus ICL-46 strain, the inhibitory effect on the release of the major factor related to allergy or immunity, β-hexosaminidase, was tested. Inhibiting the release of β-hexosaminidase means preventing the degranulation of immune cells.

[0114] Specifically, RBL-2H3 (rat basophilic leukemia cells) were seeded at 2X10 5Cells were evenly seeded into 24-well plates and stabilized for 24 hours at 37°C and 5% CO₂. After washing the cells with PBS (phosphate buffer solution), each well was treated with anti-DNP-IgE (mouse anti-dinitrophenyl monoclonal antibody) (50 ng / mL) and cultured for 24 h. The anti-DNP-IgE-sensitized cells were washed twice with MEM (minimum essential medium) medium supplemented with 1% FBS (fetal bovine serum), 160 μL of the medium was added and cultured at 37°C for 20 minutes. Then, 0.5% (v / v) of the cell lysates of Example 2 and Comparative Examples 1 to 4 were added respectively, 20 μL of DNP-BSA (dinitrophenol-bovine serum albumin) with a concentration of 1 μg / mL was added and reacted at 37°C for 1 h to stimulate the cells to form granules, and then centrifuged at 4°C and 13,000 rpm for 10 minutes. 50 μL of 0.1 M citrate buffer (added with 10 mM p-nitrophenyl-N-acetyl-β-D-glucosamine, pH 4.5) was added to 25 μL of the centrifuged supernatant and reacted at 37°C for 1 h. Then, 100 μL of the termination buffer (0.1 M Na₂CO₃ / NaHCO₃, pH 10.0) was added to terminate the reaction, and the absorbance was measured at 405 nm using a microplate reader.

[0115] Figure 3 To show the results of the β-hexosaminidase inhibitory effect according to the types of lactic acid bacteria strains derived from oats.

[0116] As a result, as Figure 3 shown, it was confirmed that the cell lysate of Lactobacillus rhamnosus ICL-46 strain had the best effect in reducing the β-hexosaminidase activity derived from oats compared with other lactic acid bacteria strains.

[0117] From the above results, it was confirmed that the cell lysate of Lactobacillus rhamnosus ICL-46 exhibited significantly excellent anti-allergic or skin immunity-enhancing effects.

[0118] Experimental Example 3. Confirmation of the effect of regulating skin microbiota

[0119] An experiment was conducted to confirm the effect of regulating skin microbiota of the cell lysate of Lactobacillus rhamnosus ICL-46 strain, which was confirmed to have the best anti-inflammatory and anti-allergic effects through Experimental Example 1 and Experimental Example 2.

[0120] Specifically, TSB (tryptic soy broth, BDDifco, USA) was used as the microbial medium. For the growth of bacteria, the strain was inoculated at 1×10 6Inoculate at a concentration of CFU / mL into an appropriate medium, and after culturing at 37 °C for 24 h, compare the growth rate by measuring the absorbance at 600 nm, or compare the growth rate by confirming the number of viable bacteria through the serial dilution method. Compare the percentage based on the experimental group that added the same amount of phosphate buffered saline as the test sample.

[0121] Add 1 mL of 10% TSB to a 12-well plate for co-culturing skin harmful bacteria and beneficial bacteria, and inoculate harmful bacteria (Pseudomonas aeruginosa, Staphylococcus aureus) at a concentration of 1×10 6 CFU / mL. Place a 0.4 μm cell culture insert on top of it, and inoculate beneficial bacteria (Staphylococcus epidermidis) at a concentration of 1×10 6 CFU / mL into 1 mL of 10% TSB. Treat 10% TSB with 0.5% (v / v) of the cell lysate of Lactobacillus rhamnosus ICL-46 of Example 2 and then conduct the experiment. As a control group, treat with the same amount of phosphate buffered saline. Then, after culturing at 32 °C for 12 h, confirm the number of each type of bacteria and compare the growth degree.

[0122] Figure 4 It is the comparison result of the growth degree according to the treatment with the cell lysate of Lactobacillus rhamnosus ICL-46 when co-culturing skin beneficial bacteria S. epidermidis (Staphylococcus epidermidis) and harmful bacteria P. aeruginosa (Pseudomonas aeruginosa).

[0123] Figure 5 It is the comparison result of the growth degree according to the treatment with the cell lysate of Lactobacillus rhamnosus ICL-46 when co-culturing skin beneficial bacteria S. epidermidis and harmful bacteria S. aureus.

[0124] As a result, as Figure 4 and Figure 5 shown, it was confirmed that when treated with the cell lysate of Lactobacillus rhamnosus ICL-46, it had no effect on the growth of beneficial bacteria (S. epidermidis). On the contrary, it inhibited or reduced the growth of harmful bacteria (P. aeruginosa, S. aureus).

[0125] From the above results, it can be confirmed that the cell lysate of Lactobacillus rhamnosus ICL-46 exhibits a significantly excellent effect on regulating skin microorganisms.

[0126] In summary, it was confirmed that Lactobacillus rhamnosus ICL-46 strain derived from oats inhibits or reduces the expression of COX-2 and iNOS; inhibits or reduces the release of the major allergy- or immunity-related factor β-hexosaminidase; has no effect on the growth of skin-beneficial bacteria (S. epidermidis), and on the contrary, reduces the growth of skin-harmful bacteria (P. aeruginosa, S. aureus). Therefore, it can be known that Lactobacillus rhamnosus ICL-46 strain derived from oats has the effects of improving skin inflammation (anti-inflammatory), improving skin allergy (anti-allergic), enhancing skin immunity, soothing the skin, and regulating skin microorganisms.

[0127] The above description is only an exemplary illustration of the technical idea of the present invention, and those of ordinary skill in the technical field to which the present invention pertains can make various modifications and deformations without departing from the essential characteristics of the present invention.

[0128] Deposit number

[0129] Name of the depositary institution: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0130] Deposit number: KCTC15702BP

[0131] Date of deposit: 20231120

[0132] Sequence listing

[0133] <p num＝"0137”>Attach the electronic file of the Sequence Listing .

Claims

1. A Lactobacillus rhamnosus ICL-46 strain, characterized in that Deposited under accession number KCTC15702BP.

2. The Lactobacillus rhamnosus ICL-46 strain according to claim 1, characterized in that Isolated from oats.

3. The Lactobacillus rhamnosus ICL-46 strain according to claim 1, characterized in that The 16S rRNA comprising SEQ ID NO:

1.

4. A method for preparing a broken product of Lactobacillus rhamnosus ICL-46 strain according to claim 1, characterized in that: include: The step of inoculating the Lactobacillus rhamnosus ICL-46 strain according to claim 1 into a culture medium and culturing the culture medium to obtain a strain culture solution; The step of centrifuging the culture solution to separate the bacterial cells and the supernatant; as well as The step of mixing the disrupted product obtained by disrupting the bacterial cells with the supernatant to obtain a disrupted product of the strain.

5. A cosmetic composition for improving skin inflammation, improving skin allergies, enhancing skin immunity or soothing the skin, characterized in that: The method comprises one or more of the Lactobacillus rhamnosus ICL-46 strain according to claim 1, its disrupted product, culture solution, culture solution extract and fermentation solution.

6. A cosmetic composition for regulating skin microorganisms, characterized in that The method comprises one or more of the Lactobacillus rhamnosus ICL-46 strain according to claim 1, its disrupted product, culture solution, culture solution extract and fermentation solution.

7. The cosmetic composition for regulating skin microorganisms according to claim 6, characterized in that The regulation of skin microorganisms is to inhibit the growth of harmful bacteria on the skin.

8. The cosmetic composition for regulating skin microorganisms according to claim 7, characterized in that The skin harmful bacteria are Pseudomonas aeruginosa or Staphylococcus aureus.

9. A pharmaceutical composition for preventing or treating skin inflammation, skin allergic diseases or immune skin diseases, characterized in that: The method comprises one or more of the Lactobacillus rhamnosus ICL-46 strain according to claim 1, its disrupted product, culture solution, culture solution extract and fermentation solution.

10. The pharmaceutical composition for preventing or treating skin inflammation, skin allergic diseases or immune skin diseases according to claim 9, characterized in that: Inhibit or reduce the expression of one or more of cyclooxygenase-2 and nitric oxide synthase; or inhibit or reduce the release of β-hexosaminidase.

11. A health functional food composition, characterized in that: The method comprises one or more of the Lactobacillus rhamnosus ICL-46 strain according to claim 1, its disrupted product, culture solution, culture solution extract and fermentation solution.

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