Lactobacillus sake strain amp5104 and uses thereof

Abstract

The present invention relates to a Lactobacillus sakei AMP5104 strain and uses thereof, which can be effectively used for preventing, ameliorating or treating skin-related conditions or inflammation-related conditions.

Description

Technical Field

[0001] This invention relates to a strain of Lactobacillus sarcopenia AMP5104 and its uses. Background Technology

[0002] Skin is the part of the body directly exposed to the external environment, protecting it from external stimuli. Human skin tissue is home to countless microbial communities, maintaining a balance between beneficial and harmful microorganisms, thus enhancing skin immunity. However, when harmful microorganisms proliferate excessively due to external stimuli, stress, or hormonal changes, disrupting this balance, the weakened skin barrier can trigger a primary inflammatory response caused by these harmful microorganisms. Furthermore, the toxic metabolites of superantigens and other substances from these harmful microorganisms can induce secondary inflammatory responses, such as itching and exacerbated inflammation. Skin problems caused by these factors not only affect appearance, but the substances produced during the inflammatory process can also lead to skin pigmentation, promote the breakdown of elastic fibers, and contribute to skin aging.

[0003] On the other hand, probiotics are the reason why kimchi, a traditional Korean food, has its health benefits, so research on probiotics is very active. In particular, lactic acid bacteria have excellent antibacterial activity due to the organic acids they secrete, thus maintaining a healthy balance of the microbial community. The skin achieves skin immunity through the balance of these microbial communities and skin immune cytokines.

[0004] Therefore, there is a need to develop a novel microbial strain that plays a role in the skin's immune balance. Summary of the Invention

[0005] This invention provides a product belonging to the genus *Lactobacillus* (…). Lactobacillus sp. Lactobacillus sarcopenia ( Lactobacillus sakei ) strain.

[0006] Another aspect of the present invention provides the dry matter, fragments, culture medium, extract or lysate of the strain.

[0007] In another aspect, the present invention provides a composition comprising the strain, its dry matter, fragments, culture medium, extract of the culture medium, lysate, or a mixture thereof.

[0008] This invention provides a strain of Lactobacillus sarcopenia (Saccharibrachiae) Lactobacillus sakei ) strain.

[0009] The Salk Lactobacillus ( Lactobacillus sakei The strain can be a strain deposited with the accession number KCTC14319BP.

[0010] The Salk Lactobacillus ( Lactobacillus sakei The strain can be a strain that includes the 16S rRNA of sequence number 1.

[0011] The strains can have skin-beautifying effects, such as inhibiting skin aging, antibacterial effects, enhancing the skin barrier, inhibiting skin inflammation, or regulating the skin's immune balance.

[0012] The strain can increase the expression of β-defensins (HBD)-2, cathelicidin (LL-37), and RNase 7. Additionally, it can increase the expression of heme oxygenase-1 (HO-1), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Furthermore, it can maintain immune homeostasis related to TNF-α and interleukin-10 (IL-10) expression. Therefore, the strain possesses the function of maintaining skin immune balance through anti-inflammatory, antibacterial, antioxidant, and skin keratinocyte protection effects.

[0013] Another aspect of the present invention provides a dry matter, fragment, culture medium, or extract of the strain.

[0014] The specific details of the strain are as described above.

[0015] In this specification, the term "culture medium" is used interchangeably with "culture supernatant," "conditioned culture medium," or "conditioned medium," and can refer to a complete culture medium comprising the strain obtained by culturing the strain in a medium that provides nutrients to enable the *Lactobacillus sarcopeniae* strain to grow and survive in vitro for a certain period of time, its metabolites, additional nutrients, etc. Additionally, the culture medium can refer to a culture medium from which bacterial cells have been removed from a bacterial culture obtained by culturing the strain. On the other hand, the liquid from which bacterial cells have been removed from the culture medium is also called "supernatant," which can be obtained by: allowing the culture medium to stand for a certain period of time, absorbing only the upper layer of liquid excluding the portion that has settled to the bottom, or removing bacterial cells by filtration, or removing the lower layer of sediment by centrifuging the culture medium and absorbing only the upper layer of liquid. The term "bacterial cells" refers to the strain of the present invention itself, including the strain itself selected by isolation from skin samples, etc., or the strain isolated from the culture medium by culturing the strain. The bacterial cells can be obtained by centrifuging the culture medium and removing the portion that has settled to the bottom, or by allowing the bacterial cells to settle to the bottom of the culture medium under gravity and then removing the upper liquid.

[0016] The culture medium may include the culture medium itself obtained by culturing the strain, its concentrate or freeze-dried form, or the culture supernatant obtained by removing the strain from the culture medium, its concentrate or freeze-dried form.

[0017] The culture medium can be obtained by culturing the *Lactobacillus sarcopene* strain in a culture medium (e.g., liquid *Lactobacillus* MRS broth, excluding lactobacillus culture media) at any temperature above 10°C or below 40°C for a certain period of time, for example, from 4 to 72 hours.

[0018] In one specific embodiment, the culture supernatant of the strain can be obtained by centrifugation or by filtering the strain culture to remove the strain.

[0019] In another embodiment, the concentrate can be obtained by concentrating the strain culture itself or the supernatant obtained after filtering the culture using a centrifuge or filter.

[0020] The culture medium and culture conditions used to culture the *Lactobacillus sarcopenia* strain can be appropriately selected or modified by those skilled in the art.

[0021] In this specification, the term "lysate" may refer to the product obtained by breaking the cell wall of the strain itself through chemical or physical forces, and may be used interchangeably with "lysate".

[0022] In this specification, the term "culture medium extract" means extracted from said culture medium or its concentrate, and may include extract, dilution or concentrate of extract, dried product obtained by drying extract, or fractions of such crude or purified products obtained by fractionation.

[0023] In another aspect, the present invention provides the use of the said strain, its dry matter, fragments, culture medium, extracts or lysates. Specifically, a composition is provided comprising *Lactobacillus sarcopenia* strain, its dry matter, fragments, culture medium, extracts or lysates of its culture medium.

[0024] The uses of the strain may include improving skin condition, improving skin aesthetics, preventing, improving or treating skin diseases, and preventing, improving or treating inflammatory skin diseases.

[0025] The improvement in skin condition or skin aesthetics may be achieved by inhibiting or improving skin aging, or by strengthening the skin barrier.

[0026] In this specification, the term "skin aging" refers to the collective term for the tangible and intangible changes that occur on the skin with age, such as thinning of the epidermis, decrease in the number of dermal cells or blood vessels, ability to recover from DNA damage, cell turnover cycle, wound healing, skin barrier function, epidermal moisture retention, sweat secretion, sebum secretion, vitamin D production, protection against physical damage, ability to remove chemicals, immune response, sensory function, and reduced thermoregulation.

[0027] The strain or its culture medium can be used to improve skin aging caused by exogenous or endogenous factors. Exogenous factors refer to various external factors such as ultraviolet (light), while endogenous factors refer to factors primarily caused by the passage of time, also known as time factors. Specifically, skin aging includes not only premature aging symptoms caused by external stimuli such as ultraviolet radiation, pollution, cigarette smoke, and chemicals, but also natural aging phenomena caused by the reduction of skin cell proliferation due to aging. This is a concept encompassing wrinkles, decreased elasticity, sagging skin, and dryness. Furthermore, wrinkles include those caused by changes in the composition of skin tissue due to alterations in the effects of both endogenous and exogenous factors.

[0028] The aging process can be caused by ultraviolet radiation, reactive oxygen species, etc. In this specification, the term "photoaging" refers to a phenomenon caused by external environmental factors, with ultraviolet radiation being the most typical. Ultraviolet radiation leads to the activation of proteases, the breaking of matrix protein chains, and damage to biological components such as abnormal cross-linking. Repeated occurrences of this mechanism result in noticeable skin aging. Reactive oxygen species cause damage to skin cells and tissues by disrupting the skin's antioxidant defense network, composed of antioxidant enzymes and non-enzymatic antioxidants, shifting the balance between oxidants and antioxidants towards an oxidative state. This damage manifests as lipid peroxidation, protein oxidation, activation of proteases that damage intercellular matrix components, the breaking and abnormal cross-linking of collagen and elastin (elastic fibers), hyaluronic acid chain breakage, promotion of melanin production, and DNA oxidation. Furthermore, it causes skin inflammation and suppresses the skin's immune function, thereby increasing the incidence of bacterial infections or cancer. Consequently, it accelerates skin aging, characterized by reduced elasticity, wrinkles and blemishes, freckles, etc.

[0029] The term "strengthening the skin barrier" can refer to any action that enhances the function of the skin barrier, which is located on the outermost layer of the skin and prevents the loss of moisture and nutrients.

[0030] The term "anti-inflammatory" can be used interchangeably with "inhibit or improve inflammation" and can refer to any action that reduces the immune response to suppress NO production.

[0031] The term "skin disease" can refer to diseases caused by damage to the skin barrier function, skin aging, skin wounds, skin scars, or skin inflammation. The term "prevention" includes suppressing the occurrence of disease. The term "treatment" includes suppressing, reducing, or eliminating the occurrence of disease.

[0032] The skin barrier function impairment can refer to all changes that occur on the skin due to a decrease or damage to the skin barrier function. For example, it can include increased skin wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, acne, etc.

[0033] The inflammatory skin disease can be selected from any one of skin wounds, dermatitis, atopic dermatitis, pruritus, eczematous skin diseases, dry eczema, erythema, urticaria, psoriasis, mild rashes, and acne.

[0034] In one embodiment, a mixture of the strain and *Lactobacillus pentosacchari* lysate was confirmed to possess significant antibacterial activity, detoxification and anti-aging effects, and skin immune-balancing effects. Therefore, in another specific embodiment, the strain can be used with another strain belonging to the *Lactobacillus* genus with skin-improving effects, namely a second *Lactobacillus* strain, to exhibit synergistic effects. The second *Lactobacillus* strain may, for example, include *Lactobacillus pentosacchari* (… Lactobacillus pentosus Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus acidophilus ( Lactobacillus acidophilus Lactobacillus bulgaricus () Lactobacillus bulgaricus Lactobacillus plantarum ( Lactobacillus plantarum Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. Bulgaricus ), Lactobacillus fermentum ( Lactobacillus fermentum ), Lactobacillus brevis ( Lactobacillus brevis Lactobacillus paracasei ( Lactobacillus paracasei Lactobacillus casei ( Lactobacillus casei ), Lactobacillus sanguinea ( Lactobacillus sanfranciscensis Lactobacillus gasseri ( Lactobacillus gasseri ), Lactobacillus kimchi ( Lactobacillus kimchi Lactobacillus reuteri ( Lactobacillus reuteri (etc., but not limited to these.)

[0035] Based on the total weight of the composition, the composition may include 0.001% to 80%, for example, 0.01% to 60%, 0.01% to 40%, 0.01% to 30%, 0.01% to 20%, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20%, 0.05% to 10%, 0.05% to 5%, 0.1% to 60%, 0.1% to 40%, 0.1% to 30%, 0.1% to 20%, 0.1% to 10%, 0.1% to 5% of bacterial strains, their dry matter, fragments, culture media, extracts of culture media, or lysates.

[0036] The term "included as an active ingredient" means that the strains, their dry matter, fragments, culture media, extracts or lysates of the culture media are added to the extent that they can exhibit the effects described above, and also includes the meaning of adding various ingredients as adjuvants and formulating them in various forms for the purpose of delivering and stabilizing the drug.

[0037] The composition may be a cosmetic composition.

[0038] The cosmetic composition may be a dosage form commonly prepared in the art, such as solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, spray, stick, etc., but not limited thereto. More specifically, it may be prepared into the following dosage forms: lotion such as softening lotion or nourishing lotion; lotion such as facial lotion, body lotion, scalp serum, hair lotion, etc.; cream such as nourishing cream, moisturizing cream, eye cream, etc.; serum; cosmetic ointment; spray; gel; face mask; sunscreen; makeup base; liquid, solid or spray foundation, etc.; and powder, etc. At this time, the cosmetic composition may contain excipients, including, but not limited to, emulsifiers, soap acids, solvents, colorants, preservatives, antioxidants, defoamers, antibacterial agents, anti-redeposition agents, enzymes, vegetable or mineral oils, fats, fluorescent substances, fungicides, hydrotropic inducers, humectants, fragrances, fragrance carriers, proteins, organosilicones, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, and waxes.

[0039] In addition to the active ingredient, the cosmetic composition may also include commonly used ingredients in cosmetic compositions, such as stabilizers, solubilizers, vitamins, pigments, and fragrances, as well as conventional auxiliaries and carriers.

[0040] Alternatively, the composition can be a topical skin preparation composition.

[0041] The topical skin agent may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, medicated bandage, lotion, or a combination thereof. The topical skin agent may, as needed, be appropriately mixed with ingredients commonly used in cosmetics or pharmaceuticals, such as aqueous components, oily components, powder components, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof. The topical skin preparation may be appropriately mixed with metal chelating agents such as disodium EDTA, trisodium EDTA, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; caffeine, tannins, pendimethalin, licorice extract, glycyrrhizin, hot water extract of Cirsium japonicum fruit; various crude drugs; tocopheryl acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts thereof; vitamin C; magnesium ascorbate phosphate; ascorbate glucoside; arbutin; kojic acid; glucose; fructose; trehalose, etc., but is not limited to these.

[0042] In another specific embodiment, the composition may be a pharmaceutical composition.

[0043] The pharmaceutical composition may further include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol; the flow aid may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, disintegrant, binder, flow aid, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethyl cellulose, sodium glycolate starch, anhydrous calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, or a combination thereof. The flow aid may be magnesium stearate, silica, talc, or a combination thereof.

[0044] The pharmaceutical composition can be formulated into oral or non-oral dosage forms. Oral dosage forms can be granules, powders, solutions, tablets, capsules, dry syrups, or combinations thereof. Non-oral dosage forms can be injections, but are not limited thereto.

[0045] In another aspect, the present invention provides a method for preventing, improving or treating an individual's condition, comprising: treating the individual in need or administering an effective amount of the composition.

[0046] The individual's condition can be a skin-related condition or an inflammation-related condition.

[0047] The terms “application,” “introduction,” and “implantation” are used interchangeably and can refer to placing a composition according to a particular embodiment into a subject body by a method or approach that results in the composition according to a particular embodiment being at least partially located at a desired site.

[0048] Application can be performed by methods known in the art. Application can be made directly to an individual in any manner, such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. The application can be systemic or local.

[0049] The individuals may be mammals, such as humans, cattle, horses, pigs, dogs, sheep, goats, or cats. The individuals may be those requiring improved skin aesthetic effects, such as skin moisturizing, strengthening the skin barrier, suppressing skin inflammation, and improving wrinkles.

[0050] The application can be the daily administration of the composition according to a specific embodiment of the invention to an individual in the following amounts: 0.1 mg to 1000 mg, for example 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. However, the dosage can be varied depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, diet, administration time, route of administration, excretion rate, and responsiveness. Those skilled in the art will understand that these factors can be considered to appropriately adjust the dosage. The frequency of administration can be once daily or more than twice daily within the clinically acceptable range of side effects. Regarding the application site, it can be applied to one or more sites, and the total number of administration days, with intervals of daily or every 2 to 5 days, can be from 1 day to 30 days in a single treatment. If necessary, the same treatment can be repeated after an appropriate period. For animals other than humans, the dosage can be the same as for humans per kg, or the dosage can be calculated based on the volume ratio of the target animal's organs (heart, etc.) to those of humans (e.g., average value).

[0051] Based on a novel strain of Lactobacillus sarcopenia, it can be effectively used to prevent, improve or treat skin-related conditions or inflammation-related conditions. Attached Figure Description

[0052] Figure 1 The factors that maintain the skin's immune balance are shown. Detailed Implementation

[0053] Preferred embodiments will be provided below to aid in understanding the present invention. However, the following embodiments are provided only to facilitate a better understanding of the present invention, and the scope of the present invention is not limited to the following embodiments.

[0054]

Example

[0055] Example 1: Isolation of strains

[0056] 10 g of Jeju traditional kimchi was placed in 100 mg of sterile water and incubated at 37°C for 1 hour, then diluted with physiological saline. Next, for each dilution step, 100 mg of the diluted solution was aliquoted into Lactobacillus MRS broth (BD, 288130) and spread evenly, then incubated at 37°C for 2 days. After aliquoting the resulting colonies, 16S rRNA gene sequence identification was performed using known primers 27F, 338F, 518F, 785F, 518R, 805R, 907R, and 1492R. PCR amplification was performed in 30 cycles of 95°C for 1 min, 55°C for 1 min, and 75°C for 1 min 30 s, followed by a final treatment at 72°C for 8 min, and then stored at 4°C. After the PCR reaction, the DNA sequence of the isolated and cultured species was determined using an ABI-3730XL (ABI, USA). The base sequence of the 16S rRNA region determined in the isolated and cultured microbial colonies was compared and analyzed with other strains registered using the BLAST procedure provided by the National Center for Biotechnology Information (NCBI). Only novel species with 97% or less homology were selected, with a novel microorganism with 94% or less homology (hereinafter referred to as "AMP5104"). AMP5104 has the 16S rRNA sequence of sequence number 1 (complementary DNA). The strain was then deposited on September 23, 2020, at the Bioresource Center of the Korea Institute of Biotechnology, with accession number KCTC14319BP.

[0057] Example 2: Lysate of Lactobacillus sarcopenia AMP5104

[0058] Lysate of the *Lactobacillus sarcopenia* AMP5104 strain identified in Example 1 was prepared. Specifically, the strain from Example 1 was inoculated into *Lactobacillus* MRS broth (BD Company, 288130) and cultured at 35°C to 37°C for 1 to 2 days, and then the cell walls of the strain were disrupted using a homogenizer.

[0059] Example 3: Lactobacillus sarcopenia AMP5104 culture medium

[0060] A culture medium was prepared to remove the strain from the culture medium of the *Lactobacillus sarcopenia* AMP5104 strain identified in Example 1. Specifically, the strain from Example 1 was inoculated into *Lactobacillus* MRS broth (BD Company, 288130) and cultured at 35°C to 37°C for 1 to 2 days, and then the strain was removed from the culture medium using a centrifuge.

[0061] Example 4: Mixed lysate of Lactobacillus sarcopenia AMP5104 and Lactobacillus pentosus HB-8023

[0062] Except for using Lactobacillus pentosus HB-8023 strain (KCTC13322BP), the strain lysate was prepared using the same method as in Example 2. It was then mixed with the Lactobacillus sarcopenia AMP5104 lysate prepared in Example 2 at a 1:1 ratio.

[0063] [Comparative Example]

[0064] Comparative Example 1: Lysate of Lactobacillus sarcopenia L35

[0065] Except that the Lactobacillus sarcopeniae L35 strain isolated from the same Jeju Island traditional kimchi as in Example 1 was used, the strain lysate was prepared in the same manner as in Example 2.

[0066] Comparative Example 2: Lysate of Lactobacillus sarcopenia L42

[0067] Except that the Lactobacillus sarcopenia L42 strain isolated from the same Jeju Island traditional kimchi as in Example 1 was used, the strain lysate was prepared in the same manner as in Example 2.

[0068]

Experimental Example

[0069] Cytotoxicity and cell proliferation

[0070] To confirm the cell viability of the *Lactobacillus sarcopenia* strain, a CCK-8 (wst-8) assay was performed. Specifically, human keratinocyte cell line (Human HaCaT) was subjected to a assay at 5 × 10⁻⁶. 4 After being dispensed into 48-well plates, Examples 2 and Comparative Examples 1 to 2 were treated at various concentrations (0.3%, 0.6%, 2.5%, 5%, and 10%). Cytotoxicity was then assessed using a cell counting kit-8 (Munich, Germany), and absorbance was measured at 450 nm using a spectrophotometer.

[0071] Table 1

[0072]

[0073] As shown in Table 1, compared with Comparative Examples 1 to 2, it can be confirmed that Example 2 has excellent cell activity at all concentrations.

[0074] That is, based on the fact that the Lactobacillus sarcopenia AMP5104 strain has the best cell activity and no cytotoxicity, it can be used in cosmetics, pharmaceuticals, etc.

[0075] Skin antibacterial activity and improvement of skin barrier

[0076] The antibacterial activity and skin barrier-improving effect of *Lactobacillus sarcopenia* strain were confirmed. Specifically, human keratinocyte cell line (Human HaCaT) was used at 7.5 × 10⁻⁶. 5 After being allocated to 6-well plates, the samples were incubated at 37°C and 5% CO2 for 24 hours. Next, after removing the culture medium and replacing it with DMEM:F12 (3:1) medium without fetal bovine serum (FBS), the samples were treated with the methods described in Examples 2 to 4 and Comparative Examples 1 to 2, and then incubated for another 24 hours. Following RNA isolation from the cells of each sample using the RNeasy microreactor kit (Hilden, Germany), RNA was quantified at 260 nm using nanodroplets, and cDNA was synthesized using 2 mg of RNA in an amplification instrument (LunaScript® RT SuperMix kit, USA). Using the synthesized cDNA as a template, SYBR Green (Luna Universal qPCR Master Mix, New England BioLabs, USA) was added along with primers and cDNA to target the antimicrobial peptide (cathelicidin) (LL-37), RNase 7, and β-defensin (HBD)-2. Real-time polymerase chain reaction (PCR) was performed in a real-time PCR instrument (BMS MIC2 instrument, Queensland). Finally, the expression level of the RPL13A gene was analyzed by calibration, and the results are shown in Table 2 below.

[0077] Table 2

[0078]

[0079] As shown in Table 2, the expression of antimicrobial peptides in Examples 2 to 4 was confirmed to be significantly higher than that in Comparative Examples 1 to 2. In particular, the expression level of antimicrobial peptides was significantly higher in the mixed lysate with Lactobacillus pentosus HB-8023 strain.

[0080] That is, by increasing the expression of antimicrobial peptides such as LL37, RNase7 and HBD-2 in keratinocytes according to a certain strain of Lactobacillus sarcopenia, the skin exhibits antimicrobial activity to prevent, treat or improve inflammatory skin diseases such as atopic dermatitis, and can improve the skin barrier function caused by said inflammatory skin diseases.

[0081] Skin detoxification and aging

[0082] The skin detoxification and aging-improving effects of one strain of *Lactobacillus sarcopenia* were confirmed. Specifically, the expression levels of antioxidant enzymes were analyzed using the same method as described above, except that heme oxygenase-1 (HO-1) primers were used, and the results are shown in Table 3 below.

[0083] Table 3

[0084]

[0085] As shown in Table 3, it can be confirmed that HO-1 expression in Examples 2 to 4 was significantly increased compared with Comparative Examples 1 to 2.

[0086] That is, according to one strain of Lactobacillus sarcopenia, it has the effect of improving skin aging by removing toxins accumulated due to ultraviolet rays, reactive oxygen species, etc.

[0087] Skin inflammation

[0088] The skin inflammation-reducing and immune-enhancing effects of one strain of *Lactobacillus sarcopenia* were confirmed. Specifically, the expression levels of cytokines were analyzed using the same methods as described above, except that primers for tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were used. The results are shown in Table 4 below.

[0089] Table 4

[0090]

[0091] The results confirm, as shown in Table 4, that for Examples 2 to 4, compared with Comparative Examples 1 to 2, the expression of inflammatory cytokines was significantly increased and the expression of LDH was significantly decreased.

[0092] That is, according to one strain of Lactobacillus sarcopenia, not only can it protect the skin without damaging skin cells, but it can also prevent, treat and improve inflammatory skin diseases.

[0093] Skin immune balance

[0094] The skin immune balance effect of one strain of *Lactobacillus sarcopenia* was confirmed. Specifically, except for the use of primers for tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10), the expression levels of cytokines were analyzed using the same method as described above, and the results are shown in Table 5 below.

[0095] Table 5

[0096]

[0097] The results confirm, as shown in Table 5, that for Examples 2 to 4, compared with Comparative Examples 1 to 2, the expression of the pro-inflammatory cytokine tumor necrosis factor-α and the anti-inflammatory cytokine interleukin-10 was significantly increased. In particular, the expression ratio of pro-inflammatory cytokines targeting anti-inflammatory cytokines (tumor necrosis factor-α / interleukin-10) showed a value close to 1, thus confirming that the ability to maintain immune homeostasis was excellent compared with Comparative Examples 1 to 2.

[0098] That is, certain strains of Lactobacillus sarcopenia can maintain immune balance, thereby effectively preventing, treating and improving allergic skin diseases such as atopic dermatitis, urticaria, pruritus, and dermatitis.

[0099] Antioxidant effects caused by ultraviolet radiation

[0100] The UV-induced intracellular antioxidant activity of a strain of *Lactobacillus sarcopenia* was confirmed. Specifically, human keratinocyte cell line (Human HaCaT) was subjected to UV-induced antioxidant activity at 1.0 × 10⁻⁶. 6 Cells were randomly assigned to 96-well plates at a concentration of 10 cells / mL and cultured at 37°C for 16 hours. Next, cells from Examples 2 to 4 and Comparative Examples 1 to 2 were added and cultured further at 37°C for 1 hour. The cells were then exposed to UVB (30 mJ / cm²). 2 Cells were then cultured at 37°C for 24 hours. Next, DCF-DA solution (25 μM) was added to each well, and the cells were placed in the dark for 10 minutes. The fluorescence of the 2',7'-dichlorofluorescein (DCF) product generated by intracellular reactive oxygen species (ROS) was then detected using a PerkinElmer LS-5B fluorescence spectrometer (PerkinElmer, Waltham, MA, USA). Green tea extract was used as a control group.

[0101] Table 6

[0102]

[0103] As shown in Table 6, the intracellular antioxidant activity levels of Examples 2 to 4 were confirmed to be significantly higher than those of Comparative Examples 1 to 2. Specifically, Examples 2 to 4 exhibited antioxidant activity levels similar to those of green tea extracts known to have excellent antioxidant activity.

[0104] That is, according to one strain of Lactobacillus sarcopenia, it has excellent antioxidant activity by reducing the increased ROS level in the skin due to ultraviolet radiation, thus being effective in preventing and improving skin aging.

[0105] Antioxidant effect caused by free radical scavenging ability

[0106] The antioxidant activity induced by free radical scavenging ability of the *Lactobacillus sarcopenia* strain was confirmed. Specifically, after adding *Examples 2 to 4* and *Comparative Examples 1 to 2* to each well of a 96-well plate at a certain volume, DPPH (2,2-Di(4-tert-octylphenyl)-1-picrylhydrazylfree radical) prepared from 100 μM methanol solution was added to make the total volume of the solution 200 mg. Next, after incubation at 37°C for 30 minutes, the absorbance was measured at 560 nm using a spectrophotometer. Green tea extract was used as a control group.

[0107] Table 7

[0108]

[0109] As shown in Table 7, the free radical scavenging activities of Examples 2 to 4 were confirmed to be significantly superior to those of Comparative Examples 1 to 2. Specifically, Examples 2 to 4 were confirmed to exhibit free radical scavenging activities at levels similar to those of green tea extracts known to have excellent antioxidant activity.

[0110] That is, based on the fact that the *Lactobacillus sarcopenia* strain has excellent antioxidant activity in scavenging free radicals, it can be used to prevent or improve skin aging.

[0111] [Collection Number]

[0112] Name of the depository: Korea Institute of Life Sciences

[0113] Collection Number: KCTC14319BP

[0114] Date of preservation: 20200923

[0115] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that it can be easily modified into other specific forms without changing the technical spirit or essential characteristics of the invention. Therefore, it should be understood that the above embodiments are exemplary in all respects and not restrictive. sequence list <110> IT'S HANBUL CO., LTD. <120> Lactobacillus sarcopenia AMP5104 strain and its uses <130> PX066652CN <150> KR10-2021-0161382 <151> 2021-11-22 <160> 1 <170> PatentIn version 3.2 <210> 1 <211> 1534 <212> DNA <213> Artificial <220> <223> Lactobacillus sakei AMP 5104 <400> 1 tggctcagga cgaacgctgg cggcgtgcct aatacatgca agtcgaacgc actctcgttt 60 agattgaagg agcttgctcc tgattgataa acatttgagt gagtggcgga cgggtgagta 120 acacgtgggt aacctgccct aaagtggggg ataacatttg gaaacagatg ctaataccgc 180 ataaaaccta acaccgcatg gtgtagggtt gaaagatggt ttcggctatc actttaggat 240 ggacccgcgg tgcattagtt agttggtgag gtaaaggctc accaagaccg tgatgcatag 300 ccgacctgag agggtaatcg gccacactgg gactgagaca cggcccagac tcctacggga 360 ggcagcagta gggaatcttc cacaatggac gaaagtctga tggagcaacg ccgcgtgagt 420 gaagaggtt ttcggatcgt aaaactctgt tgttggagaa gaatgtatct gatagtaact 480 gatcaggtag tgacggtatc caaccagaaa gccacggcta actacgtgcc agcagccgcg 540 gtaatacgta ggtggcaagc gttgtccgga tttattggc gtaaagcgag cgcaggcggt 600 660 acttgagtgc agaagaggac agtggaactc catgtgtagc ggtgaaatgc gtagatatat 720 ggaagaacac cagtggcgaa ggcggctgtc tggtctgtaa ctgacgctga ggctcgaaag 780 catgggtagc aaacaggatt agataccctg gtagtccatg ccgtaaacga tgagtgctag 840 gtgttggagg gtttccgccc ttcagtgccg cagctaacgc attaagcact ccgcctgggg 900 agtacgaccg caaggttgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 960 atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctttgaccac 1020 tctagagata gagctttccc ttcggggaca aagtgacagg tggtgcatgg ttgtcgtcag 1080 ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttat tactagttgc 1140 cagcatttag ttgggcactc tagtgagact gccggtgaca aaccggagga aggtggggac 1200 gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggatggtac 1260 aacgagttgc gagaccgcga ggtttagcta atctcttaaa accattctca gttcggattg 1320 taggctgcaa ctcgcctaca tgaagccgga atcgctagta atcgcggatc agcatgccgc 1380 ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgagag tttgtaacac 1440 ccaaagccgg tgaggtaacc ccttcggggg agccagcccg tctaaagtgg ggacagatga 1500 attaagggtg aagtcgtaag aggggggacc ccgt 1534

Claims

1. A type of Lactobacillus sarcopenia ( Lactobacillus sakei AMP5104 strain, with accession number KCTC14319BP.

2. Dry matter, fragments, culture medium or lysate of a strain of Lactobacillus sarcopenia AMP5104, with accession number KCTC14319BP.

3. A cosmetic composition comprising the strain of claim 1, its dry matter, fragments, culture medium, lysate, or a mixture thereof.

4. The cosmetic composition according to claim 3, further comprising a strain of Lactobacillus genus, its dry matter, fragments, culture medium, lysate, or a mixture thereof.

5. The cosmetic composition according to claim 3 or 4, used to improve skin condition, skin aging, skin antibacterial properties, skin inflammation, skin immunity, or skin barrier.

6. A topical skin composition comprising the strain of claim 1, its dry matter, fragments, culture medium, lysate, or mixtures thereof, and for use in improving skin condition, skin aging, skin antibacterial activity, skin inflammation, skin immunity, or skin barrier function.

7. A pharmaceutical composition comprising the strain of claim 1, its dry matter, fragments, culture medium, lysate, or mixtures thereof, and for use in the prevention or treatment of skin antibacterial, skin immune, or inflammatory skin diseases.

8. The pharmaceutical composition according to claim 7, wherein the inflammatory skin disease is selected from any one of skin wounds, dermatitis, atopic dermatitis, pruritus, eczematous skin diseases, dry eczema, erythema, urticaria, psoriasis, mild rashes, and acne.

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