Use of enterococcusfaecium EB9 bacterial strain, deposited at all-russian research institute of agricultural microbiology under registration number RCAM07244, in form of lysate or supernatant to improve and / or restore barrier function of skin

Abstract

FIELD: pharmaceuticals; cosmetology; dermatology.SUBSTANCE: use of the strain of the enterococcusfaecium EB9 bacterium, deposited at the All-Russian Research Institute of Agricultural Microbiology under registration number RCAM07244, in the form of a lysate or supernatant for improving and / or restoring the barrier function of the skin.EFFECT: improving and / or restoring the barrier function of the skin.1 cl, 6 dwg, 19 ex

Description

[0001] Scope of use

[0002] The invention relates to the field of cosmetology and dermatology, in particular to methods for cosmetic treatment and the use of lysates or supernatants of probiotic bacteria on human skin, or the use of such a lysate or supernatant of a probiotic bacterium for topical application to the skin to improve and / or restore the skin's barrier function. However, cosmetic treatment does not involve the treatment of a human or animal organism through therapy or surgery.

[0003] In addition, the said lysates or supernatants can be used in antibacterial compositions for use in infections caused by Staphylococcus spp., C. Acnes, in intimate hygiene products, scalp and hair products, antifungal products for feet and nails, in food products, and in feed additives.

[0004] State of the art

[0005] Currently, skin barrier restoration methods mainly involve antibacterial agents, which, although effective, have a number of side effects associated with the use of antibiotics to kill bacterial infections.

[0006] The prior art also recognizes the use of probiotics, including inactivated probiotics / metabolites, in skin care to prevent the harmful effects of pathogenic bacteria while actively restoring the damaged skin barrier. With consumers increasingly seeking natural, safe, and effective skin care products, the claimed invention could generate significant interest in addressing these market needs. Such use could eliminate the need for existing aggressive treatments, such as antibiotics.

[0007] The use of probiotic bacterial strains as medicinal and food products is known (RU 2547599 for the invention “Bacteriocin-producing Lactobacillus pentosus and its use in food and pharmaceutical compositions”).

[0008] The said patent discloses the use of the bacteriocin-producing Lactobacillus pentosus strain DSM 21980 as a medicinal product for combating gram-negative bacteria, preferably enterococci, coliform bacteria, and E. coli, and reducing or inhibiting their presence. The use of the strain for the prophylactic or curative treatment of gastroenteritis or gastroenterocolitis, infections caused by the aforementioned bacteria, is also proposed. The strain can be used in a pharmaceutical composition for use as a medicinal product. The group of inventions provides for the inhibition of gram-negative bacteria, preferably E. coli.

[0009] However, this product only suggests using live cells, without using lysate.

[0010] Meanwhile, the use of bacterial lysate in cosmetics has a number of advantages over the use of live bacteria, which are as follows:

[0011] 1. Security.

[0012] This is due to the lack of risk of infection, as lysates are dead bacteria, meaning they cannot cause infections or other health problems. However, live bacteria may pose a risk to people with weakened immune systems or sensitive skin. Furthermore, lysates are less allergenic than live bacteria, as they do not contain intact cells that could trigger an immune response.

[0013] 2. Efficiency:

[0014] The higher effectiveness of lysates is determined by the controlled amount of active ingredients. Lysates contain controlled amounts of active ingredients, such as enzymes, proteins, and peptides, that are beneficial for the skin. Live bacteria can produce varying amounts of these substances depending on the conditions of use.

[0015] 3. Stability:

[0016] Lysates are more stable than live bacteria and can be stored longer without losing potency. Lysates are not subject to the same limitations as live bacteria, which can be sensitive to light, heat, and other environmental factors.

[0017] 4. Ease of use

[0018] Lysates can be easily added to cosmetic products since they do not require special storage conditions.

[0019] 5. Wide range of applications and environmental friendliness.

[0020] Bacterial lysates can be used for various purposes such as moisturizing, skin brightening, acne control and improving skin barrier function.

[0021] Bacterial lysates are derived from non-pathogenic bacteria, making them more environmentally friendly than some synthetic ingredients.

[0022] Although not all bacterial lysates are qualitatively the same, as the quality of the lysate depends on the method of its production and purification, in general, bacterial lysates represent a safer, more stable and effective alternative to live bacteria in cosmetics.

[0023] The probiotic strain Enterococcus faecium L-3 - 79 VNIISKhM (patent RU No. 2220199) is known, used for the production of therapeutic and prophylactic agents and food products for therapeutic and prophylactic purposes, possessing resistance to a wide range of antibiotics, more pronounced antagonism to pathogenic bacteria, high viability and a relatively high level of production of B vitamins and folic acid.

[0024] However, the products and agents that use this strain have the above-mentioned disadvantages due to the fact that they use live cells of the probiotic bacterium Enterococcus faecium L-3, and not lysates.

[0025] From the RU patent No. 2800359, the “Enterococcus faecium L-3 strain for the treatment and prevention of bacterial and viral infections using therapeutic and prophylactic agents and food products made on its basis and a method for cultivating the Enterococcus faecium L-3 strain of enterococci” is known.

[0026] The invention relates to a method for culturing the Enterococcus faecium L-3 strain, comprising culturing the Enterococcus faecium L-3 strain at a temperature of 37°C for 19-24 hours in a growth medium. 2-16 hours after the start of cultivation, the synthetic pheromone Pher4 with the amino acid sequence KPQGKPASNLVECVFSLFKKC is added to the growth medium at a concentration of 10-5 to 10-17 mol / L. The invention enhances the antagonistic activity of the Enterococcus faecium L-3 strain.

[0027] However, this method is also aimed at obtaining live probiotic bacteria Enterococcus faecium L-3, which can also lead, as indicated above, to known disadvantages compared to the use of bacterial lysates.

[0028] The closest to the claimed group of inventions is the invention according to patent RU No. 2656152 “Use of a probiotic bacterium lysate to improve and / or restore the barrier function of the skin.”

[0029] The said patent discloses the use of a lysate of the probiotic bacterium Lactobacillus rhamnosus GG ATCC 53103 or Lactobacillus reuteri ATCC 55730 or Bifidobacterium longum ATCC 51870 for topical application to the skin to improve and / or restore the barrier function of the skin.

[0030] This lysate is used in a method for improving and / or restoring the skin barrier function, comprising topical administration of the lysate to the subject's skin to improve and / or restore the skin barrier function. This lysate is used as part of a cosmetic composition, a pharmaceutical composition for topical use, or a composition formulated for intradermal administration. This lysate is also used as part of an antibacterial composition for use in treating Staphylococcus infections. This group of inventions enhances and restores the barrier function of keratinocytes.

[0031] Meanwhile, as will be shown below, the use of Enterococcus bacterial strain is more preferable than Lactobacillus rhamnosus GG ATCC 53103, Lactobacillus reuteri ATCC 55730, or Bifidobacterium longum ATCC 51870 in cosmetics.

[0032] This is due to the fact that the strains Lactobacillus rhamnosus GG ATCC 53103, Lactobacillus reuteri ATCC 55730, and Bifidobacterium longum ATCC 51870 are representatives of the microbiota primarily of the human intestine. At the same time, bacteria of the genus Enterococcus are normal representatives of the microbiota of both the human intestine and skin.

[0033] The presence of Enterococcus bacteria on human skin in small numbers has been confirmed by numerous studies using metagenomic sequencing. This demonstrates the natural adaptation of these bacteria to the skin's biotope and their important role in maintaining skin health.

[0034] The main objective of the claimed group of inventions is to expand the arsenal of means, including lysates of bacterial strains for the treatment and prevention of dermatological conditions and diseases, improving and / or restoring the barrier function of the skin.

[0035] The technical result is the implementation of this purpose.

[0036] At the same time, the claimed group of inventions, as was established by the authors’ research, allows:

[0037] Based on the use of the lysate of the probiotic bacteria Enterococcus faecium EB9, it improves the condition of the skin, balances its microbiome, supports and accelerates the natural processes of skin restoration, and protects the skin from stress and aggressive environmental factors.

[0038] The use of the lysate of the probiotic bacteria Enterococcus faecium EB9 in cosmetology in the form of a composition with fillers also helps improve the balance of the skin microbiome, which will be expressed in an improvement in skin texture; in the implementation of the anti-inflammatory effect of the lysate to reduce inflammation on the skin, reducing redness and irritation caused by various factors, including the implementation of the antibacterial effect of the lysate to combat pathogenic microorganisms on the skin surface; in stimulating the natural processes of skin restoration - regeneration and healing of the skin.

[0039] The claimed technical result is achieved by using a lysate or supernatant of the Enterococcus faecium EB9 bacterial strain for the treatment and prevention of dermatological conditions and diseases.

[0040] The technical result is also achieved by a composition containing an effective amount of lysate or supernatant of the Enterococcus faecium EB9 bacterial strain, vitamins A, B1, B2, B6, B9, B12, C, D, E and dermatologically acceptable fillers.

[0041] Preferably, the cosmetic composition contains by weight%:

[0042] lysate or supernatant of Enterococcus faecium EB9 bacterial strain - 5 – 95;

[0043] vitamin A - 0.00515 - 0.0915;

[0044] Vitamin B1 - 0.00000093 - 0.0000183;

[0045] Vitamin B2 - 0.0000001463 - 0.000002489;

[0046] Vitamin B6 - 0.00000014 - 0.0000084;

[0047] Vitamin B9 - 0.000000435 - 0.00000855;

[0048] Vitamin B12 - 0.0000001012 - 0.000004022;

[0049] Vitamin C - 0.0000201 - 0.000808;

[0050] Vitamin D - 0.000005258 - 0.00009228;

[0051] Vitamin E - 0.00001205 - 0.0004209

[0052] Dermatologically acceptable fillers - rest.

[0053] It is preferable to use hyaluronic acids, or natural oils, or hydrolates, or a mixture of them as fillers.

[0054] It is preferable to use the composition for dermatological conditions such as dry skin, transepidermal water loss, inflammatory skin diseases and neurodermatitis.

[0055] The composition can be used in the form of cream, liquid, or serum.

[0056] The technical result is also achieved by the fact that in the method for producing the composition, the enterococcal bacteria Enterococcus faecium EB9 are lysed to obtain a lysate or supernatant, and an effective amount of the lysate or supernatant of the Enterococcus faecium EB9 strain of the enterococcal bacteria and dermatologically acceptable fillers are mixed.

[0057] It is optimal to carry out mixing at a temperature of +10 - +25°C, at a stirring speed of no more than 500 rpm, without foaming or whipping the mixture.

[0058] Stirring is carried out until complete dissolution and a homogeneous mixture is obtained for 1-2 hours, while the pH value of the resulting mixture is monitored, bringing it to a value within 4.5-5.5 units. The use of fillers in combination with lysates or supernatants of Enterococcus faecium EB9 bacteria in the production of cosmetics provides a number of additional benefits confirmed by scientific research.

[0059] Hyaluronic acid, natural oils, and hydrolates, widely used in the cosmetics industry, interact synergistically with the lysate's biologically active components, such as polypeptides, vitamins, and bacteriocins. This combination enhances the beneficial effects on the skin, improving its condition and the consumer properties of the final product.

[0060] In one preferred embodiment, it is recommended to include preservatives such as phenoxyethanol and ethylhexylglycerin, optimally up to 1% by weight, and pentylene glycol, sodium benzoate, or potassium sorbate, up to 5% by weight, as fillers to ensure long-term storage with high biological activity and stability of the finished product. The use of cold sterilizing filtration during lysate production allows for the preservation of valuable components in their native form without subjecting them to destruction.

[0061] For example, when using lysate in combination with hyaluronic acid, the optimal lysate content can be 5-20% of the total mass. This ratio allows the moisturizing properties of hyaluronic acid to be combined with the lysate's biologically active components, such as vitamins, peptides, and bacteriocins, providing a comprehensive beneficial effect on the skin.

[0062] When using the lysate in combination with natural oils, its optimal content can reach 10-30% of the total formula. The oils act as carriers and enhancers of the biologically active substances contained in the lysate.

[0063] It is preferable to use the lysate in combination with hydrolates of neroli, immortelle, lavender or other plants, in an amount of 15-40% by weight.

[0064] Hydrolates provide additional hydration and toning of the skin, harmoniously complementing the effectiveness of the lysate.

[0065] Thus, by varying the ratio of Enterococcus bacteria lysate and fillers, it is possible to create cosmetic products with optimal consumer properties and high effectiveness on the skin.

[0066] The claimed group of inventions provides controlled activity of the composition and detectable activity even at low concentrations; increased stability; and simplified manufacturing technology. All of this combined makes the inventors' technology more suitable for therapeutic and cosmetic applications, compared to live bacterial creams, which are difficult to manufacture and distribute.

[0067] This actively restores the skin barrier at the cellular level. While a decrease in barrier function / restoration is inextricably linked to a reduced ability to contain infection, the lysate or supernatant in the claimed invention represents a composition that both strengthens the skin barrier and enhances its ability to protect against pathogens.

[0068] The specified technical result is achieved due to the unique composition of the composition containing a lysate of the probiotic bacterium Enterococcus faecium EB9, which includes metabolites of the cultivation of the probiotic bacterium Enterococcus faecium EB9, containing, in particular, antimicrobial peptides bacteriocins, lactic acid, and vitamins A, B1, B2, B6, B9, B12, C, D, E.

[0069] Thus, when applied topically, vitamin A has been shown to be effective in improving skin condition by promoting epidermal cell renewal and increasing collagen production. By stimulating an increase in the thickness of both the epidermis and dermal layers, the use of vitamin A in cosmetics leads to a reduction in fine wrinkles. Furthermore, as indicated by [1] and [2], vitamin A helps regulate sebum production, which can help combat acne and other skin problems. Its antioxidant properties help protect the skin from the harmful effects of free radicals and prevent premature aging [1], [2].

[0070] B vitamins play an important role in skin health, participating in many processes necessary for its normal functioning.

[0071] Vitamin B1 (thiamine) has an antioxidant effect, fights free radical damage, is used to treat melasma, freckles, and slows down skin aging. It is used for sensitive skin with redness, irritated skin, acne-prone skin, or dry skin. Vitamin B2 (riboflavin) helps maintain collagen levels, is involved in the development of cellular tissue, regulates sebum production, and prevents acne. Vitamin B3 (niacin, niacinamide, vitamin PP) prevents melanin proliferation, reduces pigmentation, reduces sebum production, tightens pores, and plays an important role in helping the skin resist moisture loss and premature aging, thereby effectively rejuvenating the skin. Vitamin B3, depending on its concentration, has antimicrobial, vasoactive, photoprotective, sebostatic, and brightening effects. Vitamin B5 (pantothenic acid) helps moisturize the skin, improves its elasticity and stimulates regeneration processes.Vitamin B6 (pyridoxine) promotes the production of hemoglobin and antibodies, which improves blood circulation and maintains healthy skin; it plays a role in regulating sebum secretion. A deficiency of vitamin B6 can lead to seborrheic dermatitis, also known as eczema. Vitamin B9 (folic acid) has a detoxifying effect on the body, reduces acne formation, maintains healthy skin, and is involved in the process of tissue regeneration and the formation of new cells. Vitamins B7 (biotin, vitamin H) and B12 (cobalamin) are involved in metabolic processes and provide the skin with essential nutrients. Thus, the presence of B vitamins for use in skin care cosmetics can help improve skin, making it more hydrated, elastic, and healthy [3], [4], [5], [6], [7], [8], [9].

[0072] Vitamin C is a powerful antioxidant that plays a key role in promoting skin health. Vitamin C is a cofactor in collagen synthesis, which is important for skin elasticity and structure, and its antioxidant properties make it an effective UV protector. The accumulation of vitamin C in keratinocytes, especially when combined with vitamin E, provides skin protection from UV radiation. Its regular use helps stimulate collagen production, which helps reduce the appearance of wrinkles and signs of aging. Vitamin C also lightens dark spots and evens out skin tone, giving it a radiant and youthful appearance. In addition, thanks to its antioxidant properties, it protects the skin from the harmful effects of free radicals and the environment, preventing premature aging

[10] ,

[11] .

[0073] Vitamin D has anti-inflammatory properties. Its main function is to help the body absorb calcium, which is essential for healthy bones and teeth, and is also important for skin. Taking vitamin D can reduce skin inflammation, suppress sebum production, and improve skin appearance. It can also be helpful in treating skin conditions such as psoriasis and eczema due to its anti-inflammatory properties. Getting enough vitamin D through sunlight or supplements can promote healthy and beautiful skin

[12] ,

[13] .

[0074] Vitamin E plays a vital role in maintaining healthy and youthful skin, participating in the biosynthesis of collagen, elastin, and glycosaminoglycans. Vitamin E protects the lipid structures of the stratum corneum from damage, prevents inflammation caused by UV radiation, and has powerful antioxidant properties, protecting the skin from oxidative stress

[14] ,

[15] .

[0075] Thus, vitamins are not only essential components for skin but also modulate numerous biological functions. Although their importance has been widely studied, therapeutic approaches using vitamins are limited to antioxidants and wound healing. At the same time, research reveals a new role for vitamin D in stimulating the main antimicrobial peptide of the epidermis, cathelicidin, thereby activating innate immunity. Vitamins also play a more profound role in preventing skin damage, facilitating skin repair mechanisms, reducing oxidative damage, and activating barrier function, ultimately leading to slowed aging and improved skin health and appearance.The use of probiotics, prebiotics and postbiotics is an important direction for improving skin condition, as they are effective in improving the condition of the skin microbiome, which leads to improved skin hydration, a decrease in the depth of fine wrinkles and strengthening the integrity of the skin barrier

[16] ,

[17] ,

[18] ,

[19] ,

[20] ,

[21] .

[0076] The claimed group of inventions is illustrated by drawings, where:

[0077] Fig. 1 shows a chromatogram, obtained using an ultraviolet detector (hereinafter referred to as the UV chromatogram), of the test solution of the supernatant sample based on the probiotic strain Enterococcus faecium EB9, indicating the vitamins and lactic acid detected in the solution.

[0078] Fig. 2 shows a UV chromatogram of the test solution of the lysate sample based on the probiotic strain Enterococcus faecium EB9, indicating the vitamins and lactic acid detected in the solution.

[0079] Fig. 3 shows the UV chromatogram of the test solution of the bifidobacteria lysate sample Bifidobacterium longum and Bifidobacterium bifidum (Microecobeauty ME-3 brand) produced by Bloomage Biotech with an indication of the vitamins found in the solution.

[0080] Fig. 4 shows a diagram characterizing the quantitative composition of vitamins detected by high-performance liquid chromatography in the supernatant of Enterococcus faecium EB9 bacteria.

[0081] Fig. 5 shows a diagram showing the amount of vitamins detected by high-performance liquid chromatography in the lysate of Enterococcus faecium EB9 bacteria

[0082] Fig. 6 shows the quantitative composition of vitamins detected by high-performance liquid chromatography in the bifidobacteria lysate Microecobeauty ME-3 produced by Bloomage

[0083]

[0084] Implementation of the invention

[0085] The human microbiota, composed of a variety of microorganisms, plays a key role in the synthesis and metabolism of vitamins essential for normal body function. Essential dietary vitamins, such as the B vitamins (including B1, B2, B5, B6, and B12), vitamins A, C, D, E, and K, niacin (vitamin PP), and folic acid (vitamin B9), are synthesized by the microbiota in various parts of the body, including the intestines, skin, and other organs.

[0086] This physiological process is an important component of maintaining overall health and the effective functioning of the human body. Therefore, using lysate as a source of vitamins in cosmetic and medical products is a promising direction in the development of innovative and more effective products for improving skin condition, maintaining its protective functions, and stimulating regenerative processes.

[0087] According to numerous metagenomic sequencing studies of the human skin microbiome worldwide, the bacterial genus Enterococcus is present in small quantities in the normal skin microbiota. Unlike bifidobacteria, Enterococcus is a natural member of the skin microflora.

[0088] The authors selected the Enterococcus faecium EB9 strain (registration number RCAM07244 of the All-Russian Research Institute of Agricultural Microbiology) as the basis for use in cosmetics due to the fact that during the study the authors discovered that this genus of bacteria in some cases surpassed bifido- and lactobacilli in the ability to synthesize vitamins, especially B vitamins.

[0089] Research conducted using high-performance liquid chromatography confirms the presence of biologically synthesized vitamins in the Enterococcus bacterial lysate, which are in a more bioavailable form compared to vitamins synthesized industrially from chemical reagents that are often used in cosmetic products.

[0090] This circumstance makes bacterial lysates more preferable for use in cosmetic products where the emphasis is on natural and more beneficial components for the skin.

[0091] Thus, according to the authors of the invention, the use of the Enterococcus strain lysate in cosmetics not only expands the arsenal of cosmetics for improving the barrier function of the skin, but also has significant advantages over known ones, since it additionally provides the delivery of natural, biologically active vitamins synthesized by bacteria.

[0092] Bacterial lysate is the content of bacterial cells obtained through their destruction (lysis). Lysis can be caused by mechanical, chemical, or enzymatic action. The lysate contains intracellular bacterial components such as proteins, nucleic acids, enzymes, vitamins, and other biologically active substances.

[0093] Bacterial supernatant is the liquid fraction obtained after centrifugation of a bacterial culture. It contains extracellular metabolites and vitamins secreted by bacteria during their life cycle, as well as some soluble components released from the cells during their rupture. The supernatant typically contains fewer intracellular components than the lysate.

[0094] The authors of the invention studied experimental laboratory samples, which were a lysate and supernatant based on the probiotic strain Enterococcus faecium EB9, and reference standard samples of vitamins using high-performance liquid chromatography on a Shimadzu LC-30 Nexera chromatograph.

[0095] The analysis carried out using high-performance liquid chromatography on a Shimadzu LC-30 Nexera chromatograph showed that the supernatant and lysate of Enterococcus faecium EB9 bacteria contain vitamins A, B1, B2, B6, B9, B12, C, D, which follows from Fig. 1 and Fig. 2, the graphs of which indicate the detected compounds. The supernatant and lysate of Enterococcus faecium EB9 bacteria, in which the presence of vitamins A, B1, B2, B6, B9, B12, C, D, E was detected, demonstrate a greater amount of vitamins compared to the lysate of bifidobacteria Microecobeauty ME-3 produced by Bloomage Biotech, in which the presence of only vitamins B6, B9, B12 was detected (Fig. 3).

[0096] Figures 4 and 5 show diagrams characterizing the quantitative composition of vitamins in the supernatant and lysate of Enterococcus faecium EB9 bacteria.

[0097] The results presented in the diagrams indicate more pronounced vitamin-forming properties of microbial biocomponents obtained from bacteria of the Enterococcus faecium EB9 species, compared to bifidobacteria widely used in the cosmetic industry and the lysate produced with their help.

[0098] As follows from Fig. 1 and Fig. 2 and the diagrams presented in Fig. 4 and 5, the supernatant and lysate of Enterococcus faecium EB9 bacteria were found to contain vitamins A, B1, B2, B6, B9, B12, C, D3, E, with the indication of vitamins and lactic acid. Moreover, the supernatant and lysate of Enterococcus faecium EB9 bacteria were found to contain vitamins A, B1, B2, B6, B9, B12, C, D3, E, with a significantly higher content of a number of the identified vitamins compared to the lysate of bifidobacteria Microecobeauty ME-3 produced by Bloomage Biotech. Moreover, in the latter, only vitamins B6, B9, B12 were found (Fig. 3), and the diagram presented in Fig. 6.

[0099] The active agent formed according to the invention from the lysate and from the "complete" fermentation medium contains cytoplasmic and cytosolic fractions, fragments of cell walls and metabolites formed and / or released during the cellular lysis of the said microorganism, and all biological objects capable of being formed and released by the bacterium spontaneously during the process of its fermentation and therefore already present in the fermentation medium before the cellular lysis of the said bacterium.

[0100] Therefore, the active agent according to the invention, i.e. formed from the lysate of a bacterium belonging to the genus of the enterococcus strain, is clearly different from the supernatant of the fermentation medium of this bacterium, as follows from Fig. 1 and Fig. 2. Indeed, the active agent in question according to the invention, in contrast to the supernatant, contains fragments of cells of the said bacterium, represented by the lysate.

[0101] In the context of the present invention, the expression "non-aqueous components" means that water, which is the main component of a traditional fermentation medium, is not part of the components that, as such, must remain, i.e. in equal amounts, in the complete culture medium according to the invention.

[0102] Thus, the term "complete medium" also refers to a form of complete medium called concentrated due to the fact that it is obtained by partial evaporation of the water that constitutes the fermentation medium, in which the corresponding microorganism is subsequently cultivated and then subjected to cell lysis. This evaporation is carried out under operating conditions controlled so as not to compromise the integrity of the non-aqueous components that make up this complete medium.

[0103] By definition, a fermentation or culture medium is a substrate that enables the cultivation and, if necessary, growth of cells, bacteria, and yeast. In principle, cells obtain from this medium the components they need to rapidly multiply in large quantities, as well as sometimes elements that support the growth of specific bacterial genera or families—in this case, bacteria belonging to the genus Enterococcus.

[0104] In this regard, the composition of the medium must satisfy the needs of the microorganism in question for nutrients and the need for its reproduction.

[0105] As an example of a fermentation medium suitable for the growth of a microorganism according to the invention, it is possible to mention, in particular, media in the form of plant drinks based on soy, coconut, oatmeal, buckwheat, wheat, hops, malt, etc., various types of milk - cow's, goat's, etc.

[0106] An effective amount of the microorganism in question according to the invention is introduced into an absorption medium, which may be one of the herbal drinks mentioned above, and this mixture is placed under conditions suitable for the reproduction of this microorganism.

[0107] To obtain a lysate of bacteria belonging to the genus Enterococcus, a stage of culturing the said bacteria is carried out in a complete fermentation medium.

[0108] Preferably, to obtain the active agent according to the invention, i.e. the lysate and / or supernatant of enterococci bacteria in a complete fermentation medium, the biomass (bacterial cells after the growth phase, located in the medium in which they were cultured) is cooled, for example, to a temperature of +4°, and then centrifuged, preferably for 30 min.

[0109] Lysate, generally refers to the material obtained after the destruction or dissolution of biological cells through a phenomenon known as cell lysis, thus resulting in the release of intracellular and cellular biological components naturally contained in the biological cells in question.

[0110] In the context of the present invention, the term "lysate" means the entire lysate obtained by lysis of bacteria.

[0111] The lysate used is formed from all of its intracellular biological components, in particular its metabolites and components of cell walls and membranes formed during the lysis of its cell.

[0112] In the context of the invention, the term "metabolite" means any substance obtained as a result of the metabolism of the microorganism in question according to the invention.

[0113] This cell lysis can be achieved by various methods such as osmotic shock, heat shock, sonication, or alternatively by mechanical stress such as that generated by centrifugation.

[0114] In particular, this lysate can be obtained according to a method such as disclosed in U.S. Patent 4,464,362 and, in particular, according to the following protocol.

[0115] The fermentation medium used to cultivate the microorganism in question is degraded using ultrasound to release cytoplasmic and cytosolic fractions, cell wall fragments, and metabolic products from the microorganism's cells. All these components are then preserved in their natural distribution in a stabilized form within the "complete" fermentation medium.

[0116] Therefore, the active agent in question according to the invention can be obtained by a method comprising:

[0117] - culturing enterococci bacteria on a fermentation medium under conditions suitable for the reproduction of the said bacteria, and

[0118] - cell lysis of the specified bacteria in the specified fermentation medium.

[0119] As described above, the microorganism considered according to the invention, in the form of a lysate, is a strain of enterococci bacteria. The inventors unexpectedly discovered that such a bacterium, used in the form of its lysate or supernatant, prepared in a complete fermentation medium as defined in the present invention, has a composition and properties, from a skin physiology standpoint, equal to or superior to those of the biomass obtained from the same fermentation medium.

[0120] To obtain Enterococcus faecium EB9, a pure culture of this strain was cultivated in a fermentation medium. Cultivation was carried out in a thermostat at 37°C, which corresponds to optimal conditions for the growth and reproduction of Enterococcus faecium EB9 bacteria.

[0121] Various microbiological analysis methods were used to confirm the purity and identification of the obtained culture. One of the most common is plating on chromogenic nutrient media, which allows for visual differentiation of Enterococcus faecium EB9 colonies from other bacteria. Alternatively, molecular genetic methods such as polymerase chain reaction (PCR) or MALDI-TOF mass spectrometry can be used for more precise identification. These methods ensure high specificity and reliability in determining the species identity of the isolated cultures.

[0122] Thus, strict quality control at all stages of Enterococcus cultivation and identification guaranteed a highly pure final product that meets established regulatory requirements. The process of obtaining Enterococcus faecium EB9 bacterial lysate or supernatant involved several sequential steps. During lysate production, the culture medium and the Enterococcus faecium EB9 cells contained within it were sonicated to disrupt the cells and release the culture fluid using an immersible high-frequency ultrasonicator (850 kHz) for 5 minutes while maintaining a temperature of +4 to +8 degrees Celsius.

[0123] To produce the supernatant, the Enterococcus faecium EB9 culture was centrifuged at a temperature of +4 - +6 degrees Celsius for 40 minutes at a centrifuge speed of 3000.

[0124] Centrifugation allows the separation of bacterial cells and their components from the culture fluid.

[0125] Cold filtration using 0.22-μm pore-size membrane filters was used to sterilize and clarify the lysate or supernatant. This pore size effectively removes bacterial cells and insoluble components while preserving important biologically active compounds such as polypeptides, vitamins, and bacteriocins. Cold filtration was performed at low temperatures, which helped preserve the structure and biological activity of the target components in the resulting preparations.

[0126] Thus, cell sonication, centrifugation and subsequent cold sterilizing filtration are key steps in the process of obtaining high-quality Enterococcus faecium EB9 bacterial lysate and supernatant that retain their functional properties.

[0127] Below are examples of specific production of composition for cosmetic products in the form of spray, cream and serum.

[0128] Example 1.

[0129] Production technology of liquid cosmetic, skin spray based on Enterococcus faecium EB9 supernatant.

[0130] The components of the future cosmetic product were prepared in which the obtained supernatant of Enterococcus faecium EB9 as the main component constituted 5%.

[0131] The quantitative composition of vitamins in the supernatant was measured, which was:

[0132] vitamin A - 0.0075;

[0133] Vitamin B1 - 0.000015;

[0134] Vitamin B2 - 0.000001489;

[0135] Vitamin B6 - 0.0000054;

[0136] Vitamin B9 - 0.00000455;

[0137] Vitamin B12 - 0.00000225

[0138] Vitamin C - 0.000508;

[0139] Vitamin D - 0.000053;

[0140] Vitamin E - 0.0002209

[0141] The ingredients were mixed at a temperature of +22 °C.

[0142] The mixing speed was selected at 500 rpm, mixing was carried out without foaming or whipping the mixture.

[0143] Enterococcus faecium EB9 supernatant (5% of the total weight), fillers: biosaccharide resin (3% of the total weight), green tea enzyme (2% of the total weight), hyaluronic acid (1% of the total weight), D-panthenol 75% (1% of the total weight), glycerin (1% of the total weight), preservative phenoxyethanol and ethylhexylglycerin (1% of the total weight) were successively added to the prepared deionized water (86% of the total weight).

[0144] Stirring was carried out until the components were completely dissolved and the mixture was homogeneous for 1 hour.

[0145] The pH of the resulting mixture was controlled using professional equipment such as a pH meter, which was 5 units.

[0146] If the pH value was outside the range of 4.5–5.5 pH units, pH calibration was performed using a filler selected from the group including lactic acid or an alkali solution such as NaOH.

[0147] The pH of the cosmetic product was adjusted to 4.5–5.5 to ensure optimal compatibility with the skin. This pH range matches the skin's natural pH, minimizing the risk of irritation and inflammation and enhancing the product's effectiveness.

[0148] Example 2

[0149] The production of a liquid cosmetic skin spray based on the supernatant of Enterococcus faecium EB9 was carried out as in Example 1, but using the supernatant of Enterococcus faecium EB9 as the main component in a volume of 45% of the total mass and prepared deionized water - 46% of the total mass.

[0150] The pH was controlled and found to be 5.5. The finished product was dispensed into spray bottles.

[0151] Example 3.

[0152] The production of a liquid cosmetic skin spray based on Enterococcus faecium EB9 supernatant was carried out as in Example 1, but using Enterococcus faecium EB9 supernatant as the main component at a volume of 91% of the total weight. The pH was controlled and found to be 5.5 units. The finished product was poured into spray bottles.

[0153] Example 4.

[0154] Production technology of skin cream based on Enterococcus faecium EB9 supernatant

[0155] The following ingredients were prepared for Enterococcus faecium EB9 supernatant skin cream:

[0156] Enterococcus faecium EB9 supernatant, constituting 5% of the total mass of the cream with a quantitative content of vitamins as in Example 1.

[0157] Fillers:

[0158] Deionized water, which makes up 75.489% of the total mass of the cream.

[0159] - Hyaluronic acid (0.5%),

[0160] - Dissolved peptide Spec Ped® SC-AH 8 (0.1%),

[0161] - Aloe vera gel (2%),

[0162] - Immortelle hydrolate (10%),

[0163] - Jojoba oil (1%),

[0164] - Grape seed oil (1%),

[0165] - Tocobiol C (Tocobiol) (0.01%),

[0166] - Olive squalane (1%),

[0167] - Glycerin (1%)

[0168] - Preservative (0.5%),

[0169] - Carboxyvinyl polymer (1%),

[0170] - Gelling agent Aristoflex ®AVC (Aristoflex AVC) (0.5%).

[0171] - Emulsifier: EASYNOV® (Isinov) (1%).

[0172] The production of skin cream based on Enterococcus faecium EB9 lysate was carried out in stages:

[0173] Preparation of aqueous phase:

[0174] - Enterococcus faecium EB9 supernatant (5%), deionized water (75.489%), hyaluronic acid, immortelle hydrolate, Spec Ped® SC-AH 8 peptide, aloe vera gel, glycerin, preservative, carboxyvinyl polymer and Aristoflex ®AVC gelling agent were mixed.

[0175] - pH was controlled and found to be 5.5 units.

[0176] Preparation of oil phase:

[0177] - Jojoba oil, grapeseed oil, Tocobiol C, EASYNOV® emulsifier, and olive squalane were mixed. A preservative (0.5%) was added.

[0178] Phase mixing:

[0179] - At a temperature of +22 °C, the aqueous phase was mixed at 500 rpm, without foaming or whipping the mixture, using a paddle mixer.

[0180] - Mix the oil phase at 200 rpm, without foaming or whipping the mixture, using a paddle mixer.

[0181] - The aqueous and oil phases were combined and the mixture was homogenized at 1500 rpm.

[0182] - The resulting emulsion was stirred until the components were completely dissolved and the mixture was homogeneous for 60 minutes.

[0183] The finished cream was placed in 50g packages.

[0184] Example 5.

[0185] The production of skin cream based on Enterococcus faecium EB9 supernatant was carried out in stages as in Example 4, with the same components, but using the main active ingredient, Enterococcus faecium EB9 supernatant, in the composition of 40% of the total weight of the cream and deionized water in the composition of 40.489% of the total weight of the cream.

[0186] Example 6.

[0187] The production of skin cream based on Enterococcus faecium EB9 supernatant was carried out in stages as in Example 4, with the same components, but using the main active ingredient, Enterococcus faecium EB9 supernatant, in the composition of 80.489% of the total mass of the cream.

[0188] Example 7.

[0189] Production technology of skin serum based on Enterococcus faecium EB9 supernatant

[0190] The components of the future serum were prepared, in which the supernatant of Enterococcus faecium EB9 as the main component was 5% with the quantitative content of vitamins as in Example 1.

[0191] The ingredients were mixed at a temperature of +22 °C.

[0192] Stirring speed: 500 rpm

[0193] The ingredients were added sequentially. Enterococcus faecium EB9 supernatant (5% of the total weight), glycerin (2.5% of the total weight), Aristoflex ®AVC gelling agent (1% of the total weight), phenoxyethanol preservative, and ethylhexylglycerin (1% of the total weight) were added sequentially to prepared deionized water (90.5% of the total weight).

[0194] The mixture was stirred until the components were completely dissolved and homogeneous for 2 hours.

[0195] PH was monitored using professional equipment such as a pH meter. If the pH value fell outside the range of 4.5–5.5 pH units, pH calibration was performed using a filler selected from a group including lactic acid or an alkaline solution such as NaOH.

[0196] The finished product was poured into 30 ml bottles using a pipette.

[0197] Example 8.

[0198] The production of skin serum based on the supernatant of Enterococcus faecium EB9 was carried out as in Example 7, but using the supernatant of Enterococcus faecium EB9 as the main component in the amount of 45% of the total weight and prepared deionized water - 50.5% of the total weight.

[0199] The pH was controlled and found to be 5.5 units.

[0200] The finished product was poured into 30 ml bottles with a pipette.

[0201] Example 9.

[0202] The production of skin serum based on the supernatant of Enterococcus faecium EB9 was carried out as in Example 7, but using the supernatant of Enterococcus faecium EB9 as the main component in the amount of 95% of the total weight and prepared deionized water - 0.5% of the total weight.

[0203] The pH of the obtained serum was controlled and found to be 5.5 units.

[0204] The finished product was poured into 30 ml bottles with a pipette.

[0205] Example 10.

[0206] Production technology of liquid cosmetic product - skin spray based on Enterococcus faecium EB9 lysate

[0207] The components of the future cosmetic product were prepared in which Enterococcus faecium EB9 lysate as the main component accounted for 5%.

[0208] The quantitative composition of vitamins in the lysate was measured and amounted to, mass %:

[0209] vitamin A - 0.0085;

[0210] Vitamin B1 - 0.0000155;

[0211] Vitamin B2 - 0.000001889;

[0212] Vitamin B6 - 0.0000057;

[0213] Vitamin B9 - 0.00000755;

[0214] Vitamin B12 - 0.00000325

[0215] Vitamin C - 0.000558;

[0216] Vitamin D - 0.0000539;

[0217] Vitamin E - 0.0003209

[0218] The ingredients were mixed at a temperature of +22 °C.

[0219] - Mixing speed - 500 rpm, without foaming and whipping the mixture.

[0220] - The ingredients were added sequentially. Enterococcus faecium EB9 lysate (5% of the total weight), biosaccharide resin (3% of the total weight), green tea enzyme (2% of the total weight), hyaluronic acid (1% of the total weight), D-panthenol 75% (1% of the total weight), glycerin (1% of the total weight), phenoxyethanol preservative and ethylhexylglycerin (1% of the total weight) were added sequentially into the prepared deionized water (86% of the total weight).

[0221] The above ingredients were mixed until the mixture was completely dissolved and homogeneous for 1 hour.

[0222] The pH of the resulting spray was controlled and found to be 5.3 units.

[0223] The finished product was poured into spray bottles.

[0224] Example 11.

[0225] The production of a liquid cosmetic product, a skin spray based on Enterococcus faecium EB9 lysate, was carried out as in Example 10, but using Enterococcus faecium EB9 lysate as the main component in a volume of 45% of the total weight and prepared deionized water - 46% of the total weight.

[0226] The pH of the resulting liquid product was controlled and found to be 5 units.

[0227] The finished product was poured into spray bottles.

[0228] Example 12.

[0229] A liquid cosmetic skin spray was produced as in Example 10, but using Enterococcus faecium EB9 lysate as the main component in a volume that accounted for 91% of the total mass.

[0230] The pH of the resulting liquid product was controlled and found to be 4.7 units.

[0231] The finished product was poured into spray bottles.

[0232] Example 13.

[0233] Production technology of skin cream based on Enterococcus faecium EB9 lysate

[0234] Composition of skin cream based on Enterococcus faecium EB9 lysate:

[0235] The obtained Enterococcus faecium EB9 lysate, with the quantitative content of vitamins as in Example 10, was used in the composition of 5% of the total mass of the cream.

[0236] The following were used as fillers:

[0237] Deionized water, which makes up 75.489% of the total mass of the cream.

[0238] - Hyaluronic acid (0.5%),

[0239] - Dissolved peptide Spec Ped® SC-AH 8 (0.1%),

[0240] - Aloe vera gel (2%),

[0241] - Immortelle hydrolate (10%),

[0242] - Jojoba oil (1%),

[0243] - Grape seed oil (1%),

[0244] - Tocobiol C (Tocobiol) (0.01%),

[0245] - Olive squalane (1%),

[0246] - Glycerin (1%)

[0247] - Preservative (0.5%),

[0248] - Carboxyvinyl polymer (1%),

[0249] - Gelling agent Aristoflex ®AVC (Aristoflex AVC) (0.5%).

[0250] - Emulsifier: EASYNOV® (Isinov) (1%).

[0251] The technology for producing skin cream based on Enterococcus faecium EB9 lysate was carried out in stages:

[0252] Preparation of aqueous phase:

[0253] - Enterococcus faecium EB9 lysate (5%), deionized water (75.489%), hyaluronic acid, immortelle hydrolate, Spec Ped® SC-AH 8 peptide, aloe vera gel, glycerin, preservative, carboxyvinyl polymer and Aristoflex ®AVC gelling agent were mixed.

[0254] Preparation of oil phase:

[0255] - Jojoba oil, grapeseed oil, Tocobiol C, EASYNOV® emulsifier, and olive squalane were mixed. A preservative (0.5%) was added.

[0256] Phase mixing:

[0257] - At a temperature of +22 °C, the aqueous phase was mixed at 500 rpm, without foaming or whipping the mixture, using a paddle mixer.

[0258] - Mix the oil phase at 200 rpm, without foaming or whipping the mixture, using a paddle mixer.

[0259] - The aqueous and oil phases were combined and the mixture was homogenized at 1500 rpm.

[0260] - The resulting emulsion was stirred until the components were completely dissolved and the mixture was homogeneous for 60 minutes.

[0261] The pH of the resulting skin cream was controlled and found to be 5 units.

[0262] - Place the finished cream in 50g containers.

[0263] Example 14.

[0264] A skin cream was produced as in Example 13, with the same quantitative content of vitamins as in Example 10, but using Enterococcus faecium EB9 lysate as the main component in a volume that accounted for 40% of the total mass and deionized water that accounted for 40.489% of the total mass of the cream.

[0265] The pH of the resulting skin cream was controlled and found to be 5.2 units.

[0266] - The finished cream was placed in 50g packages.

[0267] Example 15.

[0268] The production of skin cream was carried out as in Example 13, with the quantitative content of vitamins as in Example 10, but using Enterococcus faecium EB9 lysate as the main component in a volume that was 80.489% of the total weight of the cream.

[0269] The pH of the resulting liquid product was controlled and found to be 5.4 units.

[0270] - The finished cream was placed in 50g packages.

[0271] Example 16.

[0272] Production technology of skin serum based on Enterococcus faecium EB9 lysate

[0273] The components for the serum were prepared in which the obtained Enterococcus faecium EB9 lysate as the main component was 5%, the quantitative content of vitamins was the same as in Example 10.

[0274] The ingredients were mixed at a temperature of +22 °C.

[0275] Stirring speed: 500 rpm

[0276] The ingredients were added sequentially. Enterococcus faecium EB9 lysate (5% of the total weight) with vitamins, glycerin (2.5% of the total weight), Aristoflex ®AVC gelling agent (1% of the total weight), phenoxyethanol preservative, and ethylhexylglycerin (1% of the total weight) were added sequentially to prepared deionized water (90.5% of the total weight).

[0277] Stir until the components are completely dissolved and the mixture is homogeneous for 2 hours.

[0278] PH was monitored using professional equipment such as a pH meter. If the pH value fell outside the range of 4.5–5.5 pH units, pH calibration was performed using a filler selected from a group including lactic acid or an alkaline solution, such as NaOH. The finished product was dispensed into 30 ml bottles using a pipette.

[0279] Example 17.

[0280] The production of skin serum was carried out as in Example 16, but using the lysate of Enterococcus faecium EB9 as the main component in a volume that was 45% of the total mass, the quantitative content of vitamins as in Example 10, and prepared deionized water - 50.5% of the total mass.

[0281] The pH of the obtained serum was controlled and found to be 4.5 units.

[0282] - The finished product was poured into 30 ml bottles.

[0283] Example 18.

[0284] The production of skin serum was carried out as in Example 16, but using the lysate of Enterococcus faecium EB9 as the main component in a volume that accounted for 95% of the total mass, the quantitative content of vitamins as in Example 10, and prepared deionized water - 0.5% of the total mass.

[0285] The pH of the obtained serum was controlled and found to be 5.3 units.

[0286] - The finished product was poured into 30 ml bottles.

[0287] The above cosmetic products were tested at the DJ Clinic Medical Cosmetology Center (OOO Estetika), where their effectiveness was assessed.

[0288] Two groups of volunteers were formed to conduct the study. The first group received a product (Product 1) based on the lysate of the Enterococcus faecium EB9 bacterium, in the form of

[0289] Serum, cream and liquid cosmetic product, skin spray.

[0290] The second group received cosmetic products (Product 2) based on the supernatant of the bacteria Enterococcus faecium EB9, also in the form of serum, cream and liquid cosmetic product skin spray.

[0291] At the beginning of the study, volunteers reported having various skin problems, including rosacea, freckles, age spots, wrinkles, abnormalities (dryness, tightness or oiliness), dark circles and puffiness under the eyes.

[0292] To objectively evaluate the effectiveness of cosmetic products, the following indicators were established:

[0293] i. pore size;

[0294] ii. pigmentation level;

[0295] iii. pore condition (under ultraviolet (UV) light);

[0296] iv. depth and width of wrinkles;

[0297] v. skin sensitivity.

[0298] These parameters were measured before and 4 weeks after the start of using cosmetic products using a dermatoscope.

[0299] Using cosmetic compositions based on the lysate resulted in increased skin hydration and a reduction in irritation after cleansing, consistent with high moisturizing effectiveness. Complaints of skin tightness after cleansing were eliminated by 66%. The use of the tested products also impacted skin elasticity, which increased by an average of 34% according to dermatoscopy data for the wrinkle severity parameter. Skin pigmentation analysis revealed a 12% reduction in pigmentation. Pore condition and size improved by 28%. The incidence of breakouts and acne decreased by 72%. Skin sensitivity decreased by 84%.

[0300] The use of cosmetic compositions based on the supernatant also demonstrated high effectiveness. An increase in skin hydration and a lack of irritation after cleansing were observed, consistent with high moisturizing efficiency. Complaints of skin tightness after washing were eliminated by 68%. The use of the tested products had a positive effect on skin elasticity, which increased by an average of 36% according to dermatoscopy data for the "wrinkle severity" parameter. Skin pigmentation analysis revealed a 14% reduction in pigmentation levels. Pore condition and size improved by 30%. The appearance of breakouts and acne decreased by 75%. Skin sensitivity decreased by 86%.

[0301] The study results were confirmed by photographic documentation, including skin images before and after use of Products 1 and 2, in accordance with established standards and the Methodology. Observation of volunteers for two weeks after the trial showed that 80% achieved optimal cosmetic results, while 20% experienced improvement requiring additional use. Improved skin elasticity was maintained in 100% of volunteers. Skin hydration levels returned to baseline in 65% of volunteers without using the products, and pigmentation levels achieved during the trial remained unchanged in 100%.

[0302] In the following study, the authors conducted a comparative analysis of the biochemical composition using high-performance liquid chromatography (HPLC) to examine the vitamin content of Enterococcus faecium EB9 bacterial lysate (Fig. 1), Enterococcus faecium EB9 bacterial supernatant (Fig. 2), and Microecobeauty ME-3 bifidobacteria lysate (Fig. 3) produced by Bloomage Biotech and widely used in popular cosmetic products.

[0303] The results of the analysis showed that Enterococcus faecium EB9 bacterial lysate and Enterococcus faecium EB9 supernatant exhibit higher vitamin content compared to Microecobeauty ME-3 bifidobacteria lysate produced by Bloomage Biotech. These results indicate the more pronounced vitamin-forming properties of microbial biocomponents obtained from Enterococcus faecium EB9 bacteria compared to bifidobacteria lysate, which is widely used in the cosmetics industry. The authors' study aimed to comparatively analyze the biological activity and vitamin composition of Enterococcus faecium EB9 lysate, Enterococcus faecium EB9 supernatant, and Microecobeauty ME-3 bifidobacteria lysate, all used in cosmetics.

[0304] The results obtained showed that Enterococcus faecium EB9 lysate and Enterococcus faecium EB9 supernatant have a higher vitamin content compared to Microecobeauty ME-3 bifidobacteria lysate, which is widely used in the cosmetics industry. These data indicate the potential for using Enterococcus faecium EB9 lysate and supernatant as biologically active components in cosmetics.

[0305] The cosmetic products given in the above examples, when tested on volunteers at the DJ Clinic Medical Cosmetology Center, showed the following results.

[0306] The first group, consisting of 7 volunteers, was given products in the form of serum, cream and liquid cosmetic product - skin spray based on the supernatant of Enterococcus faecium EB9 bacteria, in which the presence of vitamins A, B1, B2, B6, B9, B12, C, D, E was detected. The first and second participants received products with a supernatant concentration of 5% (Example 1, Example 4, Example 7), while the third and fourth participants used products with a supernatant concentration of 40% and 45% (Example 2, Example 5, Example 8,) the fifth, sixth and seventh volunteers used products with a supernatant concentration of 80% and 95% (Example 3, Example 6 (skin cream supernatant content 80%), Example 9 (skin serum supernatant content 95%)).

[0307] The second group of volunteers, which included 6 people, used cosmetics based on the lysate of Enterococcus faecium EB9 bacteria with vitamins A, B1, B2, B6, B9, B12, C, D, E and lactic acid, in the form of serum, cream and liquid cosmetic product skin mist. The first and second participants received products with a lysate concentration of 5% (Example 10, Example 13, Example 16), while the third and fourth participants used products with a lysate concentration of 40% and 45% (Example 11, Example 14, Example 17) the fifth and sixth volunteers used products with a lysate concentration of 80.489% and 91% (Example 15), Example 12, Example 18 (skin serum lysate content 95%).

[0308] Based on volunteer trials using products with varying concentrations of the active ingredients of Enterococcus faecium EB9 supernatant and lysate, it was found that the products had comparable efficacy. The above studies demonstrate that the use of Enterococcus faecium EB9 probiotic lysate improves skin condition, balances the microbiome, supports and accelerates natural skin regeneration processes, protects the skin from stress and aggressive environmental factors, and provides an anti-inflammatory effect to reduce skin inflammation, redness, and irritation caused by various factors.

[0309] List of information sources.

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Claims

1. Use of lysate or supernatant of the Enterococcus faecium EB9 bacterial strain for the treatment and prevention of dermatological conditions and diseases.

2. A composition containing an effective amount of a lysate or supernatant of the Enterococcus faecium EB9 bacterial strain, vitamins and dermatologically acceptable excipients.

3. The composition according to claim 2, characterized in that it contains, wt.%: lysate or supernatant of the bacterial strain Enterococcus faecium EB9 5 - 95 vitamin A 0,00515 - 0,0915 Vitamin B1 0,00000093 - 0,0000183 Vitamin B2 0,0000001463 - 0,000002489 Vitamin B6 0,00000014 - 0,0000084 Vitamin B9 0,000000435 - 0,00000855 Vitamin B12 0,0000001012 - 0,000004022 Vitamin C 0,0000201 - 0,000808 Vitamin D 0,000005258 - 0,00009228 Vitamin E 0,00001205 - 0,0004209 at least one of the dermatologically acceptable fillers rest 4. The composition according to claim 3, characterized in that the dermatologically acceptable fillers are selected from hyaluronic acids, or natural oils, or hydrolates, or mixtures thereof.

5. The composition according to claim 3, characterized in that it is used to restore, improve or regenerate skin in dermatological conditions such as dry skin, transepidermal water loss, diaper rash, inflammatory skin diseases, dermatitis.

6. The composition according to paragraph 3, characterized in that it is intended for use in the form of a liquid, or serum or cream.

7. A method for producing a composition according to claim 2, wherein the lysis of the enterococcal bacteria Enterococcus faecium EB9 is carried out to obtain a lysate or supernatant, and the lysate or supernatant of the strain of the enterococcal bacteria Enterococcus faecium EB9 is mixed with at least one of the dermatologically acceptable excipients, wherein the amount of components during mixing is, in wt.%: lysate or supernatant of the bacterial strain Enterococcus faecium EB9 5 - 95 vitamin A 0,00515 - 0,0915 Vitamin B1 0,00000093 - 0,0000183 Vitamin B2 0,0000001463 - 0,000002489 Vitamin B6 0,00000014 - 0,0000084 Vitamin B9 0,000000435 - 0,00000855 Vitamin B12 0,0000001012 - 0,000004022 Vitamin C 0,0000201 - 0,000808 Vitamin D 0,000005258 - 0,00009228 Vitamin E 0,00001205 - 0,0004209 at least one of the dermatologically acceptable fillers rest 8. A method for producing a composition according to paragraph 7, characterized in that mixing is carried out at a temperature of the medium of +10° - +25°C.

9. A method for producing a composition according to paragraph 7, characterized in that the mixing speed is no more than 500 rpm, and mixing is carried out without foaming and whipping the mixture.

10. A method for obtaining a composition according to paragraph 7, characterized in that stirring is carried out until complete dissolution and obtaining a homogeneous mixture within 1-2 hours.

11. A method for obtaining a composition according to paragraph 7, characterized in that the pH of the obtained composition is controlled and calibrated to a value of 4.5 - 5.5 units.

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