Probiotic-based compositions and uses thereof

Abstract

To provide a composition comprising probiotics, preferably based on bacteria, in particular of the genus Lactobacillus, to be used for the purpose of preventing and / or treating the disorders affecting the skin, in particular inflammatory disorders, in particular autoinflammatory disorders of dermatological interest such as atopic dermatitis, which is useful for preventing and / or reducing the damage induced by UV radiation, in particular skin aging.SOLUTION: Disclosed is a composition containing probiotics for use in the treatment and / or prevention of disorders and / or pathologies affecting the skin, preferably of an inflammatory type, and / or for use in mitigation and / or treatment of the symptoms related to the disorder / pathologies and / or influence, and / or for use in the promotion of the wound healing process and / or re-epithelialization and / or cicatrization processes, where the probiotics is bacterial cells of the strain Lactobacillus paracasei DG (R) CNCM 1-1572 and bacterial cells of the strain Lactobacillus paracasei LPC-S01 DSM26760.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compositions comprising probiotics, preferably bacteria, particularly Lactobacillus bacteria, used to prevent and / or treat skin disorders, especially inflammatory disorders, and especially autoinflammatory disorders of dermatological interest such as atopic dermatitis. Furthermore, the compositions of the present invention are useful in preventing and / or reducing UV radiation-induced damage, particularly skin aging. [Background technology]

[0002] Human skin is colonized by a rich microbiome. While this aspect was previously thought to represent a potential source of infection, today there is a general recognition that the human microbiome, and therefore the skin microbiome, plays important and useful functions in the host, not only because of its ability to resist the adhesion and development of skin pathogens, but also because of its ability to communicate and interact with the immune system.

[0003] When enterotoxemia occurs at the skin level, probiotics can act as modulators, restoring balance to the skin microbiome.

[0004] Over the past decade, the use of new technologies has facilitated the taxonomic analysis of the skin microbiome. The bacterial population of the skin microbiome can comprise approximately 1010 species of microorganisms belonging to over 25 phyla, the most numerous of which are Actinobacteria, Firmicutes, and Proteobacteria. Various skin diseases are linked to changes in the skin microbiome.

[0005] For example, in the case of acne, P. acnes is considered to be the main bacterium associated with it, and in these conditions, increased sebum production actually provides the optimal environment for its proliferation.

[0006] The usual approach to these problems is to use antibacterial agents, namely topical disinfectants and antibiotics.

[0007] On the one hand, antibiotics are undoubtedly effective, but they also eliminate beneficial bacteria, which carry the risk of sensitization and potential adverse events, especially with the long-term use of broad-spectrum antibiotics. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] US2005 / 158291A1 [Patent Document 2] US2013 / 095086A1 [Patent Document 3] US2010 / 226892A1 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] For this reason, the applicant has identified the use of probiotic compositions, particularly those based on bacteria belonging to the Lactobacillus genus, as a novel solution for restoring balance to the skin microbiome. In fact, the applicant has found that compositions containing probiotics belonging to the Lactobacillus genus have the ability to prevent, or at least mitigate, the effects of skin-dangerous bacteria such as P. acnes, and therefore the following is possible: 1) To contribute to the prevention and / or treatment of skin disorders; 2) To promote the normal healing process; and 3) To promote and / or improve the normal re-epithelialization and / or scar formation process. [Means for solving the problem]

[0010] One aspect of the present invention relates to a composition comprising probiotics, preferably one or more probiotic bacteria, for use in the treatment and / or prevention of dermatological, skin-affecting, preferably inflammatory, disorders and / or pathological conditions.

[0011] The present invention is described in detail below and illustrated with examples, including the attached figures. [Brief explanation of the drawing]

[0012] [Figure 1]The following graphs illustrate the adhesion of P. acnes in terms of the percentage of living, adhering cells: ·1A: Preliminary stimulation of keratinocytes by contact with the probiotic under test; ·1B: Co-incubation of keratinocytes with P. acnes and the probiotic under test; ·1C: Incubation of keratinocytes with the probiotic under test after eukaryotic cells have been loaded with the pathogen. The inhibitory ability of the tested strains is expressed as a percentage reduction in P. acnes adhesion compared to the positive control, with 100 representing P. acnes adhesion in the absence of probiotic stimulation. In particular, Figure 1A shows the ability of both probiotic strains to prevent P. acnes adhesion by similar percentages (42% for L. casei DG® and 35% for L. paracasei LPC-S01). Figure 1B shows that L. casei DG® strain reduced adhesion by 17%, while L. paracasei LPC-S01 strain reduced it by 9%. Mixing these strains resulted in a statistically significant reduction in P. acnes adhesion, a reduction of 42%, which is a significantly higher percentage than both the effects observed for the strains considered individually and the sum of their effects. Figure 1C shows a statistically significant synergistic effect related to the mixture of the two probiotics, demonstrating that the mixture has the ability to reduce P. acnes adhesion by 42%. In contrast, the individual probiotics showed an ability to reduce adhesion that was less than half the effect of the mixture, at 18% for L. casei DG and 11% for L. paracasei LPC-S01. [Figure 2] This graph shows the assay results for cytokines IL-10, IL-1 beta, IL-8, and TSLP in cell supernatant, expressed in picograms (pg) per ml of supernatant, obtained using ELISA. [Modes for carrying out the invention]

[0013] The disorder and / or condition affecting the skin is preferably dermatitis, acne, an infectious disease, skin inflammation, erythema, ulcer, psoriasis, atopic dermatitis, otitis, fissure, fistula, hemorrhoid, and any other skin disorder or lesion, with or without an ongoing inflammatory process, associated with irritation and / or excoriation, and / or a combination and / or complication and / or outcome thereof.

[0014] The disorder and / or condition affecting the skin is preferably associated with or caused by a pathogen. That is, the composition is not only for prevention / premonition purposes but also for treatment purposes, i.e., it is particularly effective even after the load with the pathogen.

[0015] According to a preferred embodiment of the present invention, the composition can also assist / promote / facilitate / accelerate the wound healing process and / or the re-epithelialization and / or scar formation process.

[0016] The pathogen is preferably selected from bacteria, fungi, yeasts, viruses, and combinations thereof.

[0017] The pathogen is preferably selected from bacteria, and the bacterial pathogen is preferably selected from the genus Propionibacterium, preferably the species acnes, the genus Staphylococcus, preferably epidermidis, aureus, warneri, pyogenes, Corynebacterium ssp of the mitis type; the genus Pseudomonas, preferably aeruginosa; the genus Acinetobacter, preferably johnsonii; the genus Streptococcus, preferably pyogenes; Micrococcus ssp., and Brevibacterium ssp.

[0018] Furthermore, a composition as described in detail below, which comprises probiotics, preferably one or more probiotic bacteria, is adapted to shield the skin and / or lips and / or conjunctiva from UV irradiation, UV-A and / or UV-B. In other words, the composition is adapted to prevent and / or reduce damage and / or effects resulting from / related to exposure to UV irradiation, UV-A and / or UV-B. The composition is preferably adapted to prevent and / or reduce skin aging.

[0019] From this perspective, a composition as described in detail below, which comprises probiotics, preferably one or more probiotic bacteria, has cosmetic purposes. The use of the composition preferably relates to / results from the immunomodulatory action of the probiotics contained in the composition, preferably the probiotics as defined and described below. The immunomodulation involves the regulation, preferably the reduction, of the expression of at least one cytokine selected from hematopoietic cytokines, preferably hematopoietic growth factors and / or CSF; primary inflammatory cytokines, preferably IL-1 and / or TNF; anti-inflammatory and / or immunosuppressive cytokines, preferably IL-10 and / or TGF-β; secondary inflammatory cytokines (chemokines); cytokines that control specific immune responses, preferably IL-2, and combinations thereof. The immunomodulation preferably comprises the regulation, preferably the reduction, of the expression of at least one cytokine selected from IL-1β, IL-10, IL-8, TSLP, and combinations thereof.

[0020] In this context, it should be noted that cytokines are antigen-nonspecific polypeptide mediators that act as information exchange signals between immune system cells and between immune system cells and various organs and tissues. Produced by different types of cells and released in the body, they induce specific responses in adjacent cells (paracrine effect), very distant cells (endocrine effect), or in the cells that produced them (autocrine effect). In particular, cytokines produced by immune system cells, such as interleukins and chemokines, play a fundamental role in regulating and activating the defense mechanisms of the inventors, as well as in inflammatory processes. The complex network of cytokines maintains a balance between pro-inflammatory and anti-inflammatory effects. An imbalance between pro-inflammatory and anti-inflammatory cytokines, coupled with uncontrolled cytokine production, can result in inflammatory diseases, allergies, or autoimmune conditions. TSLP (thymic-interstitial lymphocyte neoplastic factor) is a protein belonging to the cytokine family that plays a crucial role in the maturation of T cell populations through the activation of antigen-representing cells. TSLP is primarily produced by non-hematopoietic cells such as fibroblasts, epithelial cells, and various types of stromal cells, and its expression is associated with many pathological conditions, including asthma, inflammatory arthritis, atopic dermatitis, eczema, eosinophilic esophagitis, and other allergic conditions.

[0021] In particular, the applicant demonstrates that when keratinocytes are exposed to probiotics, the probiotics have the ability to exert immunomodulatory effects, as evidenced by the evaluation of cytokine assays on the cell supernatant. Specifically, the expression of IL1β, IL10, and IL8 was observed to be reduced by probiotic strains considered individually, particularly by L. paracasei LPC-S01. The effect of cytokine expression appeared to be more evident after loading keratinocytes with LPS.

[0022] Regarding TSLP, contact with probiotics determined a reduction in this index compared to baseline expression (in keratinocytes not stimulated by probiotics). Pre-stimulation of keratinocytes with LPS did not result in an increase in TSLP levels, which was a predictable phenomenon, as other molecules are known to be more stimulating to the Toll-like receptor of LPS. Probiotic strains, considered individually and in mixtures, were shown to be effective in modulating the expression of this index. This index is of considerable interest because it is overexpressed in several skin conditions, such as atopic dermatitis. Furthermore, this cytokine is considered an important mediator at the functional interface between keratinocytes and dendritic cells.

[0023] According to a preferred embodiment of the present invention, the composition is useful in the process of wound healing and / or in the process of re-epithelialization and / or scar formation of injured skin and / or skin affected by injury.

[0024] Therefore, the data clearly demonstrate that probiotics, particularly the strains tested, have the ability to exert anti-inflammatory and / or immunomodulatory effects on keratinocytes and, consequently, on the skin.

[0025] Generally speaking, given the effects described herein, the probiotic-based compositions described herein are useful for restoring balance to the skin microbiome.

[0026] In relation to the present invention, "skin" means the front line of defense against the external environment; in particular, this defense is carried out through the action of keratinocytes scattered in the outermost layer of skin (epidermis), where keratinocytes induce the secretion of cytokines and chemokines to deliver warning messages to the deeper layers of the skin, and thus can generate an inflammatory response. In the course of their development, keratinocytes move from deeper to more superficial layers, progressively depositing keratin, which plays a protective role for the underlying cells.

[0027] In the context of this invention, "skin aging" means a completely natural and unavoidable physiological process that occurs in all individuals. Over time, the skin undergoes structural changes caused by a series of factors of different origins, which result in loss of skin moisture, the appearance of fine wrinkles, loss of elasticity, hyperkeratosis, and the formation of hyperpigmented spots known as "age spots."

[0028] The aging described above is preferably intrinsic—or temporal—aging, which is substantially dependent on genetic (or intrinsic) factors. Alternatively, the skin aging is caused by extrinsic—or environmental—factors, i.e., external factors.

[0029] Generally speaking, intrinsic aging typically begins around age 25. It usually involves a series of changes that generally result in thinning and / or yielding of the skin structure.

[0030] Exogenous aging is preferably caused by the invasion of external agents and / or environmental factors, preferably selected from UV irradiation (which causes photoaging), smoking, alcoholism, pollution, continuous exposure to irritants, and combinations thereof.

[0031] Damage and / or effects resulting from / related to exposure to UV irradiation are preferably selected from erythema, hyperpigmentation, keratosis, hyperkeratosis, skin redness, sunburn, burns, photoaging or photoelastic fibrosis, cortical cataracts, pterygium, reactivation of oral herpes, skin damage of any nature, preferably damage to the lips and / or conjunctiva, cutaneous melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, squamous cell carcinoma of the cornea or conjunctiva, and / or combinations thereof and / or complications and / or outcomes.

[0032] In relation to the present invention, "restoring balance to the skin microbiome" means restoring the qualitative and quantitative physiological composition of the skin microbiome, understood as the set of microorganisms present on the skin as a whole, and therefore restoring the physiological skin microbial ecology.

[0033] In connection with the present invention, “loading” means any experimental test, loading, or trial that includes contamination with different species of microorganisms and subsequent evaluation of changes in microbial load, usually by means of plate counting the number of living microorganisms at regular time intervals.

[0034] In relation to this invention, "probiotics," as defined by the FAO and WHO, means "living microorganisms that, when administered in sufficient quantities, provide health benefits to the host." In other words, probiotics are microorganisms that demonstrate the ability to exert beneficial functions in the body when taken in appropriate amounts.

[0035] The microorganisms are preferably selected from bacteria, fungi, yeasts, and combinations thereof.

[0036] According to a preferred embodiment of the present invention, the bacteria belong to at least one genus selected from Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Aerococcus, and Enterococcus. More preferably, the bacteria belong to the genus Lactobacillus.

[0037] According to a further preferred embodiment of the present invention, bacteria of the genus Lactobacillus include Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus Alimentarius, Lactobacillus aviaries, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, and Lactobacillus cerobiosus. Lactobacillus cellobiosus, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus johnsonii Lactobacillus johnsonii), Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus mucosaeLactobacillus mucosae), Lactobacillus panis, Lactobacillus collinoides, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfrancisensis (Lactobacillus It belongs to at least one species selected from *sanfranciscensis*, and combinations thereof.

[0038] More preferably, the genus Lactobacillus is Lactobacillus paracasei, preferably Lactobacillus paracasei strain DG(registered trademark)CNCM1-1572 and / or Lactobacillus paracasei strain LPC-S01.

[0039] Both strains were isolated and deposited by SOFAR SpA, and in particular, the Lactobacillus paracasei DG bacterial strain was deposited on May 5, 1995, with deposit number CNCM1-1572, at the National Collection of Cultures of Microorganisms (CNCM) of the Pasteur Institute in Paris. The strain was initially called Lactobacillus casei DG subspecies casei.

[0040] The Lactobacillus paracasei LPC-S01 bacterial strain was deposited with DSMZ under accession number DSM26760.

[0041] The aforementioned strains are particularly effective for the uses described herein when used jointly and / or in combination, and in fact, they exhibit additive and / or further synergistic effects.

[0042] According to a further preferred embodiment of the present invention, bacteria of the genus Bifidobacterium include B. animalis, B. bifidum, B. breve, B. infantis, B. longum, B. adolescentis, B. catenulatum, B. angulatum, B. asteroides, B. bourn, B. choerinum, B. coryneforme, B. cuniculi, B. denticolens, B. dentium, B. gallicum (B. B. gallicum), B. gallinarum, B. indicum, B. inopinatum, B. lactis, B. magnum, B. merycicum, B. minimum, B. pseudocatenulatum, B. pseudolongum, B. pullorum, B. ruminantium, B. saeculare, B. subtile, B. thermacidophilum, B. thermophilum, and B. turmience (B.It belongs to at least one species selected from Bacillus tsurumiense, and more preferably from Bacillus clausii, Bacillus subtilis, Bacillus coagulans, Bacillus megaterium, Bacillus halodurans, Bacillus thuringiensis, Bacillus insolitus, and Bacillus marinus.

[0043] According to a further preferred embodiment of the present invention, the bacteria of the genus Propionibacterium belong to at least one species selected from P. shermanii, P. acnes, P. australiense, P. avidum, P. cyclohexanicum, P. freudenreichii, P. granulosum, P. jensenii, P. microaerophilum, P. propionicum, and P. thoenii.

[0044] According to a further preferred embodiment of the present invention, bacteria of the genus Streptococcus include Streptococcus thermophilus, Streptococcus salivarius, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus bovis, Streptococcus canis, Streptococcus constellatus, Streptococcus downei, Streptococcus dysgalactiae, and Streptococcus equinus. Streptococcus equinus), Streptococcus ferus, Streptococcus infantarius, Streptococcus iniae, Streptococcus intermedius, Streptococcus milleri, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus orisratti, Streptococcus parasanguinis, Streptococcus perolis (Streptococcus peroris), Streptococcus pneumoniae, Streptococcus pseudopneumoniaeIt belongs to at least one species selected from the following: pseudopneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus tigurinus, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus suis, Streptococcus uberis, Streptococcus vestibularis, Streptococcus viridans, and Streptococcus zooepidemicus.

[0045] According to a further preferred embodiment of the present invention, the bacteria of the genus Lactococcus belong to at least one species selected from L. chungangensis, L. formosensis, L. fujiensis, L. garvieae, L. lactis, L. piscium, L. plantarum, L. raffinolactis, and L. taiwanensis.

[0046] According to a further preferred embodiment of the present invention, the bacteria of the genus Aerococcus belong to at least one species selected from A. urinae, A. sanguinicola, A. christensenii, A. suis, A. urinaeequi, and A. urinaehominis.

[0047] According to a further preferred embodiment of the present invention, bacteria of the genus Enterococcus include Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus haemoperoxidus, Enterococcus hirae, Enterococcus malodoratus, Enterococcus moraviensis, Enterococcus mundtii, and Enterococcus pseudoavium. It belongs to at least one species selected from Enterococcus pseudoavium, Enterococcus raffinosus, and Enterococcus solitarius.

[0048] In a further preferred embodiment of the present invention, the yeast belongs to the genus Saccharomyces, more preferably to the species Saccharomyces cerevisiae and / or Saccharomyces boulardii.

[0049] Microorganisms, preferably L. casei DG® and / or Lactobacillus paracasei LPC-S01 bacterial strains, are used preferably in a living state, i.e., they are used as probiotics.

[0050] Alternatively, microorganisms, preferably L. casei DG® and / or Lactobacillus paracasei LPC-S01 bacterial strains, are dead or tyndallized.

[0051] In further embodiments, microorganisms, preferably L. casei DG® and / or Lactobacillus paracasei LPC-S01 bacterial strains, are used in the form of lysates and / or extracts, i.e., they are used as paraprobiotics. Alternatively, microorganisms, preferably L. casei DG® and / or Lactobacillus paracasei LPC-S01 bacterial strains, are used in the form of compounds containing immunogenic components selected from bacterial products, supernatants, derivatives, preferably bacterial derivatives, preferably their metabolites, metabolic bioproducts, postbiotics, cell walls and components, exopolysaccharides, or preferably ribosomes and glycoproteins, glucans and other polysaccharides, lipopolysaccharides, and any components of the supernatant, as immunostimulant derivatives.

[0052] Generally, the microorganisms mentioned above are individual microorganisms, combinations of microorganisms, or consortia of any microbial species listed in the EFSA QPS list.

[0053] In any case, the compositions of the present invention may include any type of microorganism having a probiotic effect / action / function, particularly bacteria, and / or microorganisms as defined above, preferably bacteria, that has the ability to stably colonize on the skin, intestines and / or other areas of the body, to displace pathogenic microorganisms / bacteria, and / or to directly fight them. The cosmetic compositions and / or medical compositions as described above preferably include combinations of the strains identified above with other microorganisms selected from, as described above, preferably bacteria, fungi, yeasts and combinations thereof.

[0054] Microorganisms, preferably bacteria, are present in a minimum amount sufficient to allow temporary colonization of the skin, intestines, and / or other areas of the body. The amount is preferably 10 6 ~10 11 Between microbial units, more preferably 10 8 ~10 9 This is a range between microbial units, and the amount is preferably a daily amount / dose (daily dose).

[0055] The composition further comprises excipients and / or further pharmaceutically acceptable substances and / or carriers.

[0056] The compositions of the present invention preferably further comprise substances selected from plasma, PRP, scar-forming substances, re-epithelializing substances, humectants, moisturizers, emollients, adsorbents, analgesics and hemolytic agents, anti-inflammatory agents, muscle relaxants, peptide and / or proteinaceous substances and / or proteins, such as collagen, substances belonging to connective tissue, such as glycosaminoglycans, preferably chondroitin sulfate, and / or combinations thereof.

[0057] In one embodiment of the present invention, the composition is preferably formulated for topical application, preferably in the form of a cream, gel, oil, emulsion, spray, gauze, patch, bandage, lotion, mousse, ointment, paste, or liquid formulation which is preferably added immediately before application to the skin.

[0058] In one embodiment of the present invention, the composition is used for transplantation of the skin microbiome. In the context of the present invention, transplantation of the skin microbiome means the administration, preferably topically, of the skin microbiome and / or a portion thereof of a healthy subject without skin disorders to re-establish the correct balance and thereby treat any enterotoxemia that may be present. For these purposes, the compositions of the present invention preferably include bacteria, preferably L. casei DG® strain and / or Lactobacillus paracasei LPC-S01 strain, and optionally fungi, viruses, peptides, proteinaceous and / or peptidic substances, carbohydrates, vitamins, trace elements, or any other substances that characterize the microbiome of the skin and / or other areas of the body. For such purposes, the compositions are preferably used in combination with amino acids, supplements, vitamins, trace elements such as zinc and selenium, macronutrients and micronutrients, enzymes, and / or prebiotic substances such as fructooligosaccharides (FOS), galactooligosaccharides (GOS), inulin, guar gum, and / or combinations thereof. In one embodiment of the present invention, the composition is formulated for oral administration, preferably as a solid formulation, preferably as a pill, capsule, tablet, granule, hard-shell capsule, orally dissolvable granule, sachet, or lozenge.

[0059] Alternatively, the composition may be formulated as a liquid, preferably for immediate preparation.

[0060] Alternatively, the composition may be in a form capable of exerting a topical effect, such as an enema, cream, spray, gel, patch, lotion, mousse, ointment, paste, or liquid formulation which is preferably added and / or prepared immediately before application to the skin.

[0061] According to preferred embodiments of the present invention, the compositions of the present invention are used in combination with amino acids, supplements, vitamins, trace elements such as zinc and selenium, macronutrients and micronutrients, enzymes, and / or prebiotic substances such as fructooligosaccharides (FOS), galactooligosaccharides (GOS), inulin, guar gum, or combinations thereof. [Examples]

[0062] Bacterial strains and cells As an example, the present invention has been demonstrated using the following bacterial strains: - L. casei DG (registered trademark) (Lactobacillus paracasei CNCM1-1572) - L. paracasei LPC-S01 - A 1:1 mixture of L. casei DG (registered trademark) and L. paracasei LPC-S01.

[0063] Bacterial strains were cultured in a selective medium consisting of MRS (de Man Rogosa Sharpe) at 37°C for 16 hours. Once growth reached the quiescent phase, bacterial samples were collected and used for testing. These were washed several times with PBS (phosphate-buffered saline) and centrifuged to remove consumed medium. Living cells in the quiescent phase were used because bacteria induce a smaller immune response at this stage than at the quiescent phase.

[0064] All of the following tests were conducted in a series of three.

[0065] Normal human keratinocytes maintained in culture with suitable supplements were used in the experiment.

[0066] Propionibacterium acnes (P. acnes) CCUG 50865 (purchased from CCUG (Culture Collection, University of Goteborg)) was cultured in cooked meat medium (both liquid and solid) prepared according to the supplier's instructions at 37 °C for 16 hours. Subsequently, the bacterial concentration was determined by spectrophotometry at an OD reading of 600 nm and used for the adhesion test.

[0067] Adhesion test and pathogen load in cell lines Keratinocytes were seeded in the wells of cell culture plates at 5% CO2 and 37 °C and allowed to grow until they reached confluence (i.e., approximately 10 5 cells). The seeding rate was 2.5 × 10 5 cells / 0.32 cm2 (i.e., a cell density of 7.8 × 10 5 cells / cm2). At that point in the experiment, the medium was removed, the cells were washed with antibiotic-free medium, and then bacteria were added for the test, which was conducted according to the different protocols described below.

[0068] 1) Adhesion of strains to keratinocytes After determining the number of keratinocytes per well, strains of L. paracasei were added at an MOI of 1:10 (10 5 cells and 10 6 CFU of bacteria per well). The strains were tested individually and in combination at a 1:1 ratio.

[0069] The bacteria were kept in contact with the cells maintained at 一点点振盪 at 37 °C for 60 minutes. At the end of the incubation, the medium was removed, and the cells were washed with sterile medium to remove bacteria that did not adhere to the cell monolayer.

[0070] To calculate the number of adherent bacteria, the cells were detached, lysed, and the bacteria were seeded on agar MRS medium and then counted by viable staining

[0071] 2) Adhesion exclusion test It should be noted that there is an unclear expression "一点点振盪" in the original text which is translated as "一点点振盪" in the English translation for now. You may need to clarify this part for a more accurate translation.After determining the number of keratinocytes in each well, L. paracasei strains were added to the cells in a 1:1 ratio, both individually and in combination.

[0072] The bacteria were kept in contact with the cells, which were maintained at 37°C with gentle shaking, for 60 minutes. At the end of incubation, the culture medium was removed, and the cells were washed with sterile medium to remove any bacteria that had not adhered to the cell monolayer.

[0073] Subsequently, P. acnes (at a 1:10 MOI) was placed in contact with the cells and incubated at 37°C for 60 minutes. At the end of incubation, the culture medium was removed and non-adherent bacteria were eliminated by washing with sterile medium.

[0074] To count the number of P. acnes adhering to keratinocytes, cells were detached, lysed, and the bacteria were seeded in 5% sheep blood agar under anaerobic conditions at 37°C for 48–72 hours, followed by counting by supravital staining. Wells containing keratinocytes inoculated with P. acnes alone but not pre-incubated with L. paracasei strains were used as positive controls for P. acnes adhesion (P. acnes adhesion controls), while wells containing cells not incubated with any bacteria were used as negative controls (sterile controls).

[0075] 3) Testing for adhesive competition After determining the number of keratinocytes per well, strains of L. paracasei were added simultaneously with P. acnes (both individually and in a 1:1 ratio). The bacteria were added at an overall MOI of 1:10 and a probiotic:pathogen ratio of 1:1.

[0076] The bacteria were kept in contact with the cells for 60 minutes at 37°C with gentle shaking. At the end of incubation, the culture medium was removed, and the cells were washed with sterile medium to remove any bacteria that had not adhered to the cell monolayer.

[0077] To count the number of P. acnes adhering to keratinocytes, cells were detached, lysed, and the bacteria were seeded in 5% sheep blood agar under anaerobic conditions at 37°C for 48–72 hours, after which they were counted by supravital staining. Wells containing keratinocytes inoculated with P. acnes alone were used as positive controls (P. acnes adhering controls), while wells containing cells not incubated with any bacteria were used as negative controls (sterile controls).

[0078] 4) Testing for removal of adhered pathogens After determining the number of keratinocytes per well, P. acnes was added (MOI 1:10).

[0079] The bacteria were kept in contact with the cells, which were maintained at 37°C with gentle shaking, for 60 minutes. At the end of the incubation, the culture medium was removed, and any bacteria that did not adhere to the cell monolayer were removed by repeated washing with sterile medium.

[0080] Subsequently, L. paracasei strains (both individually and in 1:1 combinations) were kept in contact with cells maintained at 37°C with gentle shaking for 60 minutes. At the end of incubation, the medium was removed, and the cells were washed with sterile medium to remove any bacteria that had not adhered to the cell monolayer.

[0081] To count the number of P. acnes attached to keratinocytes, the cells were detached, lysed, and the bacteria were seeded in 5% sheep blood agar under anaerobic conditions at 37°C for 48-72 hours, after which they were counted by supravital staining.

[0082] Keratinocytes treated with P. acnes were used as a positive control (P. acnes adhesion control), while keratinocytes not treated with bacteria were used as a negative control (sterile control).

[0083] The tests, conducted both with individual strains in their pure form and with 1:1 mixtures, were repeated twice.

[0084] 1) Adhesion of the strain to keratinocytes - Results The results of this test demonstrated the ability of the strains, both individually and in combination, to adhere to keratinocytes. In particular, the L. casei DG® strain showed greater adhesion to keratinocytes than L. paracasei LPC-S01. Therefore, the adhesion ability observed for the combination is likely attributable to the contribution provided by the L. casei DG® strain.

[0085] [Table 1]

[0086] - Adhesion exclusion test (pretreatment of eukaryotic cells with probiotics and subsequent incubation with pathogens) - Results As described above, a monolayer of keratinocytes was first exposed to contact with probiotics, then, after removal of non-adherent bacteria, to contact with P. acnes, and subsequently the non-adherent P. acnes were removed by washing; after this, the residual viability of P. acnes was quantified.

[0087] Negative controls were represented by eukaryotic cells unstimulated by bacterial contact, while positive controls were represented by the ability of P. acnes to adhere to keratinocytes without any pretreatment with probiotics. Figure 1A shows the results regarding P. acnes adhesion in terms of the percentage of live, surviving cells adhering to keratinocytes after preliminary stimulation by contact with the different probiotics tested. The inhibitory ability of the tested strains was expressed as a percentage reduction in adhesion by P. acnes compared to the positive control, with 100 representing P. acnes adhesion in the absence of probiotic stimulation.

[0088] Both probiotic strains demonstrated the ability to prevent adhesion of P. acnes by similar percentages (42% for L. casei DG® and 35% for L. paracasei LPC-S01).

[0089] - Adhesion competition test (co-incubation of eukaryotic cells with probiotics and pathogens) - Results During these studies, when co-incubated with P. acnes, the reduction in P. acnes adhesion to keratinocytes (if any) was assessed after simultaneous treatment of cell lines with probiotics and pathogens, in order to evaluate the potential competitive inhibitory effect exerted by probiotics.

[0090] Keratinocytes treated with P. acnes were used as positive controls, while keratinocytes not treated with bacteria were used as negative controls (sterile controls).

[0091] Figure 1B graphically shows the results regarding P. acnes adhesion to keratinocytes in terms of the percentage of living, adhering cells after co-incubation with P. acnes and different probiotics tested. As previously described, the inhibitory ability of the tested strains was expressed as a percentage reduction in adhesion by P. acnes compared to a positive control, with 100 representing P. acnes adhesion in the absence of probiotic stimulation.

[0092] In this mode of interaction between probiotics and pathogens, L. casei DG® strain showed the ability to reduce adhesion by 17%, while L. paracasei LPC-S01 strain showed 9%. The combination of these strains produced a statistically significant reduction in P. acnes adhesion, showing 42%, which is clearly higher than that observed for the strains considered individually, and also higher than the combined effect of their respective strains.

[0093] - Tests on the removal of adhered pathogens (pretreatment of eukaryotic cells with pathogens, followed by incubation with probiotics) - Results During these tests, any reduction in P. acnes adhesion to keratinocytes was assessed after pretreatment of the cell line with pathogens and subsequent incubation with probiotics after removal of non-adherent pathogens.

[0094] Keratinocytes treated with P. acnes were used as positive controls, while keratinocytes not treated with bacteria were used as negative controls (sterile controls).

[0095] Figure 1C graphically shows the results regarding P. acnes adhesion in terms of the percentage of adhering live cells after incubation of keratinocytes with different probiotics tested following loading with the eukaryotic cell pathogen (P. acnes). As previously described, the inhibitory ability of the tested strains was expressed as a percentage reduction in adhesion by P. acnes compared to a positive control, with 100 representing P. acnes adhesion in the absence of the probiotic.

[0096] In these tests, as in the case of the co-incubation protocol, an interesting, statistically significant synergistic effect related to the combination of two probiotics was observed, showing that the combination had the ability to reduce P. acnes adhesion by 42%. In contrast, the individual probiotics showed the ability to reduce adhesion to less than half that of the mixture: 18% for L. casei DG® and 11% for L. paracasei LPC-S01.

[0097] Immunomodulatory testing Immunomodulatory studies were conducted to investigate whether probiotic strains have the ability to regulate cytokine release by human keratinocytes exposed to negative stimulation with bacterial lipopolysaccharide (LPS).

[0098] Normal human keratinocytes were seeded onto cell culture plates as described above, and once confluence was reached, they were used in the experiment. In particular, the cell monolayer was washed and incubated in fresh medium without antibiotics. Then, L. paracasei strains were added individually and in 1:1 ratio combinations at a MOI of 1:10.

[0099] The bacteria were kept in contact with keratinocytes for 120 minutes at 37°C with gentle shaking. In some cases, the cells were pre-stimulated with LPS at a final concentration of 100 ng / ml (loaded for approximately 24 hours).

[0100] At the end of incubation, the culture medium was removed, and the cells were washed with sterile medium to eliminate any bacteria that had not adhered to the cells. Subsequently, the cells were kept in culture medium at 37°C for 24 hours.

[0101] At the end of incubation, culture medium was collected using an ELISA assay to quantify the produced cytokines (IL-10, IL-1 beta, IL-8, and thymic stromal lymphocyte generating factor [TSLP]), while the levels of cyclooxygenase-2 (COX-2) and activated NF-κB (induced by LPS or IL-1 beta) were determined by Western blotting on total protein extracted from lysed keratinocytes.

[0102] These markers were selected because they are involved in both acute inflammation (phlogosis) and allergic dermatitis, as well as in chronic-associated inflammation (phlogosis). In particular, IL-10 is considered anti-inflammatory, and its production generally increases in parallel with the production of potent pro-inflammatory cytokines such as IL-1 beta. TSLP is a cytokine that modulates lymphocyte action and is involved in the development of chronic keratitis.

[0103] result The ability of the strains under test to stimulate the immune response in keratinocytes was evaluated according to the protocol described above. The focus of the study was on determining cytokines (IL-8, IL-1 beta, and IL-10) and evaluating the activation of two markers, COX-2 and NF-κB.

[0104] COX-2 (cyclooxygenase-2) represents an inducible marker produced by a limited number of cell types in response to specific inflammatory stimuli. It is overexpressed in several neoplasms, including cutaneous neoplasms. NF-κB (nuclear factor kappa light chain enhancer of activated B cells) is a transcription factor-functioning protein complex produced by all cell types in response to a variety of stimuli, including inflammatory ones.

[0105] Cultured keratinocytes were exposed to the probiotics L. paracasei DG and L. paracasei LPC-S01, as well as a 1:1 combination of the two strains, according to the previously described procedure. The cell supernatant was then assayed to determine cytokines, particularly anti-inflammatory cytokines that could be assayed in keratinocytes placed in contact with the probiotic strains. The assay results are summarized in Figure 2, which graphically shows the assays of IL-10, IL-1 beta, and IL-8 in the cell supernatant, expressed as pg per ml of supernatant obtained using ELISA. A detailed evaluation of the results led to the following conclusions: - IL1β: Expression level was a small amount of nanograms per 1 ml of keratinocyte supernatant. Contact with probiotics did not cause a statistically significant reduction in this cytokine compared to baseline expression (in keratinocytes not stimulated by probiotics). L. paracasei LPC-S01 showed a moderately positive effect compared to L. casei DG and was comparable to that of the combination. Similar considerations apply to pre-stimulation of keratinocytes with LPS to maximize the immune response. - IL-10: Expression levels were several tens of pg per 1 ml of keratinocyte supernatant. Contact with probiotics caused a reduction in this cytokine compared to baseline expression (in keratinocytes not stimulated by probiotics). L. paracasei LPC-S01 showed a moderately positive effect compared to L. casei DG and was comparable to that of the combination. Similar considerations apply to pre-stimulation of keratinocytes with LPS to maximize the immune response. - IL-8: Expression level was a small amount of nanograms per 1 ml of keratinocyte supernatant. Contact with probiotics caused a reduction in this cytokine compared to baseline expression (in keratinocytes not stimulated by probiotics). L. paracasei LPC-S01 showed a positive effect compared to L. casei DG and was comparable to that of the combination. In the case of preliminary stimulation of keratinocytes with LPS to maximize the immune response, both probiotics showed the ability to positively influence the control of this cytokine, and both the probiotics considered individually and the mixture had similar potential. - TSLP: Expression level of a small amount of nanograms per 1 ml of keratinocyte supernatant. Contact with probiotics caused a reduction in this index compared to baseline expression (in keratinocytes not stimulated by probiotics). Probiotic strains, both individually and in combination, were shown to be effective in modulating the expression of this index. This index is of considerable interest because it is overexpressed in several skin conditions, such as atopic dermatitis. Furthermore, this cytokine is considered an important mediator at the functional interface between keratinocytes and dendritic cells.

[0106] Assessment of COX-2 expression levels showed that, apart from moderate containment by contact with L. paracasei LPC-S01, the level of this marker did not appear to undergo any particular changes after contact with the probiotic in keratinocytes compared to baseline levels. Treatment with LPS resulted in an increase in COX-2 expression levels in eukaryotic cells, and this increase was effectively suppressed and limited by the action of the probiotic. The presence of L. paracasei LPC-S01 reduced the expression of the marker to a greater extent compared to L. casei DG, while the combination of the two probiotic strains appeared to have no effect.

[0107] Since IL8 and COX-2 expression are regulated by the transcription factor NF-κB, we quantitatively assessed NF-κB release by keratinocytes exposed to probiotics. Our results demonstrate that, in the absence of inflammatory stimuli represented by LPS, the expression of its marker appears undetectable. However, it is activated by bacterial lipopolysaccharide via p65 phosphorylation. Two probiotic strains demonstrated the ability to suppress NF-κB expression, and in particular, with respect to L. paracasei LPC-S01, its presence was shown to be especially effective in suppressing LPS-induced NF-κB expression, both as a single strain and in a mixture with DG.

[0108] Evaluation of UV-induced damage reduction The ability of the probiotic strain LPC-S01 to reduce UV-induced damage was evaluated in a fully 3D in vitro reconstructed skin model, which replicates dermal and epidermal compartments and therefore allows for the study of dermal extracellular matrix modification and differentiation of viable skin layers (full-thickness skin model). This study related to the evaluation of the effect of the LPC-S01 strain on inflammasome activation in response to UV irradiation. The LPC-S01 strain was directly applied to the surface of a 3D skin model, incubated overnight, and then rinsed with saline to remove excess product. Tissue was lightly scratched and then exposed to 1 MED (minimum erythematous dose) of UV to mimic normal sun exposure. Inflammatory activation was tested 4 and 24 hours after UV irradiation exposure. Tissue treated with saline and exposed to UV irradiation was used as a positive control. Saline was used as a negative control.

[0109] Histological analysis using hematoxylin / eosin staining At the end of processing, the tissue was washed with saline solution and fixed in 10% formalin. For each sample, biological replicas (n=3) were placed in the same paraffin block, and two discontinuous 5pm sections were cut and collected. The tissue sections were stained with hematoxylin and eosin. Histological samples were analyzed under a light microscope (20× and 40× magnification) to evaluate morphological changes and cytotoxic effects of the tissue.

[0110] Immunostaining of NFκB Labeling NFκB allows for the evaluation of its translocation from the cytoplasm to the nucleus; this translocation can, in fact, activate inflammatory processes. NFκB was labeled using immunohistochemical staining techniques known in the art. Specifically, a primary anti-NFκB (Abeam) antibody and a secondary antibody stained with the fluorophore Alexa 555 were used, along with counterstaining with DAPI to highlight the cell nucleus. Images were acquired using a fluorescence microscope at 40x magnification.

[0111] Quantification of interleukin-1β by ELISA Antibodies were adsorbed onto an ELISA plate, and then the sample was incubated. A secondary antibody was added to form a "sandwich." Quantification was based on a standard curve. Data were obtained using microspectrophotometric measurement.

[0112] As a result of reducing UV-induced damage Four hours after UV exposure, untreated tissue (positive control) showed no morphological changes, but cells in the lower layers of the epidermis were damaged by UV irradiation. Four hours later, tissue treated with the LPC-S01 strain showed no substantial changes in tissue morphology or general structure, or in burn damage present in the lower layers of the epidermis. Twenty-four hours after UV exposure, the tissue showed typical epidermal changes due to burns, with concentrated nuclei, altered epidermis and dermis. Administration of the LPC-S01 strain did not show improvement in preventing morphological changes in UV-induced tissue.

[0113] Table 2 summarizes the results of the quantification of NFκB transfer 4 hours after exposure to UV irradiation.

[0114] [Table 2]

[0115] Four hours after irradiation, the positive control showed numerous NFκB transfers, particularly in the layer directly above the basal layer of the epidermis.

[0116] Treatment with the probiotic LPC-S01 significantly inhibited NFκB nuclear translocation compared to the positive control. Furthermore, the LPC-S01 strain demonstrated the ability to reduce NFκB cytoplasmic levels.

[0117] Finally, IL-1β secretion increased 4, 8, and 24 hours after UV irradiation injury. Administration of the probiotic LPC-S01 allowed IL-1β secretion to return to its original level 4, 8, and 24 hours after injury. In particular, a significant reduction was observed 8 and 24 hours after injury.

[0118] [Table 3]

[0119] Thus, the probiotic LPC-S01 demonstrated the ability to prevent the activation of inflammation by inhibiting the translocation of NFκB to the nucleus in cells exposed to UV irradiation damage. Furthermore, the S01 strain showed a direct effect in reducing inflammation levels by lowering the levels of pro-inflammatory interleukin-1β 4, 8, and 24 hours after irradiation damage. [Deposit Certificate]

Claims

1. 1. A composition for topical application to the skin comprising the Lactobacillus paracasei LPC-S01 DSM26760 bacterial strain used in a live state for use in a method for treating, preventing and / or curing skin damage and / or effects caused by / associated with exposure to UV radiation, UV-A and / or UV-B, by competing for adhesion to P. acnes and inhibiting nuclear translocation of NF-kB, The composition, wherein the skin damage and / or effects resulting from / related to exposure to UV radiation are selected from erythema, pigmentation, keratosis, hyperkeratosis, sunburn, burns, actinic photoaging or solar elastosis, cortical cataract, pterygium, reactivation of oral herpes, cutaneous melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, squamous cell carcinoma of the cornea or conjunctiva, and damage to the lips and / or conjunctiva.

2. 2. The composition for use according to claim 1, further comprising a microorganism selected from the group consisting of bacteria, fungi, yeasts, and combinations thereof, wherein the bacterium belongs to at least one genus selected from the group consisting of Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Aerococcus, and Enterococcus, and preferably the bacterium belongs to the genus Lactobacillus.

3. The bacterial strain is 6 ~10 11 Between bacterial strain units, more preferably 10 8 ~10 9 3. The composition for use according to claim 1 or 2, wherein the composition is present in an amount ranging between 1000 and 10 ...

4. 4. The composition for use according to any one of claims 1 to 3, in the form of a cream, gel, oil, emulsion, spray, gauze, patch, bandage, lotion, mousse, ointment, paste, or a liquid formulation for extemporaneous preparation.

5. 5. Cosmetic use of a composition according to any one of claims 1 to 4 for preventing and / or alleviating skin damage and / or effects caused by / related to exposure to UV radiation, UV-A and / or UV-B.

6. 6. The cosmetic use according to claim 5, wherein skin damage and / or effects caused / related to exposure to UV radiation are prevented and / or reduced skin aging.

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