Probiotic-based compositions and uses thereof

Abstract

The present invention relates to a composition comprising a probiotic, preferably a probiotic based on bacteria, in particular bacteria of the genus Lactobacillus, used to prevent and / or treat disorders affecting the skin, in particular inflammatory disorders, in particular autoinflammatory disorders of dermatological interest such as atopic dermatitis.Furthermore, the composition of the present invention is useful for preventing and / or reducing damage, in particular skin aging, induced by UV radiation.

Description

[Technical Field]

[0001] The present invention relates to a composition comprising a probiotic, preferably a probiotic based on bacteria, in particular bacteria of the genus Lactobacillus, for use in preventing and / or treating disorders affecting the skin, in particular inflammatory disorders, in particular autoinflammatory disorders of dermatological interest such as atopic dermatitis. Furthermore, the composition of the present invention is useful for preventing and / or reducing damage, in particular skin aging, induced by UV radiation. [Background technology]

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

[0003] When dysbiosis occurs at the skin level, probiotics can act as modulators and restore balance to the skin microflora.

[0004] Over the past decade, the use of new technologies has facilitated the taxonomic analysis of the skin microbiome, which may represent a total of approximately 10 species of microorganisms belonging to more than 25 phyla, the most abundant of which are Actinobacteria, Gram-positive bacteria (Firmicutes), and Proteobacteria. Various skin diseases have been linked to alterations in the skin microbiome.

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

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

[0007] On the one hand, antibiotics are undoubtedly effective, but except that they also eliminate beneficial bacteria, they carry the risk of sensitization and potential adverse events, especially in the case of 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 Summary of the Invention [Problem to be solved by the invention]

[0009] For this reason, the Applicant has identified the use of probiotic compositions, in particular based on the use of bacteria belonging to the genus Lactobacillus, as a new solution for restoring balance to the skin microflora.In fact, the Applicant has found that compositions containing probiotics belonging to the genus Lactobacillus are capable of preventing or at any rate mitigating the action of bacteria that are dangerous to the skin, such as P. acnes, and are therefore able to: 1) contribute to the prevention and / or treatment of disorders affecting the skin; 2) Promotes normal healing processes; and 3) Promoting and / or enhancing normal re-epithelialization and / or scarring processes. [Means for solving the problem]

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

[0011] The invention is described in detail below and illustrated by way of example with the aid of the accompanying figures. [Brief explanation of the drawings]

[0012] [Figure 1]Figure 1A graphically illustrates P. acnes adhesion in terms of the percentage of adherent, viable cells following: 1A: pre-stimulation of keratinocytes by contact with the probiotic being tested; 1B: co-incubation of keratinocytes with P. acnes and the probiotic being tested; and 1C: incubation of keratinocytes with the probiotic being tested after the eukaryotic cells were challenged with the pathogen. The inhibitory capacity of the tested strains was expressed as the percent reduction in adhesion by P. acnes compared to the positive control, where 100 represents 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 at similar rates (42% for L. casei DG® and 35% for L. paracasei LPC-S01). Figure 1B shows that the L. casei DG® strain exhibited a 17% reduction in adhesion, while the L. paracasei LPC-S01 strain exhibited a 9% reduction. Mixing the strains resulted in a statistically significant reduction in P. acnes adhesion, representing 42%, a percentage significantly higher than both the effects observed for the strains considered individually and the sum of their effects. Figure 1C shows a statistically significant synergistic effect associated with the mixture of the two probiotics, which demonstrated a 42% reduction in P. acnes adhesion. In contrast, each individual probiotic exhibited an adhesion-reducing ability that was less than half the effect of the mixture: 18% for L. casei DG and 11% for L. paracasei LPC-S01. [Figure 2] Graph showing the results of an assay for the cytokines IL-10, IL-1 beta, IL-8, and TSLP in the supernatant of cells, expressed in picograms (pg) per ml of supernatant, obtained using an ELISA test. DETAILED DESCRIPTION OF THE INVENTION

[0013] Said disorders and / or conditions affecting the skin are preferably selected from dermatitis, acne, infections, skin inflammation, erythema, ulcers, psoriasis, atopic dermatitis, otitis, cracks, fistulas, hemorrhoids, and any other skin disorders or lesions with or without an ongoing inflammatory process associated with irritation and / or abrasion, and / or combinations and / or complications and / or consequences thereof.

[0014] Said disorders and / or conditions affecting the skin are preferably associated with or caused by a pathogen, i.e. the composition is particularly effective not only for prevention / prophylaxis purposes but also for treatment / therapeutic purposes, i.e. after challenge with said pathogen.

[0015] According to a preferred embodiment of the invention, the composition is also capable of assisting / promoting / facilitating / accelerating the wound healing process and / or the re-epithelialization and / or scarring 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 Propionibacterium, preferably acnes spp., Staphylococcus, preferably epidermidis, aureus, warneri, pyogenes, mitis Corynebacterium spp.; Pseudomonas, preferably aeruginosa; Acinetobacter, preferably johnsonii; Streptococcus, preferably pyogenes; Micrococcus spp., and Brevibacterium spp.

[0018] Furthermore, compositions comprising probiotics, preferably one or more probiotic bacteria, as described in detail below, are adapted to shield the skin and / or lips and / or conjunctiva from UV radiation, UV-A and / or UV-B. In other words, the compositions are adapted to prevent and / or reduce damage and / or effects caused by / related to exposure to UV radiation, UV-A and / or UV-B. The compositions are preferably adapted to prevent and / or reduce skin aging.

[0019] In this regard, compositions containing probiotics, preferably one or more probiotic bacteria, as described in detail below, have cosmetic purposes. The use of the compositions is preferably related to / attributed to the immunomodulatory effect of the probiotics contained in the compositions, preferably probiotics as defined and described below. The immunomodulation involves regulating, preferably reducing, the expression of at least one cytokine selected from hematopoietic cytokines, preferably hematopoietic growth factors and / or CSFs; 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 controlling specific immune responses, preferably IL-2, and combinations thereof. The immunomodulation preferably includes regulating, preferably reducing, 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 communication signals between cells of the immune system and between cells of the immune system and various organs and tissues. They are produced by different types of cells, and once released in the body, they induce specific responses in neighboring cells (paracrine effect), in distant cells (endocrine effect), or in the cells that produced them (autocrine effect). In particular, cytokines produced by cells of the immune system, such as interleukins and chemokines, play a fundamental role in regulating and activating our defense mechanisms and in inflammatory processes. A complex network of cytokines maintains a balance between pro- and anti-inflammatory effects. An imbalance between pro- and anti-inflammatory cytokines, as well as uncontrolled cytokine production, can result in inflammatory, allergic, or autoimmune diseases. TSLP (thymic stromal lymphopoietin) is a protein belonging to the cytokine family that plays an important role in the maturation of T cell populations through the activation of antigen-presenting cells. TSLP is produced primarily 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 has shown that when keratinocytes are exposed to probiotics, the probiotics are capable of exerting immunomodulatory effects, as evidenced by cytokine assays of cell supernatants. In particular, it was observed that the expression of IL1β, IL10, and IL8 was reduced by the probiotic strains considered individually, particularly by L. paracasei LPC-S01. The effect of containing cytokine expression appeared to be more pronounced after LPS challenge of keratinocytes.

[0022] Regarding TSLP, contact with probiotics was found to reduce this marker compared to baseline expression (in keratinocytes not stimulated with probiotics). Pre-stimulation of keratinocytes with LPS did not result in increased levels of TSLP, a phenomenon that could be expected, as other molecules are known to be more stimulatory of LPS than Toll-like receptors. Probiotic strains, both individually and in mixtures, were shown to be effective in modulating the expression of this marker. This marker is of considerable interest because it is overexpressed in several skin pathologies, such as atopic dermatitis. Furthermore, this cytokine is considered a key mediator in 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 the process of re-epithelialization and / or scarring of damaged skin and / or skin affected by disorders.

[0024] Thus, the data clearly demonstrate that probiotics, and in particular the strains tested, are capable of exerting anti-inflammatory and / or immunomodulatory effects on keratinocytes and, therefore, on the skin.

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

[0026] In the context of the present invention, "skin" refers to the first line of defense against the external environment; in particular, this defense is carried out through the action of keratinocytes scattered throughout the outermost skin layer (epidermis), where they induce the secretion of cytokines and chemokines to deliver warning messages to deeper layers of the skin, thus enabling the generation of an inflammatory response. In the course of their development, keratinocytes migrate from deeper to more superficial layers, progressively depositing keratin, which exerts a protective effect on the underlying cells.

[0027] In the context of the present 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 origin, which lead to loss of skin moisture, the appearance of wrinkles, loss of elasticity, hyperkeratosis, and the formation of hyperpigmented spots called "age spots".

[0028] The aging is preferably intrinsic or chronological, which is substantially dependent on genetic (or endogenous) factors, or extrinsic or environmental, i.e., caused by external factors (exogenous factors).

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

[0030] Extrinsic aging is caused by the insult of external agents and / or environmental factors, preferably selected from UV radiation (which causes photoaging), smoking, alcoholism, pollution, continuous contact with irritants, and combinations thereof.

[0031] The damage and / or effects resulting from / associated with exposure to UV radiation are preferably selected from erythema, pigmentation, keratosis, hyperkeratosis, skin redness, sunburn, burns, actinic photoaging or solar elastosis, cortical cataract, 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 and / or complications and / or outcomes thereof.

[0032] In the context of the present invention, "restoring balance to the skin microbiota" means restoring the qualitative and quantitative physiological composition of the skin microbiota, understood as the total set of microorganisms present on the skin, and thus restoring the physiological skin microbial ecology.

[0033] In the context of the present invention, "challenge" means any experimental test or challenge or trial that involves contamination with different species of microorganisms and subsequent assessment of the change in microbial load, usually by means of plate counts of the number of viable microorganisms at regular time intervals.

[0034] In the context of the present invention, "probiotic", according to the definition given by FAO and WHO, means "live microorganisms which, when administered in sufficient amounts, confer a health benefit on the host". In other words, probiotics are microorganisms which, when taken in adequate amounts, demonstrate the ability to perform functions that are beneficial to the body.

[0035] The microorganism is preferably selected from bacteria, fungi, yeasts, and combinations thereof.

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

[0037] According to a further preferred embodiment of the present invention, the bacterium of the genus Lactobacillus is selected from the group consisting of Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus alimentarius, Lactobacillus aviaries, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus Lactobacillus cellobiosus, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus johnsonii 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 sanfranciscensis), and combinations thereof.

[0038] More preferably, the Lactobacillus genus is the species Lactobacillus paracasei, preferably the Lactobacillus paracasei DG® CNCM1-1572 strain and / or the Lactobacillus paracasei LPC-S01 strain.

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

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

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

[0042] According to a further preferred embodiment of the present invention, the bacterium of the genus Bifidobacterium is selected from the group consisting of 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. 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. turmiens.tsurumiense, and more preferably belongs to at least one species selected 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 bacterium of the genus Propionibacterium belongs 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, the bacterium of the genus Streptococcus is selected from the group consisting of Streptococcus thermophilus, Streptococcus salivarius, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus bovis, Streptococcus canis, Streptococcus constellatus, Streptococcus downei, Streptococcus dysgalactiae, Streptococcus equinus, and the like. equinus, Streptococcus ferus, Streptococcus infantarius, Streptococcus iniae, Streptococcus intermedius, Streptococcus milleri, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus orisratti, Streptococcus parasanguinis, Streptococcus peroris peroris, Streptococcus pneumoniae, Streptococcus pseudopneumoniaeThe bacterial strains belong to at least one species selected from the group consisting of Streptococcus 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 bacterium of the genus Lactococcus belongs 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 bacterium of the genus Aerococcus belongs 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, the bacterium of the genus Enterococcus is selected from the group consisting of Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus haemoperoxidus, Enterococcus hirae, Enterococcus malodoratus, Enterococcus moraviensis, Enterococcus mundtii, and Enterococcus pseudoavium. The bacteria belong to at least one species selected from Enterococcus pseudoavium, Enterococcus raffinosus, and Enterococcus solitarius.

[0048] According to 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] The microorganisms, preferably the L. casei DG® and / or Lactobacillus paracasei LPC-S01 bacterial strains, are preferably used in a live state, i.e., they are used as probiotics.

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

[0051] In a further embodiment, the microorganisms, preferably L. casei DG® and / or Lactobacillus paracasei LPC-S01, are used in the form of a lysate and / or extract, i.e., as a paraprobiotic, or in the form of a compound containing immunogenic components selected from bacterial products, supernatants, and derivatives, preferably bacterial derivatives, preferably metabolic bioproducts, postbiotics, cell walls and components, exopolysaccharides, or immunostimulant derivatives, such as ribosomal and glycoproteins, glucans and other polysaccharides, lipopolysaccharides, and any components of the supernatant.

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

[0053] In any case, the compositions of the present invention may comprise any type of microorganism, in particular bacteria, and / or microorganisms as defined above, preferably bacteria, with a probiotic effect / action / function, capable of stably colonizing the skin, the intestines, and / or other areas of the body, and capable of taking space from and / or directly combating pathogenic microorganisms / bacteria. As defined above, cosmetic and / or medical compositions preferably comprise a combination of the above-identified strains with other microorganisms, as defined above, preferably selected from bacteria, fungi, yeasts, and combinations thereof.

[0054] The microorganisms, preferably bacteria, are present in a minimal amount sufficient to allow transient colonization of the skin, intestine, and / or other areas of the body. The amount is preferably less than 10 6 ~10 11 Between microbial units, more preferably 10 8 ~10 9 The range is between 1000 and 1000 microorganism units, and the amounts are preferably amounts / doses per day (daily dose).

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

[0056] The composition of the present invention preferably further comprises substances selected from plasma, PRP, scar-forming substances, re-epithelializing substances, moisturizing agents, emollients, adsorbents, analgesic and phlebotomizing agents, anti-inflammatory agents, muscle relaxants, peptidic 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 to which the composition is preferably added extemporaneously prior to application to the skin.

[0058] In one embodiment of the present invention, the composition is used for skin microbiota transplantation. In the context of the present invention, skin microbiota transplantation refers to the administration, preferably topical administration, of the skin microbiota and / or parts thereof of a healthy subject without skin disorders to reestablish the correct balance and thereby treat any microbial incompatibility that may be present. For these purposes, the composition of the present invention preferably contains bacteria, preferably the L. casei DG® and / or Lactobacillus paracasei LPC-S01 bacterial strains, and optionally fungi, viruses, peptides, proteinaceous and / or peptidic substances, carbohydrates, vitamins, trace elements, or any other substances that characterize the microbiota of the skin and / or other areas of the body. For such purposes, the composition is preferably used in combination with amino acids, supplements, vitamins, trace elements such as zinc and selenium, macro- 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 preferably formulated for oral administration as a solid dosage form, preferably as a pill, capsule, tablet, granule, hard shell capsule, orally dissolving granule, sachet, or lozenge.

[0059] Alternatively, the composition is formulated as a liquid, preferably for extemporaneous preparation.

[0060] Alternatively, the composition is in a form capable of exerting a local effect, such as an enema, cream, spray, gel, patch, lotion, mousse, ointment, paste, or liquid preparation to which the composition is preferably added and / or prepared extemporaneously prior to application to the skin.

[0061] According to a preferred embodiment 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, macro- and micronutrients, enzymes, and / or prebiotic substances such as fructooligosaccharides (FOS), galactooligosaccharides (GOS), inulin, guar gum, or combinations thereof. [Example]

[0062] Bacterial strains and cells By way of example, the invention has been demonstrated using the following bacterial strains: - L. casei DG® (Lactobacillus paracasei CNCM1-1572) - L. paracasei LPC-S01 - A 1:1 mixture of L. casei DG® + 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 stationary phase, bacterial samples were collected and used for testing. They were washed several times with PBS (phosphate-buffered saline) and centrifuged to remove spent medium. Live cells in the stationary phase were used because in this phase, rather than in the stationary phase, bacteria induce a smaller immune response.

[0064] All of the following tests were performed in triplicate.

[0065] Normal human keratinocytes maintained in culture with appropriate supplements were used in the experiments.

[0066] Propionibacterium acnes (P. acnes) strain CCUG 50865 (purchased from the Culture Collection, University of Gothenburg) was cultured for 16 hours at 37°C in cooked meat medium (both liquid and solid) prepared according to the supplier's instructions. The bacterial concentration was then determined spectrophotometrically at OD 600 nm and used for adhesion tests.

[0067] Adhesion assay and pathogen challenge of cell lines Keratinocytes were seeded into wells of cell culture plates at 5% CO2 and 37°C until confluence (i.e., a total of approximately 10 5 The seeding rate was 2.5 × 10 5 cells / 0.32 cm (i.e., 7.8 × 10 5 At that point in the experiment, the medium was removed and the cells were washed with antibiotic-free medium, after which bacteria were added for testing, which was performed according to the following different protocols:

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

[0069] The bacteria were left in contact with the cells for 60 minutes, maintained with slight shaking at 37° C. At the end of the incubation period, the medium was removed and the cells were washed with sterile medium to remove bacteria that had not adhered to the cell monolayer.

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

[0071] 2) Adhesion Exclusion Test After the number of keratinocytes was determined for each well, strains of L. paracasei were added to the cells in a 1:1 ratio, both individually and in combination.

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

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

[0074] To calculate the number of P. acnes bacteria that adhered to keratinocytes, cells were detached and lysed, and bacteria were counted by supravital staining after plating in 5% sheep blood agar under anaerobiosis at 37°C for 48–72 h. Wells containing keratinocytes that were not preincubated with L. paracasei strains but inoculated with P. acnes alone served as a positive control for P. acnes adhesion (P. acnes adherence control), while wells containing cells that were not incubated with any bacteria served as a negative control (sterile control).

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

[0076] The bacteria were left in contact with the cells for 60 minutes with slight shaking at 37° C. 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.

[0077] To calculate the number of P. acnes adherent to keratinocytes, cells were detached and lysed, and bacteria were counted by supravital staining after plating in 5% sheep blood agar under anaerobiosis at 37°C for 48–72 h. Wells containing keratinocytes inoculated with P. acnes alone served as a positive control (P. acnes adherence control), while wells containing cells not incubated with any bacteria served as a negative control (sterile control).

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

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

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

[0081] To calculate the number of P. acnes adherent to keratinocytes, cells were detached and lysed, and bacteria were counted by supravital staining after plating in 5% sheep blood agar under anaerobiosis at 37°C for 48–72 h.

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

[0083] Tests were performed both with the individual strains in pure form and in a 1:1 mixture, and were repeated twice.

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

[0085] [Table 1]

[0086] - Adhesion exclusion test (pre-treatment of eukaryotic cells with probiotics and subsequent incubation with pathogens) - Results As described above, keratinocyte monolayers were first exposed to contact with the probiotics, followed by contact with P. acnes after removal of non-adherent bacteria, which was then removed by washing; the residual viability of P. acnes was then quantified.

[0087] The negative control was represented by eukaryotic cells without stimulation by contact with bacteria, while the positive control was represented by the ability of P. acnes to adhere to keratinocytes without any pretreatment with probiotics. Figure 1A shows the results for P. acnes adhesion in terms of the percentage of live, viable cells adhering after pre-stimulation of keratinocytes by contact with the different probiotics tested. The inhibitory potential of the tested strains was expressed as the % reduction in adhesion by P. acnes compared to the positive control, where 100 represents P. acnes adhesion in the absence of probiotic stimulation.

[0088] Both probiotic strains showed the ability to prevent adhesion of P. acnes at 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, the reduction (if any) in adhesion of P. acnes to keratinocytes was assessed after simultaneous treatment of cell lines with probiotics and pathogens, with the aim of evaluating potential competitive inhibitory effects exerted by probiotics when co-incubated with P. acnes.

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

[0091] Figure 1B graphically shows the results for P. acnes adhesion in terms of the percentage of viable, adherent cells after co-incubation of keratinocytes with P. acnes and the different probiotics tested. As previously described, the inhibitory potential of the tested strains was expressed as the % reduction in adhesion by P. acnes compared to the positive control, where 100 represents P. acnes adhesion in the absence of probiotic stimulation.

[0092] In this mode of probiotic-pathogen interaction, the L. casei DG® strain showed the ability to reduce adhesion by 17%, while the L. paracasei LPC-S01 strain showed 9%. The strain combination produced a statistically significant reduction in P. acnes adhesion, representing 42%, clearly higher than that observed for the strains considered individually, and also a higher percentage than the sum of their effects.

[0093] - Test for removal of attached pathogens (pre-treatment with eukaryotic pathogens and subsequent incubation with probiotics) - Results During these studies, any reduction in adhesion of P. acnes to keratinocytes was assessed after pretreatment of the cell line with the pathogen and subsequent incubation with the probiotic after removal of the non-adherent pathogen.

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

[0095] Figure 1C graphically shows the results for P. acnes adhesion in terms of the percentage of viable, adherent cells after incubating keratinocytes with the different probiotics tested following challenge with a eukaryotic pathogen (P. acnes). As previously described, the inhibitory potential of the tested strains was expressed as the % reduction in adhesion by P. acnes compared to the positive control, where 100 represents P. acnes adhesion in the absence of probiotics.

[0096] As with the co-incubation protocol, these studies also observed an interesting and statistically significant synergistic effect involving the combination of two probiotics, which showed the ability to reduce P. acnes adhesion by 42%. In contrast, the individual probiotics showed adhesion reduction abilities that were less than half that of the mixture: 18% for L. casei DG® and 11% for L. paracasei LPC-S01.

[0097] Immunomodulation Testing Immunomodulation studies were performed with the aim of verifying whether the probiotic strains have the ability to modulate cytokine release by human keratinocytes exposed to the negative stimulus of bacterial lipopolysaccharide (LPS).

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

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

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

[0101] At the end of the incubation, culture medium was collected to quantify the produced cytokines (IL-10, IL-1beta, IL-8, thymic stromal lymphopoietin [TSLP]) using ELISA assays, while the levels of cyclooxygenase-2 (COX-2) and activated NF-kB (induced by LPS or IL-1beta) 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 chronic inflammation (phlogosis). IL-10, in particular, 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 regulates lymphocyte function and is involved in the development of chronic keratitis.

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

[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 some neoplasms, including skin neoplasms. NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a protein complex with transcription factor function that is produced by all cell types in response to various stimuli, including inflammatory ones.

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

[0106] Evaluation of the level of COX-2 expression showed that the level of this marker did not appear to undergo any specific changes after contact of keratinocytes with probiotics compared to baseline levels, except for a moderate containment effect due to contact with L. paracasei LPC-S01. Treatment with LPS showed an increase in the level of COX-2 expression by eukaryotic cells, which was effectively suppressed and limited by the action of the probiotic. The presence of L. paracasei LPC-S01 reduced the expression of this marker to a greater extent than L. casei DG, while the combination of the two probiotic strains did not appear to have any effect.

[0107] Because the expression of IL-8 and COX-2 is regulated by the transcription factor NF-kB, we quantitatively assessed the release of NF-kB by keratinocytes exposed to probiotics. Results demonstrated that in the absence of inflammatory stimuli such as LPS, expression of this marker appears undetectable. However, it is activated by bacterial lipopolysaccharide via phosphorylation of p65. Two probiotic strains demonstrated the ability to suppress NF-kB expression, and L. paracasei LPC-S01 in particular was shown to be particularly effective in suppressing LPS-induced NF-kB expression, both as a single strain and in a mixture with DG.

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

[0109] Histomorphological analysis with hematoxylin / eosin staining At the end of treatment, tissues were washed with saline and fixed in 10% formalin. For each sample, biological replicates (n=3) were included in the same paraffin block, and two non-serial 5 pm sections were cut and collected. Tissue sections were stained with hematoxylin and eosin. Histological samples were analyzed under a light microscope (20x and 40x magnification) to assess tissue morphological changes and cytotoxic effects.

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

[0111] Quantification of interleukin-1β by ELISA Antibodies were adsorbed onto ELISA plates, after which samples were incubated. Secondary antibodies were added to form a "sandwich." Quantification was based on a standard curve. Data were acquired using microspectrophotometry.

[0112] Results in reduced UV-induced damage Four hours after exposure to UV irradiation, untreated tissue (positive control) showed unchanged morphology, but cells in the lower layers of the epidermis were damaged by UV irradiation. After 4 hours, tissue treated with the LPC-S01 strain showed no substantial changes in tissue morphology or general structure, or in the burn damage present in the lower epidermis. Twenty-four hours after exposure to UV irradiation, the tissue showed typical changes in the epidermis due to burn injury, with pyknotic nuclei, altered epidermis and dermis. Administration of the LPC-S01 strain did not show any improvement in preventing morphological changes in the tissue induced by UV irradiation.

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

[0114] [Table 2]

[0115] Four hours after irradiation, the positive control showed extensive NFkB translocation, especially in the suprabasal layers of the epidermis.

[0116] Treatment with probiotic LPC-S01 was able to inhibit the nuclear translocation of NFkB to a significant extent compared to the positive control. Furthermore, the LPC-S01 strain also showed the ability to reduce the cytoplasmic levels of NFkB.

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

[0118] [Table 3]

[0119] Thus, the probiotic LPC-S01 demonstrated the ability to prevent inflammatory activation by inhibiting nuclear translocation of NFkB in cells exposed to UV radiation injury. Furthermore, the S01 strain showed a direct effect on reducing the level of inflammation by reducing the level of pro-inflammatory interleukin-1β at 4, 8, and 24 hours after radiation injury.

Claims

1. 1. A composition for topical application to the skin comprising the Lactobacillus paracasei LPC-S01 DSM26760 and Lactobacillus paracasei DG® CNCMI-1572 bacterial strains 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. A 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 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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