Compositions comprising bacterial strain lactobacillus paracasei and hyaluronic acid and use thereof for treatment of skin

Abstract

To provide a composition and its use for preventing, alleviating, or treating skin aging caused by natural skin aging or by exposure to external factors such as ultraviolet rays.SOLUTION: A composition comprising a mixture M comprising or consisting of: (I) a bacterial strain belonging to the species Lactobacillus paracasei, identified as Lactobacillus paracasei LPC-S01 and deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the accession number DSM 26760; and (II) hyaluronic acid or a salt thereof; and, optionally, at least one acceptable pharmaceutical, cosmetic, or food-grade additive and / or excipient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition comprising a probiotic (a live or inactivated bacterial strain) and hyaluronic acid or a salt thereof for use in the prevention, treatment (therapeutic or cosmetic treatment) or attenuation of at least one sign or symptom associated with or caused by skin aging, a weakening of the skin's immune defenses, or skin inflammation / infection, such as inflammation induced or caused by ultraviolet light. [Background technology]

[0002] Skin is the outermost lining of the human body and constitutes the body's first line of defense against external attacks, acting as an anatomical barrier against potential pathogens and harmful substances. Skin changes can promote the development of skin diseases, especially those caused by pathogens. For example, as skin ages, it becomes thinner and more fragile because cell renewal slows down, from the usual 3–4 weeks to 4–6 weeks. Over time, skin undergoes structural changes caused by several factors of different origins that determine the loss of skin hydration, the development of microwrinkles, loss of elasticity, hyperkeratosis, and the formation of hyperpigmented spots.

[0003] Furthermore, the abundant microbiota colonizing human skin plays an important and useful function in combating the attachment and development of skin pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, and Propionibacterium acnes, which may face imbalances or dysbiosis due to various factors.

[0004] When dysbiosis occurs on the skin, probiotics can act as modulators, restoring balance at the level of the skin microbiota. Over the past decade, new technologies have facilitated taxonomic analysis of the skin microbiota, which contains approximately 10 species of microorganisms belonging to more than 25 phyla, the most representative of which are Actinobacteria, Firmicutes, and Proteobacteria. Various skin diseases are associated with alterations in the skin microbiota; for example, Propionibacterium acnes is considered a pathogen associated with acne. Therefore, maintaining homeostasis of the skin microbiota or restoring said homeostasis after dysbiosis is fundamental for preventing or treating skin diseases, particularly skin infections or inflammations, and more specifically, infections or inflammations caused by pathogens such as bacteria or viruses, as well as infections or inflammations caused by exposure to ultraviolet radiation or unfavorable conditions.

[0005] Classically, these problems are approached with the use of antimicrobial agents, i.e., antiseptics and topical antibiotics. While antibiotics are undoubtedly effective, in addition to eliminating beneficial bacteria, prolonged use, especially of broad-spectrum antibiotics, poses the risk of sensitization and potential adverse events.

[0006] Furthermore, the conventional approach to prevent UV radiation damage and skin aging problems is to use creams or skin-related products that reflect or absorb UV rays, commonly called sunscreens. The active ingredients of so-called "chemical" solar filters (salicylates, cinnamates, oxybenzone, octylcrylene, etc.) are able to absorb UV rays thanks to their structure. On the other hand, titanium dioxide and zinc oxide are inert mineral substances that have a strong covering power and physically reflect sunlight, and are therefore included in so-called physical screens. Summary of the Invention [Problem to be solved by the invention]

[0007] However, said solar filters can only prevent the interaction of UV rays with the skin, do not stimulate the maintenance of homeostasis of the skin microflora, and cannot restore said homeostasis in cases of dysbiosis or infections or inflammations caused by said UV rays that may not be completely shielded by the solar filters. [Means for solving the problem]

[0008] Following intensive research and development activities, the Applicant has addressed and solved the aforementioned technical problem by providing an innovative composition (in short, the composition of the present invention) comprising a mixture (in short, the mixture of the present invention) comprising or consisting of a live and viable or inactivated bacterial strain belonging to the species Lactobacillus paracasei identified as Lactobacillus paracasei LPC-S01DSM26760 and hyaluronic acid or a salt thereof (for short, HA).

[0009] Summary of the invention A first aspect of the present invention relates to the use of a composition comprising a probiotic and hyaluronic acid or a salt thereof to prevent, reduce or treat skin aging, for example natural skin aging or induced by exposure to external factors such as ultraviolet light.

[0010] A second aspect of the present invention relates to the use of a composition comprising a probiotic and hyaluronic acid or a salt thereof to prevent, alleviate or treat at least one sign or symptom associated with or caused by a weakened immune defense of the skin.

[0011] A third aspect of the present invention relates to the use of a composition comprising a probiotic and hyaluronic acid or a salt thereof for preventing, alleviating or treating at least one sign or symptom associated with or caused by skin inflammation / infection.

[0012] Particularly useful probiotics for the purposes of the present invention are bacteria belonging to the genus Lactobacillus, preferably belonging to the species Lactobacillus paracasei, for example L. paracasei strain LPC-S01 DSM 26760.

[0013] The applicant has indeed discovered that a composition comprising a probiotic, preferably a bacterium belonging to the genus Lactobacillus, and hyaluronic acid can enhance the immune defense by promoting the release of antimicrobial peptides and chemokines in the skin, enhance the normal processes of epidermal differentiation and cell replacement, and strengthen the structure of the dermis.

[0014] Furthermore, the applicant has discovered that the use of a composition comprising hyaluronic acid and a probiotic, preferably a bacterium belonging to the genus Lactobacillus, can perform an anti-inflammatory effect and prevent the activation of inflammation induced or caused by ultraviolet light.

[0015] In particular, the compositions and mixtures of the present invention comprise: - triggering skin defense and / or healing mechanisms, especially immunomodulatory mechanisms; - Prevent and / or treat inflammatory or infectious skin diseases, especially those induced by UV radiation, both UV-A and UV-B; - combats the growth of pathogenic bacteria on the skin; - Maintaining or restoring the homeostatic state of the skin and / or the balance of the skin microflora; - favoring, facilitating, promoting and / or accelerating the wound healing process and / or re-epithelialization and / or scar formation process; - Combat and / or slow down skin aging; It is possible to do this.

[0016] Furthermore, the compositions of the present invention can induce and / or promote positive effects, both short-term and long-term.

[0017] Furthermore, the mixtures and compositions of the present invention have no significant adverse effects, and they can be administered to all subjects, particularly pediatric subjects and pregnant or lactating women.

[0018] Finally, the mixtures and / or compositions of the present invention are easy and cost-effective to prepare.

[0019] These and other objects that will become apparent from the following detailed description are achieved by the mixtures and compositions (including said mixtures) of the present invention by virtue of the technical features set forth in the appended claims.

[0020] The present invention will now be described in detail and illustrated with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1 shows 20x and 40x magnification images of histological tissue sections marked with Masson's trichrome staining, treated with saline (A) and a composition containing strain LPC-S01 (B), respectively. [Figure 2] FIG. 2 shows 20x and 40x magnification images of histological tissue sections marked with Masson's trichrome staining, treated with a composition containing hyaluronic acid (A) and a composition containing LPC-S01 + hyaluronic acid strain (B), respectively. [Figure 3] Figure 3 shows quantification of NFκB nuclear translocation in tissues exposed to UV light and treated with a composition containing LPC-S01 strain (P1), a composition containing LPC-S01 strain plus hyaluronic acid (P2), and a composition containing hyaluronic acid (P3). [Figure 4] Figure 4 shows 20x magnification images of histological tissue sections marked with hematoxylin-eosin staining 4 hours after UV injury, treated with saline (A), a composition containing the LPC-S01 strain (B), a composition containing hyaluronic acid (C), and a composition containing the LPC-S01 strain and hyaluronic acid (D). [Figure 5]Figure 5 shows 20x magnification images of histological tissue sections marked with hematoxylin-eosin staining 24 hours after UV injury, treated with saline (A), a composition containing the LPC-S01 strain (B), a composition containing hyaluronic acid (C), and a composition containing the LPC-S01 strain and hyaluronic acid (D). [Figure 6a-d] Figures 6a, 6b, 6c, and 6d are images obtained under a microscope of histomorphological analysis by H&E staining in the homeostasis model at 24 hours (6a: NC at 24 hours, 6b: P3-i at 24 hours) and 48 hours (6c: NC at 48 hours, 6d: P3-i at 48 hours). [Figure 7] Figure 7 shows gene expression results (qRT-PCR) at 24 hours in the homeostasis model for P1-i, P2-i, and P3-i products (RQ calculated using NC at 24 hours = 1; RQ<0.5 deregulation, RQ>2 upregulation). [Figure 8] FIG. 8 is a pretreatment protocol for UV irradiation of "scratched" tissue with a composition of the present invention containing inactivated strains. [Figure 9] FIG. 9 is a post-treatment protocol for UV irradiation of "scratched" tissue with a composition of the present invention containing inactivated strains. [Figure 10-11] Figures 10 and 11 show the reduction in viability of the pathogen C. acnes DMS1897 expressed in Log10 CFU on skin inserts in vitro under experimental conditions (exclusion model and competition model, respectively). DETAILED DESCRIPTION OF THE INVENTION

[0022] definition In the context of the present invention, the term "skin" is used to denote the first line of defense with respect to the external environment; in particular, the defense is initiated by the action of keratinocytes scattered throughout the outer skin layer (epidermis), which induce the secretion of cytokines and chemokines to transmit warning messages to deeper layers of the skin and cause an inflammatory response.

[0023] In the context of the present invention, the expression "skin aging" is used to denote an irreversible evolutionary process; it is represented by a series of physiological changes that determine the loss of cutaneous hydration, the appearance of microwrinkles, loss of elasticity, hyperkeratosis and the formation of hyperpigmented spots called "senile freckles".

[0024] In the context of the present invention, the term "probiotics" is used to indicate according to the definition given by FAO and WHO: "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host." In other words, probiotics are microorganisms (bacterial strains) which, when ingested in adequate amounts, prove capable of exerting a beneficial function on the organism.

[0025] In the context of the present invention, the expression "hyaluronic acid" is used to denote a glycosaminoglycan consisting of repeating units of glucosamine and glucuronic acid linked together or by glycosidic bonds β1→4 and β1→3 and by intramolecular hydrogen bonds that stabilize its conformation.

[0026] Detailed Description of the Invention The object of the present invention is a composition (in short, the composition or compositions of the present invention) comprising (I) a bacterial strain belonging to the species Lactobacillus paracasei identified and deposited as Lactobacillus paracasei LPC-S01 DSM 26760 (live and viable or inactivated or derivatives thereof) and hyaluronic acid or a salt thereof; and (II) at least one acceptable pharmaceutical or cosmetic or food grade additive and / or excipient.

[0027] The object of the present invention is a composition or mixture M of the invention comprising or consisting of a bacterial strain belonging to the species Lactobacillus paracasei identified and deposited as L. paracasei LPC-S01 DMS 26760 and hyaluronic acid or a salt thereof (in any of the described embodiments), for use as a medicament.

[0028] A first aspect of the present invention relates to a composition (composition of the invention) comprising a probiotic (L. paracasei LPC-S01 DSM 26760, viable or inactivated or derivatives thereof), preferably a probiotic bacterium, and hyaluronic acid or a salt thereof, for use in the treatment (therapeutic or cosmetic), prevention or alleviation of at least one sign or symptom associated with or caused by skin ageing (intrinsic ageing or extrinsic ageing as defined below).

[0029] The signs or symptoms of skin ageing are generally associated with a series of alterations that lead to thinning and / or yielding of the skin structure.

[0030] Preferably, the aging is intrinsic or chronological aging, which is substantially dependent on genetic (or intrinsic) factors. Intrinsic aging generally begins after the age of 25. Preferably, the signs or symptoms associated with or caused by intrinsic skin aging are selected from wrinkles, skin laxity, loss or deterioration of skin integrity, loss of skin elasticity, loss of skin firmness, skin thinning, skin scaling and dehydration, formation of dark spots or hyperpigmentation of the skin, also known as "age spots."

[0031] An object of the present invention is therefore the cosmetic use of a composition or mixture M according to the invention, comprising or consisting of a bacterial strain belonging to the species Lactobacillus paracasei identified and deposited as L. paracasei LPC-S01 DMS 26760 and hyaluronic acid or a salt thereof, and optionally a first substance and / or a second substance (according to any of the described embodiments) for the maintenance of skin homeostasis and / or as an anti-aging agent for the skin, for example for the cosmetic treatment of skin wrinkles, loss of skin elasticity (solar elastosis), dry or dehydrated skin, rough skin, photoaging, redness of the skin, the presence of dilated capillaries on the cheeks, nose and / or ears, sunburn, abnormal or uneven pigmentation or hyperpigmentation, or for making the skin brighter and more natural-looking.

[0032] Alternatively, the skin aging is extrinsic, i.e., caused by external environmental factors (external factors).Extrinsic aging is caused by environmental factors such as attack by external factors and / or UV radiation (the cause of photoaging), tobacco smoking, alcohol abuse, pollution, continuous contact with irritants, cold, wind, and combinations thereof.Photoaging is particularly interesting because it is associated with many diseases and skin damage, which can lead to serious diseases such as skin tumors.

[0033] Preferably, said signs or symptoms associated with or caused by extrinsic skin ageing are selected from among erythema, photopigmentation or sunburn, keratosis, preferably hyperkeratosis, skin redness, sunburn, burns, photoaging, solar elastosis, cortical cataract, pterygium, reactivation of cold sores, skin lesions of any nature (ulcers, wounds or bruises), preferably lip and / or conjunctival lesions, cutaneous melanoma, squamoacanthoma, basal cell carcinoma (basal cell tumor), squamoacanthoma or conjunctival carcinoma.

[0034] Skin ageing is inevitably associated with changes in the structure of the skin, which leads to an increased susceptibility to inflammatory and / or infectious diseases.

[0035] In one embodiment, a composition (the composition of the present invention) comprising a probiotic (L. paracasei LPC-S01 DSM 26760, viable or inactivated or derivatives thereof), preferably a probiotic bacterium, and hyaluronic acid or a salt thereof, is used to treat (therapeutic treatment method), prevent, or alleviate at least one sign or symptom associated with or caused by a weakened immune system of the skin or an inflammatory disease and / or skin infection. In other words, the composition is used to strengthen the immune defenses of the skin.

[0036] The at least one sign or symptom associated with or caused by a weakened immune defense of the skin or caused by an inflammatory condition of the skin is preferably selected from among dermatitis accompanied by irritation or abrasion, acne, acute or chronic dermatoses (e.g., rosacea or couperosa), skin infections, skin inflammation, erythema, ulcers, psoriasis, atopic dermatitis, otitis, cracks, fistulas and hemorrhoids.

[0037] Skin affections or diseases can be associated with or caused by pathogens, which can be bacteria, fungi, yeasts, viruses, and combinations thereof.

[0038] In one embodiment, the composition or mixture M of the present invention comprising a probiotic (L. paracasei LPC-S01 DSM 26760, viable or inactivated or a derivative thereof) and hyaluronic acid or a salt thereof is used to treat (therapeutic treatment method), prevent or alleviate at least one sign or symptom associated with or caused by, for example, a pathogen.

[0039] Preferably, the pathogen is a bacterium, preferably a bacterium of the genus Propionibacterium, preferably acnes (species Propionibacterium acnes or Cutibacterium acnes, abbreviated as C. acnes); Staphylococcus, preferably Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus pyogenes, Staphylococcus mitis; Corynebacterium subsp.; Pseudomonas, preferably Pseudomonas aeruginosa; Acinetobacter, preferably Acinetobacter johnsonii; Streptococcus, preferably Streptococcus pyogenes; Micrococcus subsp., Brevibacterium subsp.

[0040] Experimental data prepared by the Applicant show that the composition or mixture M of the invention comprising hyaluronic acid and a probiotic (L. paracasei LPC-S01 DSM 26760, live and viable or inactivated or derivatives thereof) is able to exert an anti-inflammatory and / or immunomodulatory effect at the level of keratinocytes and therefore of the skin.

[0041] Without wishing to be bound by any theory, the use of the composition is due to its anti-inflammatory capabilities, immunomodulation, epidermal cell regeneration, epidermal differentiation, and enhanced skin structure favored by probiotics, as well as the hyaluronic acid contained in the composition.

[0042] In particular, the applicant has shown that when skin is exposed to a composition containing hyaluronic acid and probiotics, it is possible to increase the differentiation process of the epidermis, in particular the stratum corneum thickening, and collagen fibers are observed to be denser and more compact.

[0043] Furthermore, as evidenced by the evaluation of the expression of genes encoding chemokines and defensins, the compositions of the present invention comprising probiotics (L. paracasei LPC-S01 DSM 26760, viable or inactivated or derivatives thereof) and hyaluronic acid or a salt thereof can exert an immunomodulatory (or immunostimulatory) effect on the skin's immune system. In particular, an increase in the expression of defensin β2 has been observed both with probiotics and with the compositions of the present invention comprising hyaluronic acid and probiotics.

[0044] In an embodiment of the present invention, the probiotics (present in the composition of the present invention together with hyaluronic acid or a salt thereof) are preferably bacteria, fungi, yeasts and combinations thereof; preferably bacteria, more preferably selected from among the bacterial strains belonging to the species Lactobacillus paracasei and identified as L. paracasei LPC-S01 DSM 26760 (live, inactivated or derivatives thereof):

[0045] According to a preferred embodiment of the present invention, the bacterium belongs to at least one genus selected from among Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Aerococcus and Enterococcus.

[0046] More preferably, the bacterium belongs to the genus Lactobacillus.

[0047] According to a further preferred embodiment of the present invention, the bacteria of the genus Lactobacillus are Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus alimentarius, Lactobacillus avialis, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus coliniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus The bacterial strain belongs to at least one species selected from Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus mucosa, Lactobacillus panis, Lactobacillus corinoides, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfrancisensis, and combinations thereof.

[0048] More preferably, the lactic acid bacteria are of the species Lactobacillus paracasei, preferably of the strain Lactobacillus paracasei DG® CNCM I-1572 and / or the strain Lactobacillus paracasei LPC-S01 DSM26760.

[0049] Both strains were isolated and deposited by SOFAR SpA; in particular, the bacterial strain L. casei DG® (a trademark registered by Sofar, Italy) was deposited by SOFAR SpA with the National Collection of Microbial Cultures of the Pasteur Institute in Paris on May 5, 1995, under the accession number CNCMI-1572. Initially, the name of this strain was Lactobacillus casei DG subsp. casei (since reclassified as Lactobacillus paracasei DG® CNCM I-1572).

[0050] The bacterial strain Lactobacillus paracasei LPC-S01 was deposited at the German Collection of Microbial and Cell Cultures by SOFAR SpA under accession number DSM26760 on May 15, 2017 (date of application for modification of deposit under the Budapest Treaty; original deposit date January 11, 2013).

[0051] Lactobacillus paracasei strains were reclassified as Lacticaseibacillus paracasei.

[0052] In a preferred embodiment of the present invention, the composition comprises Lactobacillus paracasei LPC-S01 and hyaluronic acid or a salt thereof.

[0053] 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. anguulatum, B. asteroides, B. boeum, B. coelinum, B. coryneforme, B. cuniculi, B. denticollens, B. dentium, B. gallicum, B. gallinarum, B. indicum, B. inopinatum, B. lactis, B. magnum, B. mericicum, B. minimum, B. pseudocatenulatum, B. pseudolongum, B. prolum, B. ruminantium, B. saeculare, B. subtilis, B. thermophilum, B. thermophilum, B. more preferably, the bacterium belongs to at least one species selected from among Bacillus clausii, Bacillus subtilis, Bacillus coagulans, Bacillus megaterium, Bacillus halodurans, Bacillus thuringiensis, Bacillus insolitus, and Bacillus marinus.

[0054] 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. australiens, P. avidum, P. cyclohexanicum, P. freudenreichii, P. granulosum, P. jensenii, P. microaerophyllum, P. propionicum and P. soenii.

[0055] 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, Streptococcus fels, Streptococcus infantarius, Streptococcus iniae, Streptococcus intermedius, Streptococcus milleri, Streptococcus mitis, and Streptococcus genus. The bacteria belong to at least one species selected from among Tococcus mutans, Streptococcus oralis, Streptococcus ollistii, Streptococcus parasanguinis, Streptococcus peroris, Streptococcus pneumoniae, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Streptococcus latch, Streptococcus tigrinus, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus suis, Streptococcus uberis, Streptococcus bestibularis, Streptococcus viridans, and Streptococcus zooepidemicus.

[0056] According to a further preferred embodiment of the present invention, the bacterium of the genus Lactococcus belongs to at least one species selected from among L. chungangensis, L. formocensis, L. fusiensis, L. garvieae, L. lactis, L. pythium, L. plantarum, L. raffinolactis and L. taiwanensis.

[0057] 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. urinaequi and A. urinaehominis.

[0058] According to a further preferred embodiment of the present invention, the bacterium of the genus Enterococcus belongs to at least one species selected from Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hemoperoxidus, Enterococcus hirae, Enterococcus malodoratus, Enterococcus moraviensis, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus and Enterococcus solitarius.

[0059] 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.

[0060] In the mixtures or compositions of the present invention, the probiotics, preferably the bacterial strain L. casei DG® CNCM I-1572 and / or the bacterial strain Lactobacillus paracasei LPC-S01 DSM 26760, are used live (together with hyaluronic acid or a salt thereof), i.e., they are used as probiotics.

[0061] Alternatively, said probiotics, preferably the bacterial strain L. casei DG® CNCM I-1572 and / or the bacterial strain Lactobacillus paracasei LPC-S01 DSM 26760, are dead and / or inactivated and / or tyndallized.

[0062] For example, viable bacterial strains (probiotics) of the present invention (e.g., L. paracasei LPC-S01 DSM 26760) can be inactivated by heating, tindalization, gamma irradiation, or ultrasonic treatment. The inactivation by heating or tindalization can be carried out at a temperature ranging from 50°C to 120°C, preferably 65°C to 105°C, more preferably 75°C to 95°C, for example, about 85°C, for a time ranging from 30 minutes to 120 minutes, preferably 45 minutes to 85 minutes, for example, about 60 minutes; or by tindalization. The heating, tindalization, gamma irradiation, or ultrasonic treatment process is carried out according to techniques, procedures, and equipment known to those skilled in the art.

[0063] Bacteria subjected to the inactivation process by heating or tindalization are dead (control by plate count and / or cytofluorometry) with the cell walls remaining intact in a percentage of bacteria ranging from 70% to 99.5%, preferably 80% to 95%, of the total number of bacteria subjected to the heating or tindalization inactivation technique.

[0064] In a further embodiment, probiotics, preferably the bacterial strain L. casei DG® CNCM I-1572 and / or the bacterial strain Lactobacillus paracasei LPC-S01 DSM 26760, are used in the form of a lysate and / or extract (together with hyaluronic acid or a salt thereof), i.e., they are used as paraprobiotics.

[0065] Alternatively, said probiotics, preferably the bacterial strain L. casei DG® CNCM I-1572 and / or the bacterial strain Actobacillus paracasei LPC-S01 DSM 26760, are used in the form of a bacterial product (together with hyaluronic acid or a salt thereof) selected from among supernatant, metabolites, biological products, postbiotics, cell walls and their components, exopolysaccharides, ribosomes and glycoproteins, glucans and other polysaccharides, lipopolysaccharides and any component of the supernatant.

[0066] In short, in the context of the present invention, the expression "derivatives" of a bacterial strain or a viable bacterial strain (e.g. L. paracasei LPC-S01 DSM 26760) or a probiotic is any derivative and / or component (supernatant, metabolites, biologicals, postbiotics, cell wall and its components, exopolysaccharides, ribosomes and glycoproteins, glucans and other polysaccharides, lipopolysaccharides and any component of the supernatant) of said paraprobiotic or bacterial strain, which, when administered in appropriate amounts (via oral or topical route), confers a benefit to the human or animal consumer.

[0067] In the context of the present invention, the term "probiotics" denotes and includes live and inactivated bacterial strains as defined above (e.g., L. paracasei LPC-S01 DSM 26760), and derivatives of said bacterial strains, unless otherwise specified.

[0068] Generally, the probiotics are single microorganisms or combinations or consortia of microorganisms of any of the microbial species listed in the EFSA QPS list.

[0069] Preferably, said composition comprises a combination of the above reported strains with other microorganisms as described above, preferably selected from among bacteria, fungi, yeasts and combinations thereof.

[0070] The probiotics, preferably the bacterial strain L. casei DG® CNCM I-1572 and / or the bacterial strain Lactobacillus paracasei LPC-S01 DSM 26760, are present in the composition (together with hyaluronic acid or a salt thereof) in a minimal amount sufficient to allow transient colonization of the skin, intestine and / or other areas of the organism. Preferably, for a daily intake (or single administration) of the composition of the present invention, said amount is 1 x 10 6 CFU ~ 1 x 10 12 CFU range, preferably 10 8~10 12 Units of Microbiology, 10 8 ~10 12 units of microorganisms, more preferably 10 9 ~10 11 Units of microorganisms, e.g., about 1x10 9 It varies at concentrations in the range of CFU (colony forming units) and above.

[0071] The probiotics of the present invention, preferably the bacterial strain L. casei DG® CNCM I-1572 and / or the bacterial strain Lactobacillus paracasei LPC-S01 DSM 26760, are preferably administered in a dose of 10 8 ~10 12 units of microorganisms, more preferably 10 9 ~10 11 It is administered in variable amounts between units of microorganisms.

[0072] According to a preferred embodiment, probiotics and bacteria are preferably taken at least once or twice a day.

[0073] As mentioned above, the composition or mixture M of the present invention comprises hyaluronic acid or its salts, as well as combinations thereof. The hyaluronic acid or its salts (abbreviated as HA) is comprised in the composition of the present invention together with the L. paracasei LPC-S01 DMS 26760 strain and optionally at least one additional first or second substance (according to any of the described embodiments), and is preferably of a molecular weight of about 10 kDa (kilodalton = 1,000.00 daltons or 10 3The hyaluronic acid salts have an average molecular weight ranging from about 1,000 kDa to about 2,000 kDa (Daltons), e.g., about 1,300 kDa, 1,400 kDa, 1,500 kDa, 1,600 kDa, 1,700 kDa, or 1,800 kDa. In one embodiment, the hyaluronate salt (e.g., an alkali or alkaline earth metal hyaluronate) is selected from sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, and combinations thereof.

[0074] Furthermore, in the context of the present invention, the terms "hyaluronic acid or its salts" or "HA" or simply "hyaluronic acid" are used to denote hyaluronic acid per se, as well as hydrolyzed hyaluronic acid (obtained, for example, by fermentation), and the hyaluronate salts (hyaluronates) described above.

[0075] When the hyaluronic acid of the present invention is a biotechnologically hydrolyzed hyaluronic acid obtained by fermentation, it can have an average molecular weight of about 10 kDa.When the hyaluronic acid of the present invention is sodium hyaluronate (e.g., CAS No. 9067-32-7, Mw-molecular weight 1,000-1,400 kDa or Mw 1,000-1,700 kDa) or potassium hyaluronate, it can have an average molecular weight in the range of about 1,000 kDa-2,000 kDa.

[0076] In addition to L. paracasei LPC-01 DSM 26760 and HA, the compositions of the present invention advantageously further comprise a pharmaceutically acceptable excipient and / or additional substance (e.g., a first substance or a second substance described below) and / or a carrier.

[0077] In addition to L. paracasei LPC-01 DSM 26760 and HA, and optionally a second substance (defined below), the composition of the present invention preferably further comprises a first substance selected from among plasma, PRP, a cicatrizing substance, a re-epithelializing substance, a humectant, a hydrating agent, an emollient, an absorbent, an analgesic, a phlebotonic, an anti-inflammatory agent, a muscle relaxant, an antibiotic, an antibacterial agent, an antifungal agent, an antiviral agent, an insecticide, a peptide and / or protein substance and / or a protein such as collagen, a substance belonging to connective tissue such as glycosaminoglycans, preferably chondroitin sulfate, and / or combinations thereof.

[0078] In one embodiment of the invention, the composition is formulated for topical or topical application to the skin (e.g., in solid, semi-solid, or liquid form), preferably in the form of a cream, gel, oil, emulsion (or foam), solution, dispersion (solid-liquid or liquid-liquid), suspension, two-phase mixture, spray (spray liquid), gauze, plaster, bandage, lotion, mousse, mask (mask that can be applied to the skin), ointment, or paste.

[0079] In one embodiment of the invention, the composition is formulated for oral administration, preferably as a tablet, capsule, bar, granule powder, operculum, buccal dissolvable granules, sachet or pill, suspension (e.g., drinkable vial) or solution (monophasic or biphasic).

[0080] Alternatively, the composition is prepared extemporaneously by mixing the composition with water.

[0081] Alternatively, for example, to prepare a mask to be applied to the skin or a suspension for oral use (a drinkable vial), the composition is extemporaneously prepared by mixing two components, a probiotic (L. paracasei LPC-01 DSM 26760) and hyaluronic acid or its salt. In particular, a device for extemporaneously preparing the composition of the present invention may consist of a vial containing an aqueous solution of hyaluronic acid or its salt and one end of a bottle (e.g., a cap, etc., for sealing the vial) containing the bacterial strain (L. paracasei LPC-01 DSM 26760, viable or inactivated, or its derivative) in solid form such as powder, granules, or tablets; extemporaneous preparation of the composition of the present invention occurs by releasing (e.g., by pressure) the solid form of the bacterial strain from the end of the vial into the solution of hyaluronic acid or its salt contained in the vial.

[0082] According to a preferred embodiment, in addition to L. paracasei LPC-01 DSM 26760 and HA, and optionally said first substance, the composition of the invention comprises other substances (second substances) selected from among 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), xylooligosaccharides (XOS), inulin, guar gum or combinations thereof.

[0083] The composition of the present invention, comprising L. paracasei LPC-01 DSM 26760, HA, and optionally a first or second substance, further comprises (II) at least one pharmaceutical, food, or cosmetic-grade additive and / or excipient, which is a substance lacking therapeutic activity suitable for use in pharmaceuticals or foods. In the context of the present invention, additives and / or excipients acceptable for use in pharmaceuticals, foods, or cosmetics include all auxiliary substances known to those skilled in the art for preparing compositions in solid, semi-solid, or liquid form, such as diluents, solvents (e.g., water, glycerin, ethyl alcohol), solubilizers, acidifiers, thickeners, sweeteners, flavor enhancers, colorants, lubricants, surfactants, preservatives, pH-stabilizing buffers, and mixtures thereof.

[0084] The composition of the present invention comprising the L. paracasei LPC-S01 DMS 26760 strain and hyaluronic acid or a salt thereof, and optionally a first and / or second substance, may be a pharmaceutical composition (or live biotherapeutic product), a medical device composition, a dietary supplement, a food, a novel food, a probiotic product, a composition for a food for special medical purposes (FSMP), or a cosmetic composition.

[0085] An embodiment (FRn) of the present invention is outlined below: FR1. A composition comprising a probiotic and hyaluronic acid or a salt thereof for use in the treatment, prevention or alleviation of at least one sign or symptom associated with / caused by skin aging or in the treatment, prevention or alleviation of at least one sign or symptom associated with / caused by a weakened immune system of the skin. FR2. A composition for use according to FR1, wherein the skin aging is selected from among intrinsic skin aging and extrinsic skin aging. FR3. A composition according to FR2, wherein the at least one sign or symptom associated with / caused by intrinsic skin aging is selected from among wrinkles, sagging skin, loss or deterioration of skin integrity, lack of skin elasticity, lack of skin tone, thinning skin, peeling skin and skin dehydration. FR4. A composition for use according to FR2, wherein the at least one sign or symptom associated with / or caused by extrinsic skin aging is selected from among erythema, pigmentation, keratosis, preferably hyperkeratosis, skin redness, burns, cortical cataracts, pterygium, reactivation of cold sores, skin lesions of any nature, preferably lip and / or conjunctival lesions, cutaneous melanoma, cutaneous squamous epidermoid carcinoma, basal cell carcinoma (basal cell carcinoma), corneal or conjunctival squamous epidermoid carcinoma. FR5. A composition for use according to FR1, wherein the at least one sign or symptom associated with / or caused by a weakened immune system of the skin is selected from among dermatitis, preferably accompanied by irritation or abrasion, acne, infections, skin inflammation, erythema, ulcers, psoriasis, atopic dermatitis, otitis, cracks, fistulas and hemorrhoids. FR6. A composition for use according to any one of FR1 to 5, wherein the probiotic is preferably selected from among bacteria, fungi, yeasts and combinations thereof, preferably a bacterium belonging to at least one genus selected from among Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Aerococcus and Enterococcus. FR7. Bacteria of the genus Lactobacillus include Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus alimentarius, Lactobacillus aviarius, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus coliniformis, Lactobacillus crispatus, Lactobacillus culbatus, Lactobacillus delbrueckii, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus 1. A composition for use according to FR6, wherein the Lactobacillus species belongs to at least one species selected from Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus mucosa, Lactobacillus panis, Lactobacillus corinoides, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfrancisensis and combinations thereof. FR8. A composition for use according to FR6 or FR7, wherein the bacterium is of the Lactobacillus paracasei species, preferably of the Lactobacillus paracasei DG® CNCM I-1572 strain and / or the Lactobacillus paracasei strain LPC-S01 DSM 26760. FR 9. The composition for use according to any one of the preceding FRs, wherein the hyaluronate salt is selected from among sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, and combinations thereof. FR10. A composition for use according to any one of the preceding FRs, wherein said probiotic is live, and / or dead, and / or inactivated, and / or tindalized, and / or in the form of a lysate and / or extract, and / or in the form of a bacterial product selected from supernatant, metabolites, biologicals, postbiotics, cell walls and their components, exopolysaccharides, ribosomes and glycoproteins, glucans and other polysaccharides, lipopolysaccharides and any component of the supernatant. FR11. The probiotic is 10 8 ~10 12 units of microorganisms, preferably 10 9 ~10 11 A composition for use according to any one of the preceding FRs, wherein the microorganisms are present in variable amounts among the units. FR12. Compositions for use in accordance with any of the preceding FRs in the form of creams, gels, oils, emulsions, sprays, gauze, plasters, bandages, lotions, mousses, masks, ointments, pastes, or liquid preparations for extemporaneous preparation.

[0086] The terms "treatment" or "therapeutic treatment" or "therapeutic method" in the context of the present invention are used to indicate an intervention in a subject in need thereof, comprising the administration of a therapeutically effective amount of a composition or mixture M for the purpose of eliminating, alleviating / reducing or preventing a condition or disease and its symptoms or disorders.

[0087] The term "therapeutically effective amount" refers to that amount of active compound and / or bacterial strain that elicits a biological or medical response in a tissue, system, mammal, or human that is desired and defined by an individual, researcher, veterinarian, physician, or other clinician or health care professional.

[0088] In the context of the present invention, the expression "subject" is used to denote a human subject or an animal subject (e.g., a pet such as a dog or cat or other mammal). Preferably, the compositions of the present invention are for use in a method of medical treatment or for cosmetic use in human subjects.

[0089] In the context of the present invention, the term "medical device" is used in the sense according to Legislative Decree No. 46 of 24 February 1997 or according to the new Medical Device Regulation (EU) 2017 / 745 (MDR).

[0090] In the context of the present invention, the term "novel food" is used in the sense according to Regulation EC258 of 1997.

[0091] Unless otherwise specified, an expression composition or mixture or other that contains an amount of a component "ranging from x to y" is used to indicate that said component may be present in the composition or mixture or extract or other in any amount present in the end ranges of the included range, even if not specified. [Example]

[0092] Example A A cream according to the present invention comprising the L. paracasei LPC-S01 DMS 26760 strain and hyaluronic acid or a salt thereof: To prepare the cream, strain LPC-S01 DSM 26760 (viable or inactivated) - powder or powder capsules (approximately 8x10 9 The lyophilized cells (CFU) were dissolved in 13 ml of hyaluronic acid-based cream (abbreviated as HA cream).

[0093] Example of HA cream composition (wt / wt%): - Water (solvent) 100% (sufficient); - Functional substances: Octocrylene (UV-B filter) 2-8% (4.5%), encapsulated BMDBM stabilized with Octocrylene (UV-B filter) 0.1-5% (2%), Shea organic butter (butyrospermum parkii = Shea) (CAS number 194043-92-0) 0.1-4% (1%), collagen complex and panthenol 0.1-4% (1%), butyl methoxydibenzoylmethane (UV-A filter) 0.1-4% (1%), vitamin E 0.01-1% (0.3%), gardenia stem 0.01-1% (0.1%), low molecular weight hyaluronic acid (e.g. CAS number 9004-61-9) 0.005-1% (0.05%), medium / high molecular weight hyaluronic acid (e.g. CAS number 9067-32-7). 0.005-1% (0.02%); - excipients and additives (2-10% w / w, according to the technical requirements known to the skilled person): antioxidants (e.g. tocopheryl acetate), preservatives (e.g. phenoxyethanol, potassium sorbate), surfactant-emulsifiers (e.g. cetearyl glucoside, cetyl alcohol), emulsion stabilizers (e.g. carbomer), solvents (e.g. 1,2-hexanediol), abrasives (e.g. silica), binders (e.g. PVP), moisturizers (e.g. glycerin), and / or skin conditioners (e.g. dimethicone, xanthan gum, tropolone).

[0094] Example B A pack according to the invention comprising the L. paracasei LPC-S01 DMS 26760 strain and hyaluronic acid or a salt thereof: To prepare the pack, strain LPC-S01 DSM 26760 (viable or inactivated, preferably viable) - powder or powder capsules (approximately 8x10 9 lyophilized in the form of CFU (Cell Units), an aqueous solution of hyaluronic acid or a salt thereof, for example, a solution having the components set forth in Table A wherein 0.01-2% (0.1%) of low molecular weight hyaluronic acid (e.g., CAS No. 9004-61-9) and 0.005-1% (0.05%) of medium / high molecular weight hyaluronic acid (e.g., CAS No. 9067-32-7) are present in a wt / wt % (based on the total weight of the aqueous hyaluronic acid solution). Table A [Table 1]

[0095] - Heat-inactivated L. paracasei strain LPC-S01 DMS 26760:

[0096] Experimental Part-(1) Episkin T-Skin TM Model (3D skin model) Episkin T-Skin TM (abbreviated as T-Skin or "full thickness skin" or 3D skin) is an in vitro reconstructed 3D skin model that includes the dermis and epidermis (full thickness skin model). Episkin T-Skin TM is an in vitro reconstructed skin consisting of a human fibroblast-equivalent skin layered on a well-differentiated stratified epidermis derived from normal human keratinocytes cultured on an inert polycarbonate filter. In vitro reconstructed human skin models are close to in vivo human tissue in terms of morphology (multilayered epidermis), biochemical and physiological properties, and are currently the most promising alternative to animals, ex vivo explants, and immersed cell monolayers for the evaluation of the efficacy and safety of topical product application (Gordon et al. 2015, Zuang V. 2016).

[0097] The biological relevance and predictability of these models stem from the presence of tissues organized with distinct layers of living cells, which allow for the evaluation of topically applied products at realistic clinical doses and exposure conditions. Treatment of human skin with topically applied products, such as cosmetics, results in a genomic response with dynamic pathways, which represent the initial cellular signals involved in a cascade of events at the transcriptional level. 3D human tissues are relevant test systems for studying mechanisms of action and evaluating product efficacy, taking into account both the direct genomic response and the consequences of cellular and cross-talk communication via the expression of soluble mediators and specific biomarkers.

[0098] I.Episkin T-Skin TM Homeostatic model (non-inflammatory) Episkin T-Skin TM In the present in vitro study of a homeostasis model, the effectiveness of a composition according to the invention comprising hyaluronic acid (HA) and viable or inactivated bacterial strain LPC-S01 DSM 26760 in terms of benefits on skin homeostasis was evaluated to investigate its potential application and effectiveness for skin care.

[0099] The aim of the study was to study the skin tolerance profile of compositions according to the invention after exposure to high concentrations and to evaluate their effectiveness in: Strengthening the skin's self-defense by inducing antimicrobial peptides, Stimulation of innate immune responses, epidermal regeneration and differentiation of keratinocytes Induction of positive renewal of the epidermal and dermal compartments, acting as an anti-aging agent.

[0100] I.1. Viable strains - homeostasis model I.1.1. Products and Controls Under Analysis - Negative control (NC): 0.9% NaCl saline; - P1: viable strain LPC-S01 DSM 26760 resuspended in saline; - P2: hyaluronic acid-based aqueous solution (as in Example B (pack) above); - P3: viable strain LPC-S01 DSM 26760 resuspended in a hyaluronic acid-based aqueous solution;

[0101] To prepare P3, freeze-dried powder capsules of viable strain LPC-S01 DSM 26760 (approximately 8 × 10 9 The contents of the 1000 cells / ml of hyaluronic acid-based solution were dissolved in 13 ml of hyaluronic acid-based aqueous solution. To prepare P1 (strain only), freeze-dried powder capsules of viable strain LPC-S01 DSM 26760 (approximately 8 × 10 9 The contents of the 1000 cells / ml (CFU) were resuspended in 13 ml of saline.

[0102] I.1.2. Methodological evaluation of self-defense and increased cell regeneration capacity In an in vitro reconstructed complete 3D skin model ("full-thickness skin" model), which reproduces the dermal and epidermal compartments and therefore allows studying the modification of the extracellular matrix of the dermis and the differentiation of viable skin layers, the increased self-defense and cell regeneration capacity of the probiotic strain LPC-S01 (viable) plus hyaluronic acid (P3) and the probiotic strain LPC-S01 (Lactobacillus paracasei LPC-S01 DSM 26760, viable; P1) in hyaluronic acid (P2) was evaluated.

[0103] This study focused on evaluating the effects of a composition containing the LPC-S01 DSM 26760 bacteria, hyaluronic acid, and the LPC-S01 bacteria plus hyaluronic acid on immune response activation, epidermal cell differentiation, and dermal cell regeneration. A composition containing the LPC-S01 DSM 26760 bacteria, hyaluronic acid, and the LPC-S01 probiotic bacteria plus hyaluronic acid was applied directly to the surface of a 3D skin model (30 μl) and incubated for 8 hours. After rinsing with saline, excess product was removed. The tissue was cultured for an additional 16 hours before analysis, mimicking realistic exposure to a face mask at 24 hours. Saline was used as a negative control (NC).

[0104] The following parameters were analyzed relative to the negative control (according to methods known to those skilled in the art): · Histological analysis by Masson's trichrome staining; · Gene expression (qRT-PCR) of main biomarkers of skin defense (HBD2 and CCL27), innate immune response (TLR2), epidermal differentiation (KRT14, LOR and IVL), epidermal regeneration (HAS-2, CD44, collagen III, IV and XIII, KGF and EGF); · Cell damage by quantifying the release of adenylate kinase (AK for short) (Toxilight assay). Experiments were performed in biological triplicate.

[0105] I.1.2.1. Histomorphological analysis by Masson's trichrome staining At the end of the treatment, the tissues were washed with saline and fixed in 10% formalin. Histological sections were marked using a Masson's Trichrome Kit (Abcam 150686) according to the manufacturer's instructions. For each sample, three microscopic acquisitions were performed on three different parts of the section. Histological samples were analyzed under a light microscope (20x and 40x magnification) to evaluate morphological changes in the tissue.

[0106] I.1.2.2. Real-time PCR analysis At the end of the treatment, tissues were collected in lysis buffer for RNA extraction and cDNA reverse transcription. RNA integrity was assessed by loading the extracted RNA on a 1% agarose gel; 18S and 28S ribosomal bands were detected. GAPDH was used as an endogenous control gene to normalize input levels. Analysis of the resulting data was performed using methods known to those skilled in the art.

[0107] I.1.3. Consequences of activating self-defense and cell regeneration abilities I.1.3.1. Results of histomorphometric analysis using Masson's trichrome staining After incubation with saline, the negative control (Figure 1A) showed no morphological or structural changes in both the epidermis and dermis. The distribution of collagen fibers was oriented and contained fibroblasts.

[0108] Treatment with strain LPC-S01 DSM 26760 (viable) did not induce significant changes in the structure of the epidermis and dermis. Collagen fibers and their distribution showed decreased fiber thickness and density compared to the negative control (Figure 1B).

[0109] Treatment with hyaluronic acid did not induce significant changes in the structure of the epidermis and dermis: collagen fibers showed a decreased density compared to the negative control (Figure 2A).

[0110] Treatment with the probiotic strain LPC-S01 DSM 26760 (viable) plus hyaluronic acid was able to alter the morphology of the epidermis, especially that of the thinner stratum corneum, and increased the differentiation process (Figure 2B).

[0111] I.1.3.2. Real-time PCR Results (qRT-PCR) Table 1 shows the relative quantification (RQ) of the values obtained by real-time PCR with respect to the negative control for tissues treated with LPC-S01 DSM 26760 (viable), hyaluronic acid, and LPC-S01 DSM 26760 (viable) + hyaluronic acid.

[0112] Table 1 [Table 2]

[0113] Treatment with the probiotic LPC-S01 DSM 26760 induces an increase in human defensin beta 2 (HBD2) and TLR2. These data are consistent with the immunomodulatory properties of probiotics and demonstrate their ability to trigger skin defenses and enhance innate immune responses in the skin.

[0114] Treatment with hyaluronic acid fails to induce modulation of the expression of the gene targets evaluated.

[0115] Treatment with hyaluronic acid + LPC-S01 DSM 26760 leads to an increase in HBD2 expression compared to treatment with hyaluronic acid. A decrease in collagen III and HAS-2 levels was observed (as already observed with probiotic treatment alone), while a decrease in KGF was also observed.

[0116] Generally speaking, the results obtained show that the probiotic LPC-S01 DSM 26760, when applied alone, exerts a positive effect on the skin by strengthening innate immunity (based on TLR2 and HBD-2).

[0117] Hyaluronic acid applied alone does not exert any positive effect on the skin; on the contrary, LPC-S01 introduced in the same formulation as hyaluronic acid is able to maintain its main activity of strengthening the skin's natural defenses.

[0118] I.1.3.4. Adenylate Kinase Release Results (Toxilight Assay) The levels of adenylate kinase released by tissues treated with the studied products indicate good biocompatibility of the products after 8 and 16 hours of incubation.

[0119] I.1.4. Conclusion The results show that treatment with hyaluronic acid plus LPC-S01 DSM 26760 (viable and viable) is the most promising, showing a positive effect on improving the differentiation process of the skin, particularly in skin regeneration and strengthening the structure of the dermal compartment by increasing the collagen network. Therefore, the combined administration of hyaluronic acid and the probiotic LPC-S01 DSM 26760 (P3) shows a synergistic effect in strengthening the dermal structure, intervening in key factors for cell differentiation and regeneration. This synergistic effect was unexpected, since the separate administration of hyaluronic acid and the probiotic LPC-S01 DSM 26760 did not show any significant effect on skin differentiation.

[0120] I.2. Inactivated Bacterial Strains - Homeostasis Model I.2.1. Products and Controls Under Analysis - Negative control (NC): 0.9% NaCl saline; - P1-i.: Inactivated strain LPC-S01 DMS 26760 (10 9 cells / tissues), using a concentration of 2 g LPC-S01 / ml; - P2-i.: hyaluronic acid based cream (as in Example A (cream) above); - P3-i.: Inactivated strain LPC-S01 DSM 26760 resuspended in a hyaluronic acid-based cream, at a concentration of 2 g cells / ml.

[0121] To prepare P3-i., freeze-dried powder capsules of inactivated strain LPC-S01 DSM 26760 (approximately 8 × 10 9 The contents of the 1000 cells / ml of hyaluronic acid cream were dissolved in 13 ml of hyaluronic acid cream. To prepare P1-i. (strain only), freeze-dried powder capsules of inactivated strain LPC-S01 DSM 26760 (approximately 8 × 10 9 The contents of the cells (CFU) were resuspended in saline.

[0122] I.2.2. Inactivation of bacterial strains during analysis Starting material: 2 x 10 11 Viable cells of strain LPC-S01 DSM 26760 in lyophilized form with a viable cell count of cells / g powder. The day before treatment, 2 g of powder was weighed and resuspended in 4 mL of saline. 11 A suspension of bacterial cells / mL of saline was obtained. 30 μL of this suspension was used to confirm the effective bacterial load by counting on MRS agar.

[0123] On the day of treatment, a suspension of the strains prepared in saline was heat-inactivated by incubating at 85°C for 1 hour. After this period, the bacteria were transferred to four Eppendorf tubes (1 ml each, 10 11 The pellet was then resuspended in: - 1 ml of saline solution, obtain P1-i. - Obtain 1 ml of hyaluronic acid cream, P3-i.

[0124] I.2.3. Methodology The products under analysis and the negative control (P1-i., P2-i., P3-i., NC: 15 μl) were applied directly to the surface of the T-Skin model tissue for 24 and 48 hours under homeostatic conditions. 10 μl of the P1-i and P3-i suspensions were applied. 9 bacteria / tissue was applied.

[0125] The following parameters were analyzed relative to the untreated control (negative control, NC): · Histomorphological analysis by H&E (hematoxylin & eosin) staining; Gene expression (by RT-qPCR) of key biomarkers of skin defense (human defensin β2 (DEFB4)), innate immune response (TLR2, TNFα), differentiation and epidermal regeneration (TGMS-1, CCND1, TGF-β1). Experiments were performed in biological triplicate.

[0126] I.2.4. Results I.2.4.1. Results of H&E histomorphometric analysis at 24 and 48 hours Three vertical tissue sections (consisting of three replicates) were prepared on each histological slide; five microscopic acquisitions were performed on selected sections. For each biological replicate, the most representative acquisition of the selected vertical section is reported. The average thickness was calculated for the five microscopic acquisitions.

[0127] The results are reported below: CN at 24 hours (Figure 6a): - Epidermis: completely viable and with normal SC (stratum corneum) lamellar structure; - Dermal-epidermal junction: structural integrity is observed; - Distribution of fibers and collagen: oriented, contains many fibroblasts. P1-i. at 24 hours (not shown): - Epidermis: differences are observed in the same replicates, possibly related to uneven distribution; it is clear that many cells show metabolic activation and proliferation; there are few pyknotic nuclei; the SC lamellar structure does not appear to be significantly altered; P3-i. at 24 h (Fig. 6b): - Epidermis: differences observed in the same replicates, possibly related to uneven distribution; many cells showing metabolic activation and proliferation are evident; few pyknotic nuclei; SC lamellar structure Significantly altered Looks like; - Dermal-epidermal junction: structural integrity is observed; - Distribution of fibres and collagen: dense, tightly packed and oriented, containing many fibroblasts.

[0128] NC at 48 h (Figure 6c): - Epidermis: completely viable and with normal SC (stratum corneum) lamellar structure; - dermal-epidermal junction: delamination and loss of integrity (inherent fragility of the tissue) were observed; - Distribution of fibers and collagen: oriented, contains many fibroblasts. P1-i. at 48 hours (not shown): - Epidermis: A fully differentiated epidermis is observed; the tissue shows altered SCs in terms of structure and thickness due to greater proliferation and differentiation. P3-i. at 48 hours (Figure 6d): - Epidermis: differences were observed in the same replicates, possibly related to an uneven distribution; many cells showed metabolic activation and proliferation; few pyknotic nuclei; the SC (stratum corneum) lamellar structure appeared altered due to greater proliferation; - Dermal-epidermal junction: structural integrity is observed; - Fiber and collagen distribution: oriented, containing many fibroblasts; denser and more compactly packed relative to the negative control.

[0129] The above data show that after application to P3-i tissues in 24 hours, the lamellar structure of the SC (stratum corneum) appears to change, whereas, taking into account that after application of P1-i, it is necessary to wait 48 hours before observing changes in the SC lamellar structure, composition P3-i. (inactivated strains + HA) acts in a shorter time and with greater efficacy than composition P1-i (inactivated strains only).

[0130] I.2.4.2. Real-time PCR Results I.2.4.2.1. Real-time PCR Results at 24 Hours Figure 7 shows the results of gene expression (qRT-PCR) at 24 h (RQ calculated using NC at 24 h = 1; RQ < 0.5 deregulation, RQ > 2 upregulation).

[0131] The relative quantification (RQ) gene expression (qRT-PCR) results obtained in tissues treated for 24 hours with the compositions under analysis (P1-i., P2-i., P3-i.) are summarized in Figure 7 below. The results are expressed relative to the negative control (RQ = 1 calculated using NC at 24 hours; RQ < 0.5 deregulation, RQ > 2 upregulation).

[0132] As a general consideration, the non-completely homogeneous application of the compositions in the assay resulted in high biological variability among the triplicates.

[0133] 24 hours post-treatment: The inactivated probiotic strains under analysis, alone (P1-i) or in compositions containing HA (P3-i), induced an upregulation of human defensin β2 (DEFB4). These data are consistent with the immunomodulatory properties of the viable strain LPC-S01 DMS 26760 observed in Section I.1.3.1, demonstrating its ability to induce skin defenses even in a nonviable state. This immunomodulatory activity may therefore correlate with externally exposed elements within the bacterial cell wall. The composition containing only HA (P2-i.) did not modulate human defensin β2 (DEFB4): this result confirms that the upregulation of DEFB4 in P3-i. (strain + HA) is solely related to the presence of bacteria. Composition P3-i. (strain + HA) induced a higher (although not significant) upregulation of human defensin β2 (DEFB4) at 24 hours compared to P1-i. (strain only). A significant modulation of TNFα was quantified, indicating the induction of an inflammatory response, likely due to immune recognition of bacteria by the skin surface. This effect was not observed in tissues treated with P2-i. (HA only), so this result may be related to the high dose of LPC-S01 DMS 26760 strain contained in composition P3-i.

[0134] I.2.4.2.2. Real-time PCR Results at 48 Hours The induction of human defensin β2 (DEFB4) by the inactivated bacterial strains under analysis (both P1-i. and P3-i.) was confirmed at values higher than those at the 24-hour time point (Section I.2.4.2.1), indicating a stable biological response and strengthening of the skin defenses resulting in an effective protective mechanism.

[0135] I.2.5. Conclusion In a homeostatic (non-inflammatory) model, the composition P3-i. according to the invention, comprising a combination of inactivated LPC-S01 DMS 26760 strain and hyaluronic acid (inactivated strain + HA), was well tolerated in the 3D skin model and was able to stimulate the body's defense and cell differentiation processes compared to the individual components and / or negative control.

[0136] In particular, the effectiveness level of the composition P3-i. (inactivated bacterial strain + HA) of the invention is achieved in a shorter time compared to the bacterial strain not combined with hyaluronic acid (P1-i.).

[0137] II.EpiskinT-Skin TM Inflammation model in model The ability of the composition according to the invention, comprising the bacterial strain LPC-S01 DMS 26760 (viable or inactivated) and hyaluronic acid, to reduce the damage caused by UV radiation was investigated in a fully 3D in vitro reconstructed skin model (T-Skin), which reproduces the dermal and epidermal compartments and allows studying the changes in the extracellular matrix of the dermis and the differentiation of the viable layers (full-thickness skin model). TM model).

[0138] The above evaluations were performed according to two models: inflammation model A (pre-treatment on uninjured tissue) and inflammation model B (pre- and post-treatment on injured tissue), and are reported below.

[0139] II.A. Inflammation Model A for Viable or Inactivated Bacterial Strains II.A.1. Products and Controls Under Analysis - P1: viable LPC-S01 DMS 26760 resuspended in saline; - P2: hyaluronic acid-based solution (see I.1.1. Pack); - P3: viable LPC-S01 DMS 26760 resuspended in a hyaluronic acid-based solution; - P1-i.: inactivated LPC-S01 DMS 26760 resuspended in saline, - P2-i.: hyaluronic acid-based solutions (see I.1.1. Packs), - P3-i.: inactivated LPC-S01 DMS 26760 resuspended in a hyaluronic acid-based solution, - Positive Control (PC): tissue treated with 0.9% NaCl saline, scraped and exposed to UV radiation; - Negative control (NC): tissue treated with 0.9% NaCl saline without exposure to UV radiation.

[0140] P1 (viable strains only) and P3 (viable strains + HA) were cultured using lyophilized bacterial strains (CFU 8x10 9 The contents of each capsule were resuspended in 13 ml of saline containing hyaluronic acid.

[0141] P1-i. (inactivated strain only) and P3-i. (inactivated strain + HA) were cultured in lyophilized strain capsules (CFU 8x10 9 The contents of each tube were resuspended in saline and incubated at 85°C for 1 hour. After this period, the bacteria were centrifuged and the pellets were suspended in 13 ml of hyaluronic acid-based saline (or cream).

[0142] II.A. Methods for Assessing UV Damage Reduction The reduction of UV damage by the probiotic strain LPC-S01 DSM 26760 (live and viable or inactivated) was evaluated in a "full-thickness skin" model as described above. This study examined the effects of the LPC-S01 strain, hyaluronic acid, and a composition containing the LPC-S01 strain plus hyaluronic acid on tissue morphology, as marked by hematoxylin-eosin, and on inflammasome activation in response to UV irradiation. The LPC-S01 DSM 26760 strain, hyaluronic acid, and a composition containing the probiotic strain LPC-S01 DSM 26760 plus hyaluronic acid were applied directly to the surface of the 3D skin model and incubated overnight. The tissue was then rinsed with saline to remove excess product (pretreatment step). After slight abrasion, the tissue was exposed to 1 MED (minimal erythema dose) of UV light to mimic normal sun exposure. Inflammatory activation was tested 4 and 24 hours after UV exposure. Saline-treated, UV-exposed tissues served as positive controls. Saline-treated, non-UV-exposed tissues served as negative controls.

[0143] According to methods known to those skilled in the art, the effectiveness of the compositions under analysis in reducing damage caused by ultraviolet light was assessed using the following: - NFkB immunostaining; - Histomorphological analysis by hematoxylin / eosin (H&E) staining; and - IL-1β quantification method (viable strains only).

[0144] II.A.3. Results of UV Damage Reduction (Inflammation Model A) II.A.3.1. NFkB Immunostaining Results FIG. 3 summarizes the results of the quantification of NFkB translocation after 4 hours of exposure to UV light (the composition under analysis includes viable strains). Four hours after irradiation, the positive control (PC) showed numerous NFkB translocations, especially in the suprabasal layers of the epidermis. Treatment with LPC-S01 DSM 26760 (P1), strain LPC-S01 DSM 26760 plus hyaluronic acid (P2), and hyaluronic acid (P3) significantly inhibits the nuclear translocation of NFkB relative to the positive control. Thus, treatment with probiotics LPC-S01 DSM 26760, LPC-S01 DSM 26760 + hyaluronic acid, and hyaluronic acid demonstrates the ability to prevent inflammatory activation by inhibiting NFkB translocation in the nucleus of UV-damaged cells.

[0145] Furthermore, Table 2 shows the semiquantitative data of NFkB nuclear translocation determined for a pretreatment time of 16 hours (long-term) using the composition under analysis, including the viable strain (P3) or the inactivated strain (P3-i), as well as the parameter evaluation 4 hours after UV irradiation, which is useful for assessing the effect of long-term treatment.

[0146] The biological relevance and reproducibility of the above inflammasome model (UV exposure at 1 MED, minimal erythema dose) was confirmed by increased NFkB translocation in cell nuclei of irradiated samples (positive control) compared to non-irradiated samples (negative control).

[0147] The relative increase (percentage difference) in NFkB translocation in the study with composition P3-i containing inactivated strains (+70.7%, w = 0.01) is comparable to that quantified in the study with composition P3 containing viable strains (+83.7, w = 0.01).

[0148] Table 2 [Table 3]

[0149] Furthermore, after 24 h, P3 showed a decrease in the cytoplasmic content of NFkB compared to the positive control (data not determined for P3-i).

[0150] II.A.3.2. Histomorphological Results by H&E Staining Figures 4 and 5 show tissues treated with saline (positive control) (A), probiotic LPC-S01 DSM 26760 (B), hyaluronic acid (C), and LPC-S01 DSM 26760 (viable) + hyaluronic acid (D), respectively, 4 and 24 hours after UV injury (histological morphology by H&E staining).

[0151] As can be seen in both figures, treatment with hyaluronic acid and probiotic LPC-S01 DSM 26760 fails to reduce UV-induced damage. Signs of UV-induced sunburn are particularly evident in the basal and spinous layers of the epidermis. Furthermore, the dermal-epidermal junction is damaged by UV rays, and the epidermis is not fully attached to the dermis, a sign of altered skin structure (Figures 4A, 4B, 5A, and 5B).

[0152] Treatment with hyaluronic acid plus probiotic LPC-S01 DSM 26760 can reduce UV-induced damage 4 and 24 hours after the induction of damage. Notably, the structures of both the dermis and epidermis are more compact compared to treatment with hyaluronic acid and probiotic LPC-S01 DSM 26760 individually. Furthermore, the structure of the dermal-epidermal junction is better maintained, promoting better attachment of the epidermis to the dermis (Figures 4D and 5D).

[0153] This synergistic effect of the combined administration of hyaluronic acid and the probiotic LPC-S01 DSM 26760 was unexpected, since neither hyaluronic acid nor the probiotic administered alone was able to reduce the "shedding" of epidermis from the dermis and, therefore, maintain the physiological structure of the skin.

[0154] II.A.3.3. Results of Il-1β quantification IL-1β was quantified at 4 hours for the composition according to invention P3 containing viable strains and hyaluronic acid, against the negative and positive controls (Table 3). Considering that the signal was below the detection limit of the kit (3.91 pg / mL limit), the results are not considered quantitative data. The results are presented to show the overall trend. Table 3 [Table 4]

[0155] II.A.4. Conclusion T-skin based inflammatory pathways induced by UVA+UVB (1MED dose) TM An experimental model (full thickness skin) was used to evaluate the efficacy of compositions from the present invention P3 and P3-i, comprising bacterial strain LPC-S01 DMS 26760 (viable or inactivated, respectively) and hyaluronic acid, when applied before the induction of inflammasome stress (pretreatment).

[0156] Compositions P3 (viable strain + HA) and P3-i (inactivated strain + HA) according to the invention showed good efficacy in reducing NFkB translocation in long-term pretreatment (16 hours).

[0157] Furthermore, composition P3 according to the invention (viable strains + HA) showed a good ability to protect the structure of the dermal-epidermal junction from UV rays in histomorphological studies by H&E staining.

[0158] II.B. Inflammation Model B for Inactivated Bacterial Strains T-skin based inflammatory pathways induced by UVA+UVB (1MED dose) TM An experimental model (full thickness skin) was used to evaluate the effectiveness of composition P3-i. according to the invention, comprising the inactivated strain LPC-S01 DMS 26760 and hyaluronic acid, applied to damaged tissue before or after the induction of inflammasome stress (pre- or post-treatment with UV irradiation).

[0159] The composition under analysis (see II.B.1) was prepared by two protocols, with the aim of investigating its potential use and efficacy in the T-Skin inflammasome model, by using a high concentration of the inactivated bacterial strain under analysis (10 7 or 10 9 cells / tissue) were assessed using:

[0160] BI pretreatment protocol: T skin was abraded by mechanical stress on the epidermal surface and pretreated with the composition under test for 45 minutes or 4 hours, followed by UVA and UVB irradiation (1 MED). After 4 hours of irradiation (post-incubation), tissues were collected and analyzed.

[0161] B.II. Post-treatment protocol: T skins are abraded by mechanical stress at the epidermal surface, subjected to UVA and UVB irradiation (1 MED), treated with the composition under test for 45 minutes or 4 hours, and immediately collected for analysis.

[0162] The aim of the study was to investigate the effectiveness of high doses (alone or mixed with HA) of the inactivated bacterial strain in the assay in modulating the activation and translocation of NFkB in the nucleus.

[0163] II.B.1. Compositions Under Analysis and Controls - P3-i.-10 9 :Inactivated LPC-S0 1DMS 26760(10 9 cells / tissue) were resuspended in a hyaluronic acid-based cream (see I.2.1) corresponding to 30% of the final composition; - P3-i.-107 :Inactivated LPC-S0 1DMS 26760(10 7 cells / tissue) were resuspended in a hyaluronic acid-based cream (see I.2.1) corresponding to 0.03% of the final composition; - Positive control (PC): tissue treated with 0.9% NaCl saline, scraped, and exposed to UV light; - Negative control (NC): tissue treated with 0.9% NaCl saline without exposure to UV light.

[0164] P3-i. (inactivated strain + HA) was prepared by resuspending the contents of a freeze-dried bacterial strain capsule (10 CFU) in saline and incubating at 85°C for 1 hour. After this period, the bacteria were centrifuged and the pellet was suspended in 13 ml of hyaluronic acid-based cream.

[0165] II.B.2. Research Plan II.B.2.1. Preparation of the Composition Under Analysis The number of cells is 2 × 10 11 The inactivated strains under analysis in lyophilized form at 100 cells / g of powder were weighed and resuspended in a suitable solvent as follows: - 2 g in 4 ml of HA-based cream (see I.2.1.), P3-10 9 Get 10 9 bacteria / tissue were applied by applying 15 μL of suspension. - 0.02 g in 4 ml of HA-based cream (see I.2.1.), P3-10 7 Get 10 7 bacteria / tissue were applied by applying 15 μL of suspension.

[0166] II.B.2.2. Induction and treatment of inflammasome T-skin II.B.2.2.1. Pretreatment Protocol The experimental design is summarized in Figure 8. On the day of the experiment, tissue was injured by slight mechanical stress (n strokes of Algerbrush = 2) and T-Skin was applied. TMThe tissue was treated with 15 μL of the assay composition (P3-i.) applied directly and evenly to the tissue and incubated for 45 minutes or 4 hours.

[0167] Subsequently, in PBS, a xenon arc lamp and irradiance WG320 [mW / cm 2 ] Oriel 1KW solar simulator with erythema filter (0.035 mW / cm 2 , according to calibration certificate number 16121 issued by Opto.Cal GmbH) to measure 1 MED (0.025 J / cm 2 Tissues were irradiated at 1000 kJ / cm (equivalent to 1000 kJ / cm). After inflammasome induction, the tissues were post-incubated under homeostatic conditions for 4 hours, fixed in formalin, and then embedded in paraffin (FFPE) for NFκB immunostaining. Tissues were also collected for further RT-qPCR analysis. The carriers were collected and stored at -20°C.

[0168] II.B.2.2.2. Post-treatment Protocol The experimental design is summarized in Figure 9: On the day of the experiment, tissues were injured by slight mechanical stress (Algerbrush n strokes = 2) and treated with 1 MED (0.025 J / cm) in PBS. 2 After inflammasome induction, the tissues were treated with 15 μL of the composition under analysis and incubated for 45 minutes or 4 hours. Immediately after treatment, the tissues were collected and fixed in formalin for NFκB immunostaining. The tissues were also collected for further RT-qPCR analysis.

[0169] II.B.3. NFkB Immunostaining Results Table 4 below shows the results of NFkB translocation (expressed as the total number of nuclei detected in three biological replicates) in the negative control versus the positive control for each protocol. As reported in Table 4, induction of the inflammasome model was confirmed by an increase in NFkB translocation in cell nuclei of irradiated samples compared to the negative control.

[0170] However, in this T-skin batch, although translocation was observed at each time point, earlier NFkB activation was observed after irradiation, with the highest induction of NFkB observed at 45 min after irradiation.

[0171] The protocol adopted is based on readings after UV irradiation in a post-treatment model and readings after incubation in a pre-treatment model, when the aim is to evaluate the effectiveness of the product on recovery from an acute inflammatory process. Table 4 [Table 5]

[0172] A) 45 minutes of pretreatment Table 5 shows the semiquantitative analysis of NFκB nuclear translocation (expressed as the total number of nuclei detected in all biological replicates) for 45 min pretreatment + 4 h UV irradiation post-incubation. Table 5 [Table 6] * Composition of the present invention

[0173] B) 4-hour pretreatment Table 6 shows the semi-quantitative analysis of NFκB nuclear translocation (expressed as the total number of nuclei detected in all biological replicates) for 4 hours pretreatment + 4 hours post-UV irradiation incubation. Table 6 [Table 7] * Composition of the present invention

[0174] Analysis of the results in Tables 5 and 6: - When applied as a pretreatment, composition P3-i., containing the inactivated strain LPC-S01 DMS 26760 and hyaluronic acid, is able to reduce nuclear NFkB translocation and exhibits a preventive effect in protecting the skin from UV-induced inflammatory stress. - Strain concentration 10 9 In the case of a 45-minute pretreatment model, composition P3-i. (inactivated strain + HA) was able to significantly reduce NFkB translocation in the nucleus, indicating a synergistic and / or highly effective effect between the inactivated LPC-S01 DMS 26760 strain and hyaluronic acid. - Composition P3-i. (10 9 ) and P3-i.(10 7 The tissue response to pretreatment with ) suggests a dose-response mechanism: fewer NFkB-positive nuclei were detected at increasing concentrations of the strain in the composition.

[0175] C) 45-minute post-treatment Table 7 shows the semiquantitative analysis of NFκB nuclear translocation (expressed as the total number of nuclei detected in all biological replicates) for 45 min post-treatment after UV irradiation. Table 7 [Table 8] * Composition of the present invention

[0176] D) 4-hour post-treatment Table 8 shows the semiquantitative analysis of NFκB nuclear translocation (expressed as the total number of nuclei detected in all biological replicates) for 4 hours post-treatment after UV irradiation. Table 8 [Table 9] * Composition of the present invention

[0177] - Composition P3 (inactivated strain + HA) showed a rapid and effective restoration of basal levels of NFKB, especially in the short term (when the inflammatory response is at its maximum level), which suggests a synergistic / improved effect of the combination of inactivated strain LPC-S01 DMS 26760 + hyaluronic acid in restoring homeostasis in inflamed tissues.

[0178] II.B.4. Conclusion These results confirm that composition P3-i, comprising the combination of inactivated LPC-S01 DMS 26760 + hyaluronic acid, is effective both in homeostatic conditions (see section I.2) and in inflammatory conditions of the skin (e.g. caused by UV irradiation), especially in the acute phase of inflammation, considering that it is particularly effective in the short term of the onset of inflammation.

[0179] Experiment Part (2) Evaluation of the adhesion of Cutibacterium acnes DSM 1897 to a 3D "full thickness skin" model in the presence of a composition according to the invention (strain Lactobacillus paracasei LPC-S01 DSM 26760 and hyaluronic acid) 1. Research Objective The aim of this study was to evaluate the ability of the probiotic strain L. paracasei LPC-S01 DSM 26760, alone and / or in combination with hyaluronic acid (HA), to counteract in vitro adherence of C. acnes to a "full-thickness skin" model.

[0180] Cutibacterium acnes (abbreviated C. acnes, also known as Propionibacterium acnes or P. acnes (Douglas et Gunter, 1946)) is a slow-growing, Gram-positive, anaerobic bacterium associated with certain skin diseases such as acne; it can also cause blepharitis and endophthalmitis.

[0181] To assess the various possible infection scenarios, a competition and exclusion model was implemented based on an adaptation of the method described by Coman et al. in 2015.

[0182] 2. Experimental Design The strain C. acnes DSM 1897 was used to simulate infection in a 3D "full thickness skin" model purchased from Phenion (Henkel) with a total of 30 inserts. .

[0183] The study was carried out considering different treatment conditions listed below: 1) evaluate the effective adhesion capacity of C. acnes DSM 1897 in no treatment, two exclusion, and competition models; 2) 24-hour prophylactic or concomitant treatment with L. paracasei LPC-S01 DSM 26760; 3) 24-hour prophylactic or concomitant treatment with 0.5% hyaluronic acid (Sigma-Aldrich 41897); 4) 24-hour prophylactic or combined treatment with a homogenous mixture of hyaluronic acid and L. paracasei LPC-S01 DSM 26760; 5) 24-hour prophylactic or concomitant treatment with benzoyl peroxide (Benzac 10%, positive control).

[0184] A suspension of the strain L. paracasei LPC-S01 DSM 26760 was prepared and 50 μl of the suspension was contacted with the surface of the insert.

[0185] A 0.5% hyaluronic acid suspension was prepared, and 50 μl of the suspension was contacted with the surface of the insert.

[0186] Additionally, 50 μl of strain L. paracasei LPC-S01 DSM 26760 was mixed with 0.25 mg of hyaluronic acid to obtain a probiotic + hyaluronic acid combination formulation with the same concentration of hyaluronic acid (0.5%) and the same initial loading as those used for testing the individual substances.

[0187] As a positive control, 50 μl of Benzac gel 10% (benzoyl peroxide) was placed in contact with the insert. All five conditions above were tested in duplicate, and 10 inserts were incubated at 37°C in the presence of CO2 for 24 hours.

[0188] 2.a. Performing an Exclusion Test Pretreatment of inserts with probiotic strains (or hyaluronic acid or a mixture of the two), subsequent infection with pathogens, and exclusion tests provided for the subsequent verification of a possible reduction in the % adhesion of pathogens to the inserts with respect to ideal conditions for infection (in vitro model of preventive probiotic treatment).

[0189] 2.b. Conducting competitive testing The simultaneous treatment of the inserts with probiotic strains (or hyaluronic acid or a mixture of the two) and the pathogen, and the exclusion test provided for the subsequent verification of the possible reduction in the % adhesion of the pathogen to the insert with regard to the ideal conditions of infection (in vitro model of probiotic treatment during the course of infection).

[0190] 3.Results 3.1 Exclusion Testing Figure 10 shows the loss of viability of the pathogen C. acnes DMS 1897 expressed in Log10 CFU, while Table 9 shows the same situation with the percent loss of pathogen viability under the various test conditions. Table 9 [Table 10]

[0191] As is evident from the reported results, treatment with 10% Benzac, LPC-S01 DSM 26760, and a combination of LPC-S01 DSM 26760 in the presence of 0.5% hyaluronic acid reduced the viability of C. acnes DSM 1897 by approximately 1.0-1.4 Log10, which corresponds to an approximately 20% reduction in pathogen viability. Treatment with 0.5% hyaluronic acid alone does not appear to reduce pathogen viability in any way.

[0192] 3.2. Competitive Exams Figure 11 and Table 10 show a chart of the survival numbers, expressed as log CFU, of the percentage reduction obtained in the competition test, of the mean values obtained for the replicates of each test condition, of C. acnes DSM 1897 on the inserts. Table 10 [Table 11]

[0193] Based on the data presented, it was confirmed that treatments performed with Benzac 10%, LPC-S01 DSM 26760, and a combination of LPC-S01 DSM 26760 in the presence of 0.5% hyaluronic acid reduced the viability of C. acnes DSM 1897 by 1.0-1.3 Log10 CFU. Similar to the exclusion test, treatment with 0.5% hyaluronic acid alone does not appear to reduce the viability of the pathogen.

[0194] 4. Conclusion All in vitro tests performed demonstrated the effectiveness of the Benzac 10% positive control in suppressing infection with C. acnes, reducing the viability of the pathogen population by 15% to 23%.

[0195] Exclusion and competition tests showed that treatment carried out with the combination of LPC-S01 + 0.5% hyaluronic acid (composition according to the invention) reduced infection with C. acnes DSM 1897 by approximately 18-19%.

Claims

1. (I) A bacterial strain belonging to the species Lactobacillus paracasei identified as LPC-S01 and deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the accession number DSM 26760; and (II) hyaluronic acid or a salt thereof; A composition comprising a mixture M comprising or consisting of: Optionally, the composition comprises at least one acceptable pharmaceutical or cosmetic or food grade additive and / or excipient.

2. 2. The composition of claim 1, wherein the bacterial strain Lactobacillus paracasei LPC-S01 DSM 26760 is a viable bacterial strain.

3. 2. The composition according to claim 1, wherein the bacterial strain Lactobacillus paracasei LPC-S01 DSM 26760 is heat inactivated or tindalized or sonicated or gamma irradiated, preferably heat inactivated.

4. The composition according to any one of claims 1 to 3, wherein the composition is formulated for topical skin use, preferably in a cream or pack.

5. The composition according to any one of claims 1 to 3, wherein the composition is formulated into a preparation for oral use, preferably for the formation of a suspension or extemporaneous suspension.

6. A composition according to any one of claims 1 to 5 for use as a medicine.

7. 6. A composition according to any one of claims 1 to 5 for use in a method for the preventive and / or curative treatment of skin inflammation and / or infection, or related diseases or conditions, wherein the treatment comprises administration of the composition to a subject in need thereof.

8. 8. The composition of claim 7, wherein the skin inflammation and / or infection is induced by ultraviolet light.

9. 8. The composition of claim 7, wherein the skin inflammation and / or infection is caused by Cutibacterium acnes or Propionibacterium acnes.

10. ultraviolet radiation; and / or adverse weather conditions for the skin, preferably sunlight, cold or wind; and / or Living conditions harmful to the skin, preferably pollution, smoking or drinking alcohol; damage caused or induced by, 6. A composition according to any one of claims 1 to 5 for use in a method for the prophylactic and / or curative treatment of rhesus malabsorption and / or associated diseases or conditions, said treatment comprising administration of the composition to a subject in need thereof.

11. 11. The composition for use according to any one of claims 7 to 10, wherein the skin inflammation and / or infection or skin-induced damage or related disease or condition comprises or is selected from the group consisting of acute or chronic skin inflammation or infection, bacterial, viral or fungal infection of the skin, abscess, aposteme, empyema, cellulitis, whitlow, furuncle, carbuncle, hidradenitis suppurativa, erysipelas, psoriasis, atopic dermatitis, acne, acute or chronic dermatosis, rosacea, couperosa, erythema, skin redness, burns, sunburn, reactivation of oral herpes, pressure sores, ulcers, fissures, fistulas, sores, wounds, bruises, abrasions, ecchymoses, hematomas, excoriations, keratosis, hyperkeratosis, keloids.

12. Topical, cosmetic, dermatological use of the composition according to any one of claims 1 to 4, which use comprises: To maintain skin homeostasis, and / or As an anti-aging skin agent That is the purpose.

13. 15. Cosmetic use according to claim 14, wherein the composition is a topical cosmetic use in the treatment of wrinkles, loss of skin elasticity or solar elastosis, dry skin, rough skin, photoaging, redness of the skin, presence of dilated capillaries on the cheeks, nose and / or ears, age spots, abnormal or uneven pigmentation or hyperpigmentation of the skin.

Images