bacterial strains for topical skin care

Abstract

The present invention relates to selected bacterial strains belonging to the species Lactobacillus paracasei (recently reclassified by J. Zheng et al., 2020), as well as mixtures thereof, compositions thereof, and their use in the prevention and / or treatment of skin diseases and disorders.

Description

[Technical Field]

[0001] This invention relates to selected bacterial strains belonging to the Lactobacillus paracasei species (recently reclassified by J. Zheng et al., 2020), mixtures thereof, compositions thereof, and their use in the prevention and / or treatment of skin diseases and disorders. [Background technology]

[0002] Many topical skincare products are available, including facial cleansers, toners, moisturizers, and anti-aging serums. These formulations very often contain chemicals that are relatively aggressive to the skin and can cause significant damage over time. These products typically contain chemical components that, alone or in combination with other ingredients, can harm the skin instead of benefiting it, and therefore are not suitable for all skin types. Furthermore, these formulations can cause irritation in certain skin types. [Overview of the project] [Problems that the invention aims to solve]

[0003] Therefore, there is a strong need to identify new skincare treatments that can alleviate skin defects, skin irritation / redness, dryness, and / or inflammation, as alternatives to conventional chemical cosmetic compositions and treatments. [Means for solving the problem]

[0004] As a result of intensive and long-term research and development, the applicant has surprisingly discovered that a specific bacterial strain of the genus Lactobacillus, belonging to the species Lactobacillus paracasei, identified as Lactobacillus paracasei LPC-S01 (registered trademark), deposited with the German Microbial and Cell Culture Collection GmbH (DSMZ) by Sofar SpA on January 11, 2013, under depositary number DSM 26760, and transferred to deposit under the Budapest Convention on May 15, 2017, can solve the above technical problems.

[0005] After intensive and long-term research and development, the applicant has surprisingly discovered that a specific bacterial strain of the genus Lactobacillus, belonging to the species Lactobacillus casei spp. casei, identified as Lactobacillus casei DG® and deposited with the National Microbial Culture Collection (CNCM) on May 5, 1995, by Sofar SpA under deposit number CNCM I-1572, can solve the above technical problems.

[0006] The applicant, after intensive and long-term research and development, has surprisingly discovered that another specific bacterial strain of the genus Lactobacillus, identified as Lactobacillus paracasei m. biome LIVESKIN88 (e.g., Lactobacillus paracasei or L. paracasei) and deposited with the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) under depositary number DSM 33788 (filed by LAC2BIOME Srl, Italy on January 20, 2021, and subsequently renamed Lactobacillus paracasei, DSM 33788), can also resolve several skin disorders.

[0007] The aforementioned bacterial strain is deposited in accordance with the provisions of the Budapest Convention; the depositor of the aforementioned bacterial strain described in and claimed in this patent application, and the applicant, express their agreement to make the strain available for use during the term of the patent. [Brief explanation of the drawing]

[0008] [Figure 1] Adhesion of P. acnes expressed as the percentage of viable and viable cells that adhered to keratinocytes after pre-contact stimulation with various probiotics tested (in the figure, (*) indicates statistically significant data (p<0.05)). LP125=LPC-S01;LC48=DG [Figure 2] Adhesion of P. acnes expressed as the percentage of surviving and viable cells that adhered to keratinocytes after co-incubation with P. acnes and various probiotics tested (in the figure, (*) indicates statistically significant data (p<0.05)). [Figure 3] Adhesion of P. acnes, expressed as the percentage of surviving and viable cells after incubation of keratinocytes with various probiotics tested following eukaryotic cell challenge by pathogens. (In the figure, (*) indicates statistically significant data (p<0.05)). [Figure 4] Immunomodulatory effects of probiotic strains on IL1β, IL10, and IL8. [Figure 5] Western blotting of COX-2 and NF-kB. [Figure 6] Average measurement of dermal thickness. [Figure 7] Visualization of the inflammasome model. [Figure 8] Block diagram reporting 4-hour VS 42-18 NF-κB translocation. CN: Negative control, CP: Positive control, P1: LPC S01, P2: Hyaluronic acid mask + LPC S01, P3: Hyaluronic acid mask. [Figure 9-10] Inflammatory aging models in T skin at 4 hours and 24 hours. [Figure 11A-B] Gene expression results after 24 hours (left) and 48 hours (right). RQ calculated with NC24 hours = 1, RQ calculated with NC48 hours = 1. RQ < 0.5 downregulation, RQ > 2 upregulation. [Figure 12] skin structure. [Figure 13-15] Protocol for the test in 4b.2. Figure 14 represents the pretreatment protocol; Figure 15 represents the post-treatment protocol [Figure 16A-B] Decrease in the survival of C. acnes (formerly, P. acnes) DSM 1897 expressed as Log10 CFU / insert (left column of Figure 16). In the right column, the percentage decrease in the survival of C. acnes DSM 1897 under various conditions tested in the probiotic pretreatment and subsequent pathogen infection model [Figure 17] Panel test [Figure 18A-B] Decrease in the survival of C. acnes (formerly, P. acnes) DSM 1897 expressed as Log10 CFU / insert (left column of Figure 18). In the right column, the percentage decrease in the survival of C. acnes DSM 1897 under various conditions tested in the competition test [Figure 19A-B] Decrease in the survival of C. acnes (formerly, P. acnes) DSM 1897 expressed as Log10 CFU / insert (left column of Figure 19). In the right column, the percentage decrease in the survival of C. acnes DSM 1897 under various conditions tested in the replacement test [Figure 20] Showing the skin surface hydration values of the treatment area obtained from 29 volunteers [Figure 21] Representing the clinical evaluation of the expansion of the facial area involved and showing the delta values obtained from 29 volunteers[[ID=xx]] [[ID=xx]] [Figure 22] Showing the delta values obtained from 29 volunteers, indicating a statistically significant decrease with respect to erythema [Figure 23] [[ID=2))]]Showing the delta values obtained from 29 volunteers, indicating a statistically significant decrease with respect to the number of papules and / or pustules [Figure 24] Showing the delta values obtained from 29 volunteers, indicating a statistically significant decrease with respect to skin dryness [Figure 25] Showing the results of the treatment based on 29 cases per sample after 14 days of treatment and after 28 days of treatment [Figure 26] It should be noted that in the above translation, there is an unclear "2))" in the original text at ID 25, which is retained as it is in the translation for the sake of consistency with the original. If this is an error in the original, it may need to be corrected for a more accurate translation.This study demonstrates that Ha mask + Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) promoted epidermal cell proliferation. Quantification of HaCa T cell wound closure after incubation at different time points using different dilutions of the complete mask. [Figure 27] This shows the quantification of epidermalization using different bacterial cell concentrations resuspended in excipient masks diluted with water from 1 to 20. [Figure 28] This image shows the adhesion of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) strain to the HaCaT epidermal cell layer, as observed by Giemsa staining under a light microscope. [Figure 29] This study demonstrates the activation of NF-κB transcription factors in HaCaT cell layers transfected with an alkaline phosphatase (SEAP) reporter vector. [Figure 30] This shows a comparison of the elimination effects of various probiotic formulations. [Figure 31] This study demonstrates the regulation of intracellular ROS in HaCaT cells by the antioxidant effect of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788). [Figure 32] This study demonstrates the antipathogenic effect of the Lactobacillus paracasei m. biome LIVESKIN88 (DSM 33788) formulation compared to various Lactobacillus suspensions. [Modes for carrying out the invention]

[0009] Description of the Invention Interestingly, two specific bacterial strains were found to exert a specific effect on P. acnes, making them highly effective against several skin diseases and functional disorders.

[0010] According to a first aspect thereof, the present invention relates to bacterial strains for use in the preventive or therapeutic treatment of infections and / or inflammations caused by pathogenic bacteria belonging to the species Propionibacterium acnes; wherein the strains consist of or are selected from the group including: - Lactobacillus paracasei LPC-S01 (DSM 26760) - Lactobacillus casei DG (registered trademark) (CNCM I-1572), and mixtures thereof; The aforementioned preventive or therapeutic treatment is characterized by being exerted through a specific action against P. acnes.

[0011] In another embodiment, the present invention relates to a composition comprising at least one of the above-mentioned strains and optionally at least one pharmaceutically acceptable excipient, characterized in that the composition is intended to inhibit inflammation through a specific action against P. acnes.

[0012] In another aspect, the present invention relates to a cosmetic method for treating acne, comprising administering a bacterial strain comprising or selected from the group consisting of: - Lactobacillus paracasei LPC-S01 (DSM 26760) - Lactobacillus casei DG (registered trademark) (CNCM I-1572), and mixtures thereof; The method described above is characterized by its intent to suppress inflammation through a specific action against P. acnes.

[0013] According to a preferred embodiment, the present invention includes, or is a bacterial strain selected from the group consisting of: - Lactobacillus paracasei LPC-S01 (DSM 26760) - Lactobacillus casei DG (registered trademark) (CNCM I-1572), and mixtures thereof; However, this refers to the above-mentioned cosmetic method, which is administered once a day to those who need it.

[0014] The use of at least one of the above-defined bacterial strains for preparing a cosmetic or dermatological composition for treating or preventing acne, characterized in that the cosmetic or dermatological composition acts through a specific action against P. acnes, represents another subject of the present invention.

[0015] In another embodiment, the present invention is -Bacterial strains including the following, or selected from the group consisting of the following: Lactobacillus paracasei LPC-S01 (DSM 26760), Lactobacillus casei DG (registered trademark) (CNCM I-1572), and mixtures thereof; - Hyaluronic acid or its salts; Regarding compositions containing, The composition is characterized by its intent to suppress inflammation through a specific action against P. acnes.

[0016] In another aspect, the present invention relates to a cosmetic method for enhancing skin hydration, comprising the following: - Apply the composition defined above to the skin and maintain the application for at least 6 hours; - Removing the applied composition with water; The application is characterized by being carried out for a period of at least 14 days, preferably 14 to 28 days.

[0017] In another embodiment, the present invention relates to a kit comprising: - The compositions defined above; and - A dispenser with separate compartments for bacterial strains.

[0018] In another aspect, the present invention relates to a composition comprising the following for use in protecting human skin from ultraviolet light and enhancing skin hydration: -Bacterial strains including or selected from the group consisting of: Lactobacillus paracasei LPC-S01 (DSM 26760), Lactobacillus casei DG® (CNCM I-1572), and mixtures thereof; - Hyaluronic acid or a salt thereof.

[0019] In another aspect, the present invention relates to a composition for use in promoting wound healing and epidermalization, comprising the following: - Lacticaceibacillus paracasei m. biome LIVESKIN88 (DSM 33788); - Hyaluronic acid or a salt thereof.

[0020] In another aspect, the present invention relates to a cosmetic composition for preventing skin aging, comprising the following: - Lacticaceibacillus paracasei m. biome LIVESKIN88 (DSM 33788); and Depending on the circumstances, - Hyaluronic acid or a salt thereof.

[0021] In another aspect, the present invention relates to a bacterial strain for use in the preventive or therapeutic treatment of infections and / or inflammations caused by Staphylococcus aureus; wherein the strain is Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788).

[0022] In another aspect, the present invention relates to a composition for use in the prophylactic or therapeutic treatment of infections and / or inflammations caused by Staphylococcus aureus, comprising Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) and optionally at least one pharmaceutically acceptable excipient.

[0023] The bacterial strains and compositions of the present invention are intended for topical use at least once a day, preferably once a day.

[0024] Preferably, the strain of the present invention is in a lyophilized form and administered in a pharmaceutical, dermatological, or cosmetic composition for topical use, sometimes in the presence of conventional excipients. Such compositions may be pharmaceuticals or cosmetics, may be prepared according to methods known in the art, and may be in single dosage form or multiple dosage form. For example, the compositions of the present invention may be prepared in the form of a gel, cream, emulsion, ointment, foam, powder, aqueous solution or aqueous suspension, oil solution or oil suspension, or two-phase solution or two-phase suspension, which are stirred before use. Masks are also suitable for the treatment of the present invention.

[0025] The composition contains 10 per gram of composition. 3 ~101 2 For example, 10 5 ~10 10 It may contain CFUs (colony-forming units). Such a composition can be administered in an appropriate amount over the area to be treated. Treatment is preferably extended for at least 14 days, preferably until the desired dermatological or cosmetic results are obtained.

[0026] The compositions of the present invention may also contain, in addition to conventional excipients, further active ingredients beneficial for the treatment of diseases or cosmetic disorders. Alternatively, the administration of the bacterial strain or composition of the present invention may be carried out in combination with any other convenient treatment, either topical or parenteral. [Examples]

[0027] Experiment Section Probiotics vs. P. acnes (Cutibacterium acnes, formerly known as Propionibacterium acnes) Two strains of Lactobacillus paracasei, namely L. paracasei LPC-S01 and L. casei DG, were used to evaluate their efficacy in the following: • Inhibition of P. acnes proliferation; • Regulation of the inflammatory response of keratinocytes after challenge with inflammatory stimuli (LPS). To conduct these experiments, normal human keratinocytes preserved in culture along with appropriate supplements were used.

[0028] Cultured human keratinocytes have been identified as a suitable substrate for preliminary measurements needed to evaluate the efficacy of probiotics intended for application in reducing skin inflammation. In fact, keratinocytes are the first line of defense for the skin against the external environment, seeded in the outer layers of skin (epidermis), and can induce the secretion of cytokines and chemokines to transmit warning messages to deeper layers of the skin, generating an inflammatory response. In the course of their evolution, migration occurs from the deepest layers to the most superficial layers, and keratin is progressively deposited, playing a protective role.

[0029] Human primary keratinocytes can be cultured in vitro in the laboratory, and bacterial culture tests can be conducted to identify the nature of the immune response induced by bacterial strains.

[0030] The bacterial strains used are listed below: L. casei DG (registered trademark) (L. paracasei CNCM I-1572) L. paracasei LPC-S01 (registered trademark) (DSM 26760) L. casei DG (registered trademark) + L. paracasei LPC-S01, 1:1 mixture

[0031] The following test was conducted: 1. Adhesion and challenge testing of cell lines by pathogens; 2. Immunomodulatory testing;

[0032] 1.2.1 Adhesion and Challenge Test These experiments aim to verify the ability of specific strains of Lactobacillus paracasei, either alone or in a 1:1 mixture, to inhibit the adhesion of Propionibacterium acnes to normal human keratinocytes in cultures.

[0033] In exclusion tests (pretreatment of eukaryotic cells with probiotics followed by incubation with the pathogen), the results revealed that both probiotic strains showed similar percentages of ability to prevent adhesion of P. acnes (42% for L. casei DG® and 35% for L. paracasei LPC-S01).

[0034] The results reported in Figure 1 represent the adhesion of P. acnes, expressed as the percentage of viable and viable cells that adhered to keratinocytes after pre-contact stimulation with various probiotics tested. After P. acnes adhered in the absence of probiotic stimulation, the inhibitory ability of the test strains is expressed as the percentage reduction in adhesion by P. acnes compared to the positive control.

[0035] In competitive trials (co-incubation of eukaryotic cells and probiotics, and pathogens and probiotics), the results showed that L. casei DG® strain had a 17% adhesion reduction ability, while L. paracasei LPC-S01 strain had a 9% adhesion reduction ability. When the strains were mixed, P. acnes adhesion was statistically significantly reduced, reaching 42%, which was significantly higher than that observed with the individually examined strains and clearly demonstrated a synergistic effect. The results are shown in Figure 2.

[0036] Figure 2 shows the adhesion of P. acnes, expressed as the percentage of viable and viable cells that adhered to keratinocytes after co-incubation with P. acnes and the various probiotics tested. As previously mentioned, in the case of P. acnes adhesion in the absence of probiotic stimulation, the inhibitory ability of the test strains was expressed as the percentage reduction in adhesion by P. acnes compared to the positive control.

[0037] In substitution studies (pretreatment of eukaryotic cells with pathogens followed by incubation with probiotics), the results showed an interesting and statistically significant synergistic effect, which was associated with a mixture of the two probiotics and was found to reduce P. acnes adhesion by 42%. Conversely, the individual probiotics demonstrated adhesion reduction capabilities equivalent to 18% for L. casei DG (trademarked) and 11% for L. paracasei LPC-S01.

[0038] The results reported in Figure 3 show the percentage of viable and viable P. acnes adhesion after stimulating eukaryotic cells with pathogens and then incubating keratinocytes with the various probiotics tested. As previously mentioned, in the case of P. acnes adhesion in the absence of probiotics, the inhibitory ability of the test strains was expressed as the percentage of P. acnes adhesion reduction compared to the positive control.

[0039] 1.2.2 Immunomodulatory Tests The objective of these experiments was to verify the ability of specific strains of Lactobacillus paracasei, either alone or in a 1:1 mixture, to inhibit the adhesion of Propionibacterium acnes to normal human keratinocytes in culture.

[0040] The study focused on measuring cytokines (IL-8, IL-1 beta, and IL-10) and evaluating the activation of two markers, COX-2 and NF-κB.

[0041] COX-2 (cyclooxygenase-2) represents an inducible marker produced by a small number of cell types in response to specific inflammatory stimuli. It appears to be overexpressed in several tumors, including skin tumors.

[0042] NF-κB (nuclear factor kappa light chain enhancer of activated B cells) is a transcription factor-functioning protein complex produced by all cell types in response to a variety of stimuli, including inflammatory ones.

[0043] The results are as follows: Cytokine assays in the supernatant of cells exposed to probiotics highlighted the immunomodulatory effects of individually examined probiotic strains, particularly L. paracasei LPC-S01, by reducing the expression of IL1β, IL10, and IL8. The results are shown in Figure 4.

[0044] The immunomodulatory effects after LPC-S01-induced activation are also confirmed by Western blotting results for COX-2 and NF-κB. While L. paracasei LPC-S01 strain has been associated with certain potential, L. casei DG® strain is also interesting, although it exhibits inferior overall anti-inflammatory efficacy against keratinocytes. The results are shown in Figure 5.

[0045] 1.3 Summary of Results The synergistic effect of mixed strains is important in eliminating pathogens and, in the presence of pathogens in contact with keratinocytes, or when the mixture is used following infection, it is used to contain their growth.

[0046] From an immunomodulatory perspective, L. paracasei LPC-S01 strain was often more effective than L. casei DG® in regulating the inflammatory response of keratinocytes induced by inflammatory stimuli (LPC-S01).

[0047] 2. Probiotics + Mask - Homeostasis Model The objective of the study was to evaluate a series of samples based on hyaluronic acid, collagen, or jojoba oil, with or without the probiotic strains according to the present invention. The study was performed on a complete 3D model (full-thickness skin model) reconstructed in vitro, including the dermis and epidermis, to investigate its potential applications and efficacy in skincare.

[0048] 2.1 Background In vitro reconstructed human skin models closely resemble in vivo human tissue in terms of morphological (multilayered epithelium), biochemical, and physiological properties, and today represent the most promising alternative to animals, ex vivo explants, and immersed cell monolayers for in vitro safety and efficacy evaluation of topical application products (Gordon et al., 2015; Zuang V. 2016).

[0049] The biological relevance and predictiveness of these models stem from the presence of organized tissue containing different living cell layers, allowing for localized evaluation of products under realistic clinical doses and exposure conditions.

[0050] The treatment of human skin with topical application products such as cosmetics involves dynamic pathways and results in genomic responses that represent initial cellular signals at the transcriptional level, leading to an event cascade. 3D biological tissue is a suitable testing system for investigating the mechanism of action and evaluating the efficacy of products, considering both direct genomic responses and the consequences of cellular communication and crosstalk mediated by soluble mediators and specific biomarker expression.

[0051] A homeostatic model of a "full-thickness skin model" (T-skin) that replicates the compartments of the dermis and epidermis was developed for cosmetic testing; this model has the unique characteristics to enable the study of dermal extracellular matrix modification and dermal differentiation using a multiparametric approach.

[0052] 2.2 Exam To explore the potential applications and efficacy in skincare, a series of novel products based on hyaluronic acid, collagen, or jojoba oil in a reference formulation (mask), along with the products in the presence of the probiotic strain LPC-S01, were evaluated in vitro in a complete 3D model (full-thickness skin model) reconstructed with the dermis and epidermis.

[0053] The products tested were as follows: [Table 1]

[0054] The product was applied directly to the surface of a 3D model for 8 hours at a physiological daily exposure level, then gently washed with saline to remove excess product, and post-incubated for 16 hours to simulate realistic exposure from a mask.

[0055] To define skin tolerance and efficacy in the following, the following parameters were examined in a complete 3D model (FT-skin model) reconstructed in vitro, including the dermis and epidermis: • Strengthening the skin's self-defense mechanism through the induction of antimicrobial peptides; • To stimulate the innate immune response of keratinocytes, epidermal regeneration, and differentiation; • It induces active regeneration of the epidermal and dermal compartments, acting as an anti-aging agent.

[0056] [Table 2]

[0057] The main positive results obtained by comparing the three main ingredients (jojoba oil, hyaluronic acid, and collagen) of different mask-based products with or without the bacterial strain Lactobacillus paracasei LPC-S01 are reported in the following table for each product.

[0058] [Table 3]

[0059] The best results were obtained with the combination of hyaluronic acid and L. paracasei LPC-S01 strain.

[0060] Morphological analysis assigns a qualitative evaluation score to the control group: + → No significant changes ++ → Significant improvement in morphological descriptors - → Slightly altered form - - → Changed form

[0061] [Table 4]

[0062] 2.3 Summary of Results Overall, the results suggest that the probiotic LPC-S01, when applied alone, has a positive effect on the skin by enhancing innate immunity (based on TLR2 and HBD-2).

[0063] Products based on hyaluronic acid, collagen, or jojoba oil that do not contain probiotic strains have not had a positive effect on the T-skin model; on the contrary, the same products that also contain the bacterial strain LPC-S01 show a positive effect in strengthening the skin's self-defense function.

[0064] The combination of hyaluronic acid mask and Lactobacillus paracasei LPC-S01 proved to be the most promising combination, showing positive effects in the following areas: • To promote the differentiation process of skin and skin regeneration; • To strengthen the overall structure of the dermal compartment by increasing the collagen network.

[0065] 3a. Probiotics + Mask - Inflammasome Model - Live Cells the purpose This study evaluated the effects of Lactobacillus paracasei LPC-S01 strain on an inflammasome model, either alone or in combination with hyaluronic acid ("mask"), to investigate its potential applications and efficacy in reducing inflammation. This study focused on the effects of the product on inflammasome activation in response to UV irradiation, i.e., inflammation.

[0066] [Table 5]

[0067] 3a.1 Background In vitro reconstructed human skin models closely resemble in vivo human tissue in terms of morphological (multilayered epithelium), biochemical, and physiological properties, and today represent the most promising alternative to animals, ex vivo explants, and immersed cell monolayers for in vitro safety and efficacy evaluation of topically applied products (Gordon et al., 2015; Zuang V. 2016).

[0068] The biological relevance and predictiveness of these models stem from the presence of organized tissue containing different living cell layers, allowing for topical evaluation of products under realistic clinical doses and exposure conditions. Treatment of human skin with topical products such as cosmetics has a dynamic pathway and triggers genomic responses that represent initial cellular signals at the transcriptional level, leading to an event cascade.

[0069] 3D biological tissue is a suitable testing system for investigating the mechanism of action and evaluating product efficacy by considering both direct genomic responses and the results of cell communication and crosstalk mediated by soluble mediators and specific biomarker expression.

[0070] This study utilized a "full-thickness skin model" (T-skin) specifically developed by VitroScreen for cosmetic testing, which replicates the compartments of the dermis and epidermis. This model enabled the study of dermal extracellular matrix modification and epidermal differentiation under UV-induced stress conditions using a multi-parameter approach.

[0071] 3a.2 Exam To investigate the potential applications and efficacy of a novel product based on hyaluronic acid itself from a reference formulation ("Mask"), and a formulation in the presence of the probiotic bacterial strain Lactobacillus paracasei LPC-S01, in an inflammasome model, we evaluated them. The products tested are as follows:

[0072] [Table 6]

[0073] The product was applied directly to the surface of a 3D model and incubated overnight, after which excess product was gently washed off with saline. After lightly scraping the tissue, it was exposed to 1 MED of UV light to mimic normal sunlight exposure. Activation of the inflammasome pathway was assessed 4 and 24 hours after 1 MED irradiation.

[0074] The following parameters were analyzed compared to the untreated control: • Immunostaining of NF-κB • Hematoxylin-eosin staining • Quantification of secreted IL-1β by ELISA

[0075] The positive control (inflammasome induced by 1MED) exhibited the following characteristics: [Table 7]

[0076] From a morphological standpoint, as expected, after epidermal exfoliation, all irradiated tissues exhibited sunburn cells after 4 hours, and significant epidermal and dermal damage was observed after 24 hours.

[0077] The main results of the test items obtained by comparing masks (hyaluronic acid), probiotic strain LPC-S01, and combinations of both with radiation-positive controls are reported in the following table.

[0078] [Table 8]

[0079] The results obtained after 4 hours are shown in Figure 8. A significant reduction in inflammation was observed in all products within 4 hours.

[0080] The mask likely acts as a film-forming agent that protects against ultraviolet light. However, as shown in Figures 9 and 10, the tissue morphology of the samples treated with P2 differed significantly between 4 hours and 24 hours.

[0081] Product Hyaluronic Acid + LPC-S01 exhibits a clear synergistic effect compared to hyaluronic acid masks without bacterial strains according to the present invention, better preserving the integrity of the dermal-epidermal junction and the overall skin structure and firmness against UV damage.

[0082] 3a.3 Summary of Results Probiotic LPC-S01 alone (P1) and probiotic LPC-S01 in combination with HA (P2) showed remarkable effects in mitigating the effects of biologically relevant UV doses (1MED) on NF-β on κB nuclear translocation and subsequent accumulation in the cytoplasm of keratinocytes when the skin barrier is impaired: they first reduced nuclear translocation (initial effectiveness visible at 4 hours), then reduced cytoplasmic NF-κB content (visible at 24 hours), demonstrating their delayed positive efficacy against inflammasome reduction.

[0083] The probiotic LPC-S01(P1) induced a further decrease in cytoplasmic NF-κB content after 4 hours.

[0084] Under the specific exposure conditions employed in this study (overnight treatment, gentle cleansing of residues, abrasion to reduce barrier function and induce greater sensitivity to UV radiation, and UV radiation exposure), P1 and P2 were shown to act through anti-inflammatory mechanisms that were visibly active 4 hours after stress induction and still active 24 hours later.

[0085] Despite similar anti-inflammatory effects, P2 showed a synergistic effect in protecting the dermal-epidermal junction structure from UV damage. Furthermore, when used in combination with the HA mask (P2), the effects of probiotics were more efficient and lasted longer (24 hours).

[0086] 3b. Probiotics + Mask - Inflammasome Model - Live and Inactivated Cells The efficacy of applying the probiotic strain LPC-S01 (surviving and inactivated) before inflammasome stress induction was evaluated using an experimental T-skin (full-thickness skin) model based on the inflammatory pathway induced by UVA + UVB (1 MED dose).

[0087] This examination was planned and conducted with the following two objectives: • Evaluate the effectiveness of Viable LPC-S01 itself, or its effectiveness when introduced into a reference product (mask) after a short pretreatment time (45 minutes) compared to a long pretreatment time (16 hours overnight as in previous studies), and the final readings after 4 hours; • To evaluate the effectiveness of inactivated LPC-S01 under short-term (45 minutes) and long-term (16 hours overnight) pretreatment periods.

[0088] 3b.1 Background In this study, the VitroScreen "Full-Thickness Skin Model" (T-skin), which replicates the dermal and epidermal compartments, was used. This model enabled the study of dermal extracellular matrix modification and epidermal differentiation under UV-induced stress conditions using a multi-parameter approach.

[0089] 3b.2 Exam A study was conducted to evaluate the efficacy of Lactobacillus paracasei probiotic strain LPC-S01 itself, or its efficacy when introduced into a topical formulation (mask), compared to a formulation placebo (3 products): This protocol is based on the inflammatory pathway induced by UVA + UVB (1 MED dose).

[0090] In this study, the product was applied for an overnight pretreatment period, then removed, and the T-skin was irradiated. Readouts (NF-κB translocation and morphology) were quantified 4 and 24 hours after irradiation.

[0091] The tested samples and experimental conditions (the product was applied directly to the surface of the 3D model) are shown in the following table.

[0092] [Table 9]

[0093] 16-hour long-term pre-treatment protocol: Comparison of live and inactivated probiotics

[0094] The table below shows a semi-quantitative analysis of NF-κB nuclear translocation during a 16-hour (long-term) pretreatment period. Current data are compared with previously obtained data (see previous studies) to evaluate the effects of long-term treatment.

[0095] [Table 10]

[0096] The inactivated LPC-S01 series (inactivated LPC-S01 itself or in the formulation) did not induce a significant reduction in NF-κB measured after 16 hours of pretreatment, but the survival series LPC-S01 (LPC-S01 itself or in the formulation) (ref. VS 42-18) was effective in returning all test items to negative control levels of nuclear NF-κB due to a substantial reduction in NF-κB translocation. • Considering the placebo mask, efficacy was detected in this study even after 16 hours of pretreatment, as reported in previous studies (-28.4% p=0.00).

[0097] The following table reports the results of the histological analysis: [Table 11]

[0098] Scoring the effectiveness of treatments that reduce inflammasomes (i.e., NF-κB positive nuclei and SBCs) Legend: (+) Slightly effective; (+++) Significantly effective; (-) No effect

[0099] 3b.3 Summary of Results The biological relevance and reproducibility of the inflammasome model (UV exposure at 1MED, minimum erythemal dose) have been confirmed by the increase in NF-κB translocations in the cell nuclei of irradiated samples compared to negative controls.

[0100] The relative increase (percentage difference) in NF-κB translocation is comparable to that quantified in a previous study (+83.7, p=0.01- vs 42-18) (+70.7%, p=0.01- vs 75-18). Products containing the live probiotic LPC-S01 were shown to have a lower effect in reducing NF-κB translocations when pre-treated for a short period (45 minutes) compared to when pre-treated for a longer period (16 hours). • When the inactivated probiotic LPC-S01 is introduced either directly or into a formulation (mask), it has been shown to have little effect in reducing UV-induced damage (NF-κB translocation) in both short-term and long-term pretreatment. • Hyaluronic acid mask alone (mask placebo) demonstrated its effect as a physical shield in both short-term (-27.2%, p=0.001) and long-term (-28.4%, p=0.00) pretreatment settings.

[0101] 4. Inactivated Probiotics + Yaluage (Registered Trademark) Cream This research is divided into three different phases: 1. To explore potential applications and efficacy in skincare, we will evaluate the efficacy of inactivated probiotic strain LPC-S01, either alone or included in a cream formulation (Yaruage), on a fully reconstructed in vitro 3D model including the dermis and epidermis (full-thickness skin model) (homeostasis model).

[0102] 2. The previously studied doses will be tested for efficacy in the inflammasome model using 45-minute and 4-hour pretreatments (with respect to irradiation) in three forms: a) by itself, b) in the Yaruage formulation, and c) Yaruage as itself (control). (Inflammasome model).

[0103] 3. The previously studied doses will be applied in three forms: 45 minutes and 4 hours after irradiation (post-treatment), and their efficacy will be tested in the inflammasome model: a) itself (probiotic strain LPC-S01), b) in the Yaruage preparation, and c) Yaruage (inflammasome model).

[0104] Yaruage Cream is an anti-aging face cream containing hyaluronic acid, collagen, vitamin E, gardenia stem cells, free and bond chemical filters, and shea butter.

[0105] 4a. Homeostasis Model The objective of this study was to investigate the skin tolerance profile after high-concentration exposure to the inactivated bacterial strain (LPC-S01) and to evaluate its efficacy (alone or in cream form) in the following ways: • Strengthening the skin's self-defense mechanism through the induction of antimicrobial peptides. • Stimulates the innate immune response of keratinocytes, as well as epidermal regeneration and differentiation.

[0106] 4a.1 Background This study utilized a "full-thickness skin model" developed by VitroScreen. VitroScreen specifically developed a homeostatic model of the "full-thickness skin model" (T-skin) that replicates the dermal and epidermal compartments for cosmetic testing: this model possesses specificity that enables the study of dermal extracellular matrix modification and viable layer differentiation using a multiparametric approach.

[0107] 4a.2 Research The effects of inactivated probiotic strain LPC-S01 on skin homeostasis, either alone or included in a cream formulation (Yaluage), were evaluated using the T-Skin model.

[0108] This study investigates the potential applications and effectiveness of high concentrations of inactivated bacteria (10) in skincare. 9 The procedure was performed using cells / tissues.

[0109] Specifically, the products are as follows: - Inactivated LPC-S01 (10) resuspended in physiological saline 9 Cells / Tissues) (Univocal Code = P1), - Yaruage Cream (Univocal Code = P2), - Inactivated LPC-S01 (10) resuspended in Yaruage cream 9 Cells / tissues), equivalent to 30% of the final formulation (Univocal code = P3), The solution was applied directly to the surface of the 3D model for 24 and 48 hours.

[0110] The following parameters were analyzed compared to the untreated control: • Release of IL-1α in culture medium; • Gene expression of important biomarkers for skin defense (DEFB4), innate immune responses (TLR2, TNFα), and epidermal differentiation and regeneration (TGMS-1, CCND1, TGF-β1); • Histological analysis by H&E. The biological significance of the above markers is as follows:

[0111] [Table 12]

[0112] The results are explained below. This study found very high concentrations of inactivated bacteria (10 9 The procedure was performed using cells / tissues. Specifically, the products tested: - Inactivated LPC-S01 resuspended in physiological saline (10 9 cells / tissues) (labeled as P1), - Yal-Arg cream (labeled as P2), - Inactivated LPC-S01 resuspended in Yal-Arg cream (10 9 cells / tissues), corresponding to 30% of the final formulation (labeled as P3), were directly applied to the surface of the T-skin model for 24 hours and 48 hours.

[0113] Yal-Arg cream is an anti-aging face cream containing hyaluronic acid, collagen, vitamin E, keratinocytes stem cells, free and bond chemical filters, and shea butter, and has the following properties: anti-aging, prevention of wrinkles and fine lines, UVA and UVB protection, smoothness of the epidermal layer, moisturizing, skin softening, antioxidant. The specific parameters and their biological meanings are shown in the following table:

[0114]

Table 12b

[0115] The main results obtained are reported below:

[0116]

Table 13

[0117] The results regarding human defensin β are reported in Figure 11. On the other hand, the effects of the combination on the skin differentiation process are reported in Figure 12.

[0118] 4a.3 Summary of Results In this homeostasis model, the effects observed with the combination of inactivated LPC-S01 and Yal-Arg cream can be summarized as follows: • Continuous (24-hour and 48-hour) upregulation of human β2-defensins is considered, suggesting increased effectiveness in enhancing host defense capabilities through the aforementioned combination. • Metabolic activation that induces an accelerated differentiation process in a dynamic pattern of 24 to 48 hours, accompanied by highly differentiated tissue and changes in the stratum corneum (see diagram below).

[0119] Therefore, this combination was well-tolerated in 3D skin models and was able to stimulate the body's defense and cell differentiation processes.

[0120] 4b. Inflammasome Model The objective of this study was to investigate the efficacy of two different administrations (alone or in cream) of the inactivated bacterium LPC-S01 in regulating NF-κB activation and nuclear translocation when applied before or after inflammasome stress induction.

[0121] This study utilized a "full-thickness skin model" developed by VitroScreen. VitroScreen specifically developed a homeostatic model of the "full-thickness skin model" (T-skin) that replicates the dermal and epidermal compartments for cosmetic testing: this model enables the study of dermal extracellular matrix modification and epidermal differentiation under UV-induced stress conditions using a multiparametric approach.

[0122] 4b.2 Research As shown in Figures 13-15, in order to investigate the potential application and efficacy of the inactivated probiotic strain LPC-S01 to the T-skin inflammasome model, two different concentrations of inactivated bacteria (10) were used, either alone or in a cream formulation (Yaruage), according to two protocols: pretreatment and posttreatment. 7 or 10 9 Evaluation was performed using cells / tissues.

[0123] Specifically, the product was tested on T-skin according to the following two protocols: • Inactivated LPC-S01 (10) resuspended in physiological saline 9 Cells / tissues (labeled as P1); • Yaluage Cream (labeled as P2); • Yaruage Cream comes in two concentrations (10 7 or 10 9 Inactivated LPC-S01 resuspended in cells / tissues, corresponding to 30% and 0.03% of the final formulation, respectively (P3-10) 9 and P3-10 7 (and labeled); • Pretreatment protocol: T-skin samples, abraded by mechanical stress on the epidermal surface, were pretreated for 45 minutes and 4 hours using the test parameters, and then subjected to UVA and UVB (1MED) irradiation. After 4 hours of post-incubation, tissue samples were collected for analysis (Figure 14). • Post-treatment protocol: T-skin samples abraded by mechanical stress on the epidermal surface were subjected to UVA and UVB (1MED) irradiation, followed by treatment for 45 minutes and 4 hours according to the test parameters, and immediately collected for analysis (Figure 15). The results are reported below.

[0124] [Table 14]

[0125] [Table 15]

[0126] In the table above, the results corresponding to the effectiveness of inactivated LPC-S01 in reducing the inflammasome pathway are: Bold The best synergistic effect observed between inactivated LPC-S01 and Yaruage is: Underline and bold This is shown.

[0127] 4b.3 Summary of Results In this experimental model of T-skin based on the inflammatory pathway induced by UVA+UVB, the inactivated probiotic strain LPC-S01, when applied alone or as a cream before or after inflammasome stress induction, shows the following: • Previous text: 10 9 The combination of LPC-S01 at cell / tissue concentrations with the cream demonstrated a synergistic anti-inflammatory and prophylactic effect compared to the individual components. Pretreatment at two different concentrations suggests a positive dose-response mechanism: the highest dose had a greater effect in reducing bacteria, thus reducing inflammation.

[0128] These results confirm the immunomodulatory properties of the probiotic strains at both a physiological level and as a prophylactic treatment against UV-induced inflammatory stress. Simultaneously, synergistic effects ensure local skin tolerance and induction of host defense mechanisms.

[0129] • Post-treatment: When included in a cream formulation, LPC-S01 was found to restore NF-κB to baseline levels more efficiently and rapidly, especially after short-term treatment (when the inflammatory response is at its peak), suggesting that this synergistic effect is effective in restoring tissue homeostasis.

[0130] These results support the effectiveness of the combination of cream and inactivated LPC-S01 in maintaining homeostasis and in treating UV-induced inflammasome skin.

[0131] 5. Probiotics + Mask vs. P. Acnes The objective of this study was to evaluate the ability of the probiotic Lactobacillus paracasei LPC-S01 strain, alone and / or in combination with hyaluronic acid, to inhibit the adhesion of Cutibacterium acnes (formerly Propionibacterium acnes) to a full-thickness in vitro skin model. To extend the evaluation to all possible infection scenarios, competition, exclusion, and replacement models were evaluated within the framework of this project, based on the adaptation of the method described by Coman et al. in 2015.

[0132] 5.1 Research Using an in vitro model of infection with full-thickness skin inserts by C. acnes, we evaluated the potential effects of high molecular weight hyaluronic acid and / or the probiotic strain L. paracasei LPC-S01 on the pathogen's infectivity.

[0133] The potential effects of probiotic strains and / or hyaluronic acid on pathogens were evaluated using three different in vitro models: • Full-thickness skin pretreatment with probiotics and / or hyaluronic acid (exclusion model) • Simultaneous treatment of the entire skin with probiotics and / or hyaluronic acid and pathogens (competitive model) • Post-treatment (replacement model) of full-thickness skin primarily infected with pathogens using probiotics and / or hyaluronic acid.

[0134] Specifically, the tests were conducted considering a variety of treatment conditions, including the following: 1) Evaluate the effective and uninterrupted adhesive capacity of the untreated C. acnes DSM 1897 using three models: exclusion, competition, and substitution; 2) 24-hour prophylaxis, concurrent treatment, or post-treatment with L. paracasei LPC-S01; 3) 24-hour prophylaxis, concurrent treatment, or post-treatment with 0.5% hyaluronic acid; 4) 24-hour prophylaxis, concurrent treatment, or post-treatment with a homogeneous mixture of hyaluronic acid and L. paracasei LPC-S01; 5) 24-hour prophylaxis with benzoyl peroxide, either concurrently or as a post-treatment (Benzac 10%, positive control).

[0135] The research results are reported in Figure 16.

[0136] a) Exclusion assay Based on the results reported in Figure 16, treatment with Benzac 10%, LPC-S01, and LPC-S01 combination in the presence of 0.5% hyaluronic acid can be said to reduce the life charge of C. acnes to approximately 1.0–1.4 Log10, which corresponds to a reduction of approximately 20% in the survival rate of the pathogen. However, treatment with 0.5% hyaluronic acid alone does not appear to reduce the survival rate of the pathogen by any means.

[0137] It should be emphasized that the L. paracasei LPC-S01 strain exhibits affinity for 3D full-thickness skin models. In fact, the L. paracasei LPC-S01 strain can grow actively during incubation with 3D full-thickness skin models, and its vital charge increases to slightly above half a log in pre-treated inserts.

[0138] Therefore, the decrease in the pathogen C. acnes DSM 1897 after pretreatment with the probiotic strain L. paracasei LPC-S01 is thought to be due to the acidification of the culture medium by the active proliferation of the probiotic.

[0139] The slight degradation observed in inserts at the end of the incubation period (yellowing of the insert's culture medium) may also be attributable to the metabolic activity of the probiotics (Figure 17).

[0140] b) Competitive assay As shown in Figure 18, treatment with Benzac 10%, LPC-S01, and a combination of LPC-S01 in the presence of 0.5% hyaluronic acid resulted in a 1.0-1.3 Log10 CFU reduction in the biocharge of C. acnes DSM 1897. Treatment with 0.5% hyaluronic acid alone did not appear to reduce the survival rate of the pathogen.

[0141] It should be noted that the L. paracasei LPC-S01 strain showed very low affinity to the 3D full-thickness skin model during incubation. The number of L. paracasei LPC-S01 strains showed a very slight increase from 8.0 Log10 CFU to 8.2 Log10 CFU after 24 hours of incubation with the insert. Consequently, in the competing model, the decrease in the pathogen C. acnes DSM 1897 following the coexistence of the probiotic strain L. paracasei LPC-S01 is clearly not attributable to the proliferation of the probiotic, and therefore, a different mechanism of action can be hypothesized.

[0142] c) Replacement assay As shown in Figure 19, only treatment with Benzac 10% was able to reduce the growth of pathogens by approximately 2 logarithmic scale (approximately 23%).

[0143] All other post-infection treatments in the C. Acnes DSM 1897 do not appear to reduce the survival rate of the pathogen.

[0144] It should be noted that the L. paracasei LPC-S01 strain did not grow actively during the 24-hour substitution test. In fact, the number of L. paracasei LPC-S01 strains decreased after 24-hour incubation with the insert. Consequently, in the substitution model, the slight decrease in the pathogen C. acnes DSM 1897 after the co-existence of the probiotic strain L. paracasei LPC-S01 is clearly not attributable to the proliferation of the probiotic.

[0145] 5.2 Exam Overview In all in vitro studies, the effectiveness of 10% Benzac positive control in containing infection by C. acnes has been demonstrated, with the percentage of pathogen population vitality reduced from 15% to 23%. As shown in replacement studies, once infection with C. acnes DSM 1897 begins within an insert, only medical devices have been proven to effectively inhibit its replication.

[0146] Exclusion and competition studies showed that treatment with LPC-S01 and LPC-S01 combined with 0.5% hyaluronic acid reduced C. acnes DSM 1897 infection by approximately 18%–19%. The efficacy of the probiotics is thought to be reasonably attributable to the ability of the strain to grow in the insert medium and cause slight acidification of the medium in the in vitro exclusion model, whereas this effect was not observed in the competition model due to the shorter incubation time, and therefore the significant pathogen inhibition by the probiotics must be attributable to a different mechanism.

[0147] The results of the substitution model showed a very slight positive effect of probiotics (a 3% reduction in pathogen population survival), although the effect was much lower than that observed in the two models previously examined.

[0148] The data obtained suggest that 0.5% hyaluronic acid does not cause any antagonistic effects against the probiotic strain LPC-S01, and that when combined with hyaluronic acid, it maintains its ability to effectively combat C. acnes DSM 1897 without any significant alteration.

[0149] Experiment Part Research objectives The objective of this study was to evaluate the effectiveness of the product "Infinite Skin Microbiome Serum" after 14 and 28 days of treatment through clinical assessment, instrumental measurement of skin moisture content, and self-report questionnaires.

[0150] Product and Instructions for Use One embodiment of the present invention is called "Infinite Skin Microbiome Serum" and comprises: water, propanediol, sodium hyaluronate, phenoxyethanol, lactic acid ferment, maltodextrin, 1,2-hexanediol, caprylyl glycol, sodium anate, and hydrolyzed hyaluronic acid. Volunteers were asked to apply the cosmetic as follows: Product revitalization: • Tear off the tab. • Push the plunger in with your palm (it is easier to do this on a hard, flat surface). Shake the bottle for 5-10 seconds to completely disperse the powder in the liquid.

[0151] keep: • Always store in the refrigerator after reactivation and after each application.

[0152] How to use: • Apply the product in the evening and leave it on overnight (as the final step in your nightly routine). Shake the bottle for 2-3 seconds before each application. Take two pipettes of serum, place it in your palm, and apply it to your face and neck; let it dry (the product will dry in a few minutes). I wash my face in the morning.

[0153] the purpose: The therapeutic effect after 14 and 28 weeks of use will be evaluated from the following perspectives: • Changes in skin moisture content as evaluated using a device with MoistureMeter SC (Delfin). Through clinical evaluations by dermatologists, we will examine changes in each volunteer's skin regarding the extent of acne-related facial areas, changes in the number of acne lesions, areas of redness (erythema), and skin dryness. • Evaluate the effects experienced by volunteers regarding treatment and side effects after 14 and 28 days of use. • Record images of the skin condition before and after treatment using a digital camera.

[0154] subject: 30 volunteers; male and female. Ages: 18-40 years. Healthy subjects with no skin conditions; they have no allergies and / or intolerances to cosmetics or medications. Characteristics: Subjects with acne-prone skin and mild to moderate acne.

[0155] subject: Volunteers participating in the study were selected from a panel of healthy subjects in the Modena area according to the following inclusion / exclusion criteria.

[0156] Inclusion criteria: Participants must be between 18 and 40 years old, have acne-prone skin, and exhibit mild to moderate acne. They must be healthy and free from skin conditions or allergies / intolerances to cosmetics or medications. Participants must agree to follow research procedures and respect administrative procedures.

[0157] Exclusion criteria: Subjects receiving topical or systemic treatment with drugs that may affect the results of efficacy studies; subjects with skin conditions; pregnant or breastfeeding women; subjects with intolerance to drugs and / or cosmetics.

[0158] limit: Throughout the study period, volunteers were asked not to apply any cosmetics other than those being studied to the skin area being analyzed.

[0159] dropout: One participant was eliminated.

[0160] Before the start of the study, investigators provided volunteers with the product to be tested and an information form. Each participant read and signed an informed consent form at the start of the study to participate. The following assessments were conducted at the start of treatment, 14 days later, and 28 days later: - The surface hydration level of the skin in a specified area of ​​the face is evaluated using instrumental measurement with MoistureMeter SC (Delfin). - A dermatologist will perform a clinical evaluation regarding the areas of the face where acne is a concern, the number of pimples, skin redness (erythema), and skin dryness. - Take pictures of your skin condition with a digital camera and record them.

[0161] Volunteers were asked to evaluate the treatment in terms of perceived efficacy and side effects, and completed questionnaires 14, 28, and 30 days after the procedure, following the end of the study.

[0162] Surface hydration evaluation: Instrumental measurements were performed on a designated area of ​​the face treated with the product during the test. For each volunteer, the following probe was used, and each step was repeated three times: MoistureMeter SC (Delfin). The probe measures the hydration level of the skin surface at the stratum corneum level.

[0163] Clinical evaluation: Clinical evaluations were performed by dermatologists according to the clinical scores shown in the table below. Clinical classification of facial region dilations: No area involved 0 * <25% of the face 25% to 50% of the face 2 > 50% of the face 3

[0164] Clinical classification of erythema: No evidence of erythema 0 * Almost inconspicuous erythema 0.5 Mild erythema 1 Moderate redness 2 Uniform, intense redness 3 Severe redness 4 * In T0, a value of 0 is considered an exclusion criterion.

[0165] Clinical classification of dry skin: No drying 0 Very slight dryness 0.5 Slight dryness 1 Moderate dryness 2 Severe dryness 3 Severe dryness with visible peeling 4

[0166] 1. Results from the instrument: Skin surface hydration (MoistureMeter SC-Delfin) The graph reported in Figure 20 shows the delta of instrumental evaluation data recorded for each volunteer from T0 to 14 and 28 days of cosmetic treatment. A Wilcoxon test was performed on the obtained data. This test can confirm whether there is a statistically significant therapeutic effect for nonparametric data (P=95%).

[0167] The graph in Figure 20 shows the skin surface hydration values ​​of the treatment area obtained from 29 volunteers.

[0168] Figure 20 shows the difference in mean values ​​between T14 and T28 compared to T0 for each volunteer. Statistically significant improvements in skin surface hydration were observed 14 and 28 days after treatment.

[0169] 2.Clinical results: Clinical evaluation of facial region expansion The graph in Figure 21 shows the delta of clinical evaluation data recorded by dermatologists for each volunteer, between T0 and 14 and 28 days after cosmetic treatment. A Wilcoxon test was performed on the obtained data. This test can confirm whether there is a statistically significant therapeutic effect for nonparametric data (P=95%).

[0170] The graph in Figure 21 (clinical evaluation of facial region dilation involved) shows the delta values ​​obtained from 29 volunteers.

[0171] 3.Clinical results: erythema The graph in Figure 22 shows the delta values ​​obtained from 29 volunteers. Clinically, a statistically significant reduction in erythema is recorded after 14 and 28 days of treatment.

[0172] 4.Clinical results: Number of papules and / or pustules The graph in Figure 23 shows the delta values ​​obtained from 29 volunteers. Clinically, a statistically significant reduction in the number of papules and / or pustules is recorded after 14 and 28 days of treatment.

[0173] 5.Clinical results: Dry skin The graph in Figure 24 shows the delta values ​​obtained from 29 volunteers. Clinically, a statistically significant reduction in skin dryness is recorded after 14 and 28 days of treatment.

[0174] 6. Results of self-awareness Based on 29 cases per sample, Figure 25 shows the results after 14 days of treatment and after 28 days of treatment.

[0175] Volunteer evaluations were subjected to analysis of variance (P=95%) and least significant difference (LSD P=95%). From a graphical perspective, the results of the LSD test are marked in the graph by applying one or more letters near the mean: for each item, if the same letter is not applied, the two steps are considered statistically distinguishable with a probability of P ≥ 95%.

[0176] conclusion (i) Instrumental and clinical conclusions Instrumental measurements performed using MoistureMeter SC (Delfin) in a panel of participating volunteers at both 14 and 28 days post-treatment showed a statistically significant increase in skin hydration. Clinical assessments performed by dermatologists showed a statistically significant reduction in the extent of acne-related facial areas, as well as statistically significant reductions in skin redness, the number of papules and / or pustules, and skin dryness.

[0177] (ii) Conclusion of self-awareness Overall, the volunteers 14 days and 28 days after the treatment highly evaluated the treatment. The volunteers fully agreed to recognize the improvement of the skin regarding the redness / inflammation in the area with acne. The degree of agreement to the opinions of "feeling that the skin condition has improved" and "acne has decreased and looks better" is good. This treatment also showed good tolerance to all the subjects who participated in the study (no harmful skin reactions were observed during the entire 28-day test period).

[0178] Effects of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) + Hyaluronic Acid (HA) Dry Cap Mask on Wound Healing, NFκB Expression, and Skin Aging Purpose of the study The purpose of this project is to investigate the effectiveness of hyaluronic acid mask (HA) as a skin care product in the presence of probiotic Lactobacillus paracasei m. biome LIVESKIN88 (DSM 33788). In particular, the synergistic effect of the two components is evaluated in the following aspects: - Wound healing - Adhesion / colony formation characteristics - NFkB activation - Antioxidant properties - Antipathogenic properties

[0179] Materials and methods HaCaT Adhesion Assay The adhesion of Liveskin88 to the HaCaT cell layer was evaluated using changes in light, as described (Guglielmetti, 2008). Briefly, HaCaT cells were grown in Dulbecco's modified Eagle medium (MEM) supplemented with 10% (v / v) heat-inactivated fetal bovine serum, 100 U ml-1 penicillin, 100 mg ml-1 streptomycin, 0.1 mM non-essential amino acids, and 2 mM L-glutamine, and incubated at 37°C under a 95% air and 5% carbon dioxide atmosphere. The monolayer was washed three times with pH 7.3 phosphate-buffered saline (PBS) to release unbound bacteria, and the cells were fixed by incubation with 3 ml of methanol at room temperature for 8 minutes. Subsequently, the cells were stained with 3 ml of Giemsa stain (1:20; Carlo Erba, Milano, Italy) and left in the dark at room temperature for 30 minutes. Finally, the monolayer was washed three times with PBS, dried in an incubator for one hour, and examined under a microscope (magnification 400x) under oil immersion.

[0180] In vitro HaCaT wound healing Wound healing in response to HA mask stimulation was evaluated by a scratch assay against a HaCaT cell monolayer. Briefly, 5 × 10 5 HaCaT cells were seeded into each well of a culture insert and incubated at 37°C in a humidified atmosphere containing 5% CO2. After 24 hours, the culture insert was gently removed using sterile forceps and wounded into a single layer. Photographs of the wound area were taken immediately before irritation (at hour 0) and at selected incubation times to monitor wound closure. The percentage of wound closure was calculated as (initial area - final area) / initial area × 100.

[0181] NF-κB activation assay Activated nuclear factor κB (NF-κB) was studied using recombinant HaCaT cell lines stably transfected with the vector pNiFty2-Seap (InvivoGen, Labogen, Rho, Italy). Briefly, recombinant Caco-2 monolayer (approximately 5 × 10⁻¹⁴) 5 50 μg ml of cells per well-1 The cells were cultured in the presence of zeosin, washed with 0.1 M Tris-HCl buffer (pH 8.0), and then suspended in fresh DMEM containing 100 mM HEPES (pH 7.4) in a z5 × 10⁶ solution. 7 Cells were incubated with the Lacticaseibacillus paracasei m. Biome LIVESKIN88 (DSM 33788) product, and an MOI of approximately 100 was obtained. To evaluate the immunomodulatory properties of each product component, HA and the complete mask (HA + Lacticaseibacillus paracasei m. Biome LIVESKIN88 (DSM 33788)) were diluted 1:20 in PBS and added to a HaCa T cell monolayer.

[0182] The stimulation is 20 ng ml -1 This was performed by adding TNF-α. After incubation at 37°C for 4 hours, SEAP in the supernatant was elucidated using Quanti-Blue reagent (Invivogen) according to the manufacturer's protocol and quantified at 655 nm OD. All measurements were performed using a microplate reader (Multiskan SkyHigh, Thermo Fisher Scientific). Two independent experiments were performed three times for each condition.

[0183] (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity The free radical scavenging activity of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) was investigated using several modified DPPH assays. Briefly, 1x10 9 A probiotic suspension at a concentration of cells / mL was added to a 1 mM DPPH solution in a 96-well plate. The mixture was incubated at room temperature for 30 minutes and protected from light. DPPH changes color from purple to pale yellow when it accepts hydrogen atoms from the antioxidant sample. Absorbance was measured at 517 nm using a microplate reader. 1 μg / mL ascorbic acid was used as a positive control.

[0184] ROS generation in HaCaT cells Intracellular ROS concentrations were evaluated using the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. HaCaT cells were placed in 96-well plates in a 3.0 × 10⁶ format. 4 Cells were seeded in wells for 24 hours. After incubation, the culture medium was removed. Cells were washed with excess PBS and pretreated with 10 μmol / L DCFH-DA in serum-free medium at 37°C for 45 minutes. Next, cells were washed with excess PBS and treated with a 1500 μmol / L probiotic suspension in serum-free medium and H2O2 at 37°C. Finally, ROS levels were measured after 3 hours using a microplate reader at excitation wavelengths of 485 nm and emission wavelengths of 485 nm and 530 nm.

[0185] Antimicrobial assay Antimicrobial activity was measured using the agar spot test method. Briefly, each isolate from LAB was spotted onto MRS agar and incubated under anaerobic conditions at 37°C for 72 hours. Next, 6 ml of soft MRS agar (0.9% agar) inoculated with 1 ml of Staphylococcus aureus strain cultured overnight was placed on a plate. The activity of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) was studied in comparison to the activity of the following strains: L. rhamnosus GG, L. acidophilus LA5, and L. paracasei Shirota. Chlorexidine was used as a control.

[0186] Results and Discussion The effect of the Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) mask on wound healing. Skin injuries can be caused by a variety of reasons, including trauma (cuts, abrasions, chemical burns, burns, cold, heat, radiation, surgery, etc.) or as a result of underlying conditions such as diabetes. The most effective wound management strategies are to prevent infection, promote healing, and prevent excessive scarring.

[0187] Traditional monolayer cultures are simple, inexpensive, and yield results relatively quickly. However, monolayers of cells such as human epidermal keratinocytes are typically destroyed using sterile wound instruments (scratch assays), which is a common drawback in understanding the mechanisms governing wound healing. One of the most commonly used cell line models in wound healing research is HaCaT, a keratinocyte cell line spontaneously mutated from immortalized adult skin.

[0188] Several pieces of evidence support the idea that HA is a key component of the extracellular matrix involved in various biological processes, including proliferation and migration. To address the hypothesis of a synergistic effect between HA and Lacticaseibacillus paracasei m. Biome LIVESKIN88 (DSM 33788) cosmetic in the regeneration process of human skin, we tested its effect on proliferation in vitro using HaCa T cells. While there is some published literature on the use of models to simulate wound healing, there is currently no published literature on the use of probiotics.

[0189] First, complete mask (Lacticaseibacillus paracasei m. Biome LIVESKIN88 (DSM 33788) freeze-dried powder (8x10) 9 The wound healing-promoting capacity of CFU / g was measured. To do this, different dilutions of the finished product were used to assess the toxicity of the excipients and to assess their ability to promote tissue regeneration after mechanical stress (Figure 26).

[0190] The results show that the application of the complete mask (HA + Lactobacillus paracasei m. biome LIVESKIN88 (DSM 33788)) promotes wound healing by scratching the confluent monolayer of damaged HaCaT cells. Despite the promotion of monolayer regeneration by a 1:10 dilution, the decrease in the % closure after 24 hours indicates toxicity towards cell proliferation / migration. The best results regarding the % closure of the HaCaT monolayer were obtained with a 1:20 dilution, with slower but higher tissue regeneration being achieved. Furthermore, this dilution mimics the exposure of the facial mask to the actual face, considering 7 applications of the product per bottle.

[0191] To investigate whether the effect of regeneration is due to the effect of bacterial cells or HA, several experiments were performed using the 1:20 HA mask alone and Lactobacillus paracasei m. biome LIVESKIN88 (DSM 33788) at various bacterial concentrations.

[0192] To investigate the effect of probiotic intoxication in the HA mask, the promotion of wound healing after 4 hours was monitored using various concentrations of Lactobacillus paracasei m. biome LIVESKIN88 (DSM 33788) resuspended in the 1:20 diluted mask.

[0193] Figure 27 As shown, the use of high concentrations of cells (9 and 8 logCFU / mL) resuspended in the 1:20 mask resulted in no or a lower degree of wound closure of the tissue. Presumably, this is due to the effect of the low pH (between 4.5 and 5) of the tested suspension, caused by the mask excipient in which a large amount of lyophilized Lactobacillus paracasei m. biome LIVESKIN88 (DSM 33788) was dissolved.

[0194] Good results were obtained at lower concentrations (7 and 6 logCFU / mL). These data, in particular, demonstrate the synergistic effect of the combination of HA masks and Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788). Specifically, 7 logCFU / mL corresponds to the bacterial cell concentration of a complete mask diluted 1:20. No significant difference was detected between the last two dilutions.

[0195] Adhesion ability of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) to HaCaT cells Adhesion to the host is a classic selection criterion for potential probiotic bacteria, potentially leading to transient colonization that enhances immunomodulatory effects and stimulates skin barrier and metabolic functions. Probiotic bacteria are well known to play a potential protective role against pathogens through various mechanisms, including the production of antimicrobial compounds, reduction of pathogenic bacterial adhesion, and competition for host cell binding sites.

[0196] Competitive exclusion, that is, competition among microorganisms for nutrients and binding sites, and the production of antimicrobial substances, are important functions of a normal and healthy skin microbiome in inactivating pathogens. Probiotics have been shown to have a similar effect, potentially preventing the binding of some pathogens by adhering to the skin.

[0197] To evaluate the "enhancing effect" of HA on the adhesion of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788), the adhesion characteristics of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) to HaCaT cells were evaluated in the presence and absence of an HA mask (Figure 28).

[0198] The results showed that resuspension of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) in an HA mask ensured improved adhesion properties of the bacterial cells. In fact, the adhesion index obtained with bacterial cells resuspended in the presence of HA was higher compared to the value obtained with cells resuspended in PBS. This improvement is likely due to the mechanical effect of HA, which creates a layer of HaCaT skin model that enhances the adhesion stability of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) cells.

[0199] The Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) mask modulates NF-κB activation of HaCa T cells under inflammatory stimuli. NF-κB constitutes a family of inducible transcription factors that are important regulators of the host's immune and inflammatory responses. Because NF-κB activates the transcription of several pro-inflammatory cytokines, it is closely involved in inflammatory processes in the skin. Skin pathologies include epidermal hyperplasia, hyperkeratosis, parakeratosis, loss of the granular layer, T cell infiltration, and microabscess formation. These features are considered characteristic of psoriasis, a human inflammatory skin disease, and the central role of T cells is well established.

[0200] The anti-inflammatory properties of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788), resuspended in HA products, were evaluated using a HaCaT / NF-κB reporter system. Experiments were performed by stimulating recombinant HaCaT cell layers for 4 hours in the presence of TNF-α-induced pro-inflammatory stimulation at baseline.

[0201] The addition of TNF-α approximately doubled the activation level of NF-κB (Figure 29). In particular, in both cases, the presence of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) or HA reliably reduced NF-κB activation compared to the inflammatory state (a decrease of 27% and 13%, respectively). The combined use of both components yielded potent and statistically significant effects, suggesting an additive or synergistic effect between Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) and HA.

[0202] The effect of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) on ROS production in HaCaT cells under oxidative stress. The term reactive oxygen species (ROS) refers to free radicals, primarily derived from oxygen molecules, and several other chemically reactive molecules that are generated during the stepwise reduction of oxygen molecules. Probiotic strains have been reported to remove hydroxyl radicals and superoxide anions, producing antioxidants. The most widely studied strains today are Bifidobacterium and Lactobacillus.

[0203] The DPPH assay is based on the ability of a sample to scavenge DPPH radicals. In this study, different probiotic suspensions (Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788), L. paracasei DG, and L. paracasei Shirota) were used to evaluate ROS scavenging activity compared to ascorbic acid, which was used as a positive control.

[0204] Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) and L. paracasei DG showed the best performance in terms of scavenging effect. These results suggest that Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) has a potent radical scavenging effect that contributes to reducing oxidative stress in the skin (Figure 30). To confirm the effect of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) on intracellular ROS levels, HaCaT keratinocytes treated with a probiotic suspension were induced with H2O2. (Figure 31) Subsequently, ROS levels were analyzed using DCF-DA solution. In untreated cells, intracellular ROS levels in HaCaT keratinocytes were significantly increased by H2O2 at a concentration of 1500 μmol / L (Ctrl+). Cells treated with Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) showed an inhibitory effect on H2O2-induced ROS. The evidence suggests that Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) protects HaCaT keratinocytes by removing ROS generated in response to H2O2 exposure.

[0205] The effect of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) on Staphylococcus aureus infections. Antimicrobial activity against pathogens is another important characteristic to consider when selecting potential probiotic strains to maintain a healthy microbial balance in the skin.

[0206] In this analysis, five Lactobacillus strains were examined, all of which showed high antagonistic activity against pathogenic Staphylococcus aureus strains that are pathogenic to human skin. Lactobacillus paracasei m. biome LIVESKIN88 (DSM 33788) showed high antagonistic activity against the tested pathogenic strains and demonstrated higher growth inhibition ability than the reference strain L. casei Shirota (Figure 32).

[0207] conclusion The data obtained showed a synergistic effect between the HA mask and Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788): The mask + probiotic combination showed that the probiotic Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) had a positive effect on HaCaT epidermal formation, and simultaneously, HA had a positive effect on the adhesion properties of Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788).

[0208] The same synergistic effect was also demonstrated in the regulation of NFκB expression, highlighting the excellent immunomodulatory activity of the probiotic product. Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) also showed antioxidant properties due to its potent scavenging effect against ROS normally produced by skin aging. Furthermore, Lacticaseibacillus paracasei m. biome LIVESKIN88 (DSM 33788) exhibits a potent antibacterial effect against Staphylococcus aureus, making it a valuable agent for the treatment and prevention of infections.

Claims

1. - Lacticaceibacillus paracasei m. biome LIVESKIN88-DSM 33788; and - Hyaluronic acid or its salts; A pharmaceutical composition for wound healing, comprising:

2. A bacterial strain for use in the prophylactic or therapeutic treatment of skin infections and / or inflammations caused by Staphylococcus aureus, wherein the bacterial strain is Lacticaseibacillus paracasei m. biome LIVESKIN88-DSM 33788.

3. To prevent skin aging, - Lacticaceibacillus paracasei m. biome LIVESKIN88-DSM 33788; and, if applicable, - Hyaluronic acid or its salts; Dermatological or cosmetic compositions, including those mentioned above.

4. A pharmaceutical composition for preventing or treating skin infections and / or inflammations caused by Staphylococcus aureus, comprising Lacticaseibacillus paracasei m. biome LIVESKIN88-DSM 33788.

5. The pharmaceutical composition according to claim 4, further comprising at least one pharmaceutically acceptable excipient.

6. A cosmetic composition containing Lacticaseibacillus paracasei m. biome LIVESKIN88-DSM 33788 for removing radicals and reducing oxidative stress on the skin.

7. A cosmetic composition containing hyaluronic acid and Lacticaseibacillus paracasei m. biome LIVESKIN88-DSM 33788 for promoting the regeneration of human skin.

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