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    • 专利摘要:
    The present invention is directed to the direct application of beneficial or probiotic bacteria to the skin to aid in maintaining a healthy skin microbiota and repairing an unbalanced skin microbiota. This application is based on the use of selected Lactobacillus strains as antipathogenic agents, in particular L. plantarum, L. pentosus and / or L. rhamnosus against common skin pathogens, the acids produced such as lactic acid being important antimicrobial factors.
    公开号:BE1024425B1
    申请号:E2016/5454
    申请日:2016-06-21
    公开日:2018-02-20
    发明作者:Sarah Lebeer；Ingmar Claes；Eline OERLEMANS；DEN BROECK Marianne VAN
    申请人:Yun NV；
    IPC主号:
    专利说明:

    DERMATOLOGICAL PREPARATIONS FOR MAINTENANCE ΕΝ OR REPAIR OF HEALTHY SKIN MICROBIOTA
    DOMAIN OF THE INVENTION
    The present invention is directed to the direct topical application of beneficial or probiotic bacteria to the skin to aid in maintaining a healthy skin microbiota and repairing an unbalanced skin microbiota. This restoration of a healthy microflora falls under the term probiotherapy, defined as the use of useful microorganisms or probiotics to restore a healthy microflora at the place where the microbial imbalance or dysbiosis is situated. This application is based on the use of selected Lactobacillus strains as antipathogenic agents, in particular L. plantarum, L. pentosus and / or L. rhamnosus against common skin pathogens, the acids produced such as lactic acid being important antimicrobial factors.
    BACKGROUND OF THE INVENTION
    Consequently, it was an object of the present invention to provide a solution to persons suffering from skin disorders that are linked to a deviant microbial balance on the skin. For this it was established that the topical application of L. plantarum, L. pentosus and / or L. rhamnosus to the skin is very effective in repairing and / or maintaining a healthy skin microbiota, and is therefore very suitable for improving skin conditions in patients who need it.
    Oral formulations with Lactobacillus strains have previously been used in the treatment of skin conditions such as atopic dermatitis. However, oral administration versus direct local administration are different routes of administration and each have a very different underlying mechanism. With oral administration, a beneficial effect on general health is expected through immunostimulation, in contrast to direct dermatological or topical (skin) administration where competition with 'unwanted' microorganisms will occur.
    Just like the gastrointestinal tract, our skin contains a unique microbial ecosystem. The type of microorganisms that occur on the skin depends on a combination of host factors, environmental factors but also the topographic location. The role of the microbiota in skin disorders is still not fully unraveled. However, it appears that some skin conditions are linked to a disturbed microbiota as antimicrobial treatments can improve clinical symptoms (Grice & Segre 2011). For example, a correlation with the presence of Propionibacterium acnes has been found with acne vulgaris (Beylot et al. 2014). Although acne vulgaris is a multifactorial disorder, influenced by hormonal factors, among other things, this P. acnes appears to induce inflammation resulting in inflamed pimples, also called papules or pustules. Since P. acnes is also found on healthy skin where the bacteria does not cause acne, this implies that other factors play a role, and together they cause the composition of the skin microbiota to be disrupted towards an excessive growth of this bacterium.
    Another example of a skin condition where the microbiota appears to be important is dandruff (Wang et al. 2015; Sugita et al. 2015; Grice & Segre 2011). In people with dandruff, the Malassezia fungus is often over-represented. Indications that this fungus is a possible cause of the condition come from the fact that antimycotic treatment improves symptoms. In contrast, antibacterial therapies do not reduce dandruff. Again, other factors are expected to be involved in this skin condition, but the correlation with Malassezia is intriguing. Similar to dandruff, fungal skin infections with Candida albicans or dermatophytes such as Trichophyton spp. Skin disorders that are linked to skin microbiota dysbiosis since these species are also present on healthy subjects. In the case of Tinea pedis or "Athlete's Foot", overgrowth of Trichophyton rubrum and T. mentagrophytes is often observed.
    The production of lactic acid in combination with any other antimicrobial components such as bacteriocins appears to protect against the above infections and dysbioses and lactic acid appears to be effective against bacteria, fungi and even viral pathogens. It is for this reason that lactobacilli are considered important in the homeostasis of the dynamic dermatological ecosystem. Potential health promotion mechanisms of lactobacilli are i) maintaining a healthy skin pH (+ / - 5.5), in particular through the production of lactic acid; ii) the production of antimicrobial components and competitive exclusion of pathogens; iii) modulation of the immune response and iv) the enhancement of the epithelial barrier.
    Therefore, it was an object of the present invention to provide a solution for persons suffering from dermatological conditions linked to a deviant microbial balance of the skin. For this it was established that topical use on the skin of L. plantarum, L. pentosus and / or L. rhamnosus species is very effective in repairing and / or maintaining a healthy microflora on the skin, and is therefore very suitable for relieving of dermatological disorders in patients in need thereof.
    Oral formulations with Lactobacillus strains have previously been used in the treatment of dermatological conditions. However, oral administration versus direct local administration are different routes of administration and each have a very different underlying mechanism. With oral administration, a beneficial effect on general health is expected through immunostimulation, in contrast to direct dermatological or topical (skin) administration where competition with 'unwanted' microorganisms will occur.
    SUMMARY OF THE INVENTION
    In a first aspect, the present invention provides a topical dermatological formulation with one or more living Lactobacillus species; wherein at least one of the Lactobacillus species is L. plantarum; more particularly, a L. plantarum strain with at least 97% sequence identity with SEQ ID NO: 4 in the 16S rRNA gene.
    In a further aspect, the present invention provides a living Lactobacillus species for use in restoring and / or maintaining a healthy skin microbiota, via topical administration, with L. plantarum as Lactobacillus species; more particularly, a L. plantarum strain with at least 97% sequence identity with SEQ ID NO: 4 in the 16S rRNA gene.
    In yet another aspect, the present invention provides the use of one or more living Lactobacillus species, in the preparation of a topical skin formulation for repairing and / or maintaining a healthy skin microbiota; wherein at least one of the Lactobacillus species is L. plantarum; more particularly, a L. plantarum strain with at least 97% sequence identity with SEQ ID NO: 4 in the 16S rRNA gene.
    The present invention also provides a method for repairing and / or maintaining a healthy skin microbiota; with at least one step by topically administering to an individual an effective amount of one or more living Lactobacillus species; wherein at least one of the Lactobacillus species is L. plantarum; more particularly, a L. plantarum strain with at least 97% sequence identity with SEQ ID NO: 4 in the 16S rRNA gene.
    In yet another aspect, the present invention provides a formulation containing one or more living Lactobacillus species for use in restoring and / or maintaining a healthy skin microbiota, by topical route, wherein Lactobacillus species is selected from the list of L. plantarum, L. pentosus and L. rhamnosus; more in particular a L. plantarum strain with at least 97% sequence identity with SEQ ID N ° 4 in the 16S rRNA gene, an L. pentosus strain with at least 97% sequence identity with SEQ ID N ° 1 in the 16S rRNA gene and a L. rhamnosus strain with at least 97% sequence identity with SEQ ID NO: 5 in the 16S rRNA gene.
    The present invention further provides a Lactobacillus strain that was deposited as L. rhamnosus YUN-S1.0 under accession number LMG P-29611 (deposited at BCCM May, 12, 2016).
    In one aspect, the present invention provides a formulation containing one or more Lactobacillus strains as defined above.
    In a specific dosage form, the formulation of the present invention is a topical dermatological composition, more particularly in the form of a gel, cream, foam, lotion or ointment.
    In another specific embodiment, the present invention provides the Lactobacillus strain as defined above or a formulation as defined above; for use in repairing and / or maintaining a healthy skin microbiota, via topical administration.
    In one aspect, the present invention provides a topical application to the skin of one or more living Lactobacillus species via probiotherapy; said Lactobacillus species being selected from the list consisting of L. plantarum, L. pentosus and L. rhamnosus; more in particular, said probiotherapy consists of restoring and / or maintaining a healthy skin microbiota in a patient in need thereof.
    In another particular application, said Lactobacillus species are in the topical applications, methods and compositions as described herein, Lactobacillus strains selected from the list consisting of L. plantarum YUN-V2.0 deposited under deposit number LMG P-29456 ( deposited with BCCM on March 9, 2016), L. pentosus YUN-V1.0 deposited under deposit number LMG P-29455 (deposited with BCCM on March 9, 2016); and L. rhamnosus YUN-S1.0 deposited under "deposit nunber" LMG P-29611 (deposited at BCCM on May 12, 2016).
    BRIEF DESCRIPTION OF THE FIGURES
    Referring to the accompanying figures, it is emphasized that the details shown are merely exemplary and illustrative of the discussion of the various embodiments of the present invention. They are provided in the context of what is considered the most useful and easy description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description with the drawings is intended to show those skilled in the art how the various forms of the invention can be practiced.
    FIG. 1: Characteristics of lactobacilli with respect to growth, production of D and L lactic acid (LA) and lowering of the pH of the medium.
    FIG. 2: Analysis of the antipathogenic effect of culture supernatant ('spent culture supernatant' or SCS) of selected lactobacilli against Propionibacterium acnes. Growth of the bacteria (optical density at 600 nm; Y axis) is measured as a function of time (X axis). Each graph shows repetitions of the growth of P. acnes. It is clear to see that without the addition of antibiotics or SCS, P. acnes begins to grow rapidly (NC1).
    By analogy with adding erythromycin to 50pg / ml, SCS of all lactobacilli causes inhibition of P. acnes growth as opposed to SCS of Streptococci and staphylococci that does not inhibit P. acnes growth. * Erythromycin (50ug / ml); # Erythromycin (5ug / ml); § Minocycline (20 pg / ml) NC1 = medium control; NC2 = MRS at pH4.3; Numbers 1 to 22 = lactobacilli strains (for details see Table 1); St = Streptococcus thermophilus; Ss = Streptococcus salivarius; Se = Staphylococcus epidermidis; 10.5 = 0.5% Tween 80; T1 = 1% Tween 80.
    DETAILED DESCRIPTION OF THE INVENTION
    The present invention is based on the discovery of specific Lactobacillus strains that can compete with the growth of Propionibacterium acnes, Candida albicans, Malassezia spp., Trichophyton spp. and bacteria or fungi associated with skin conditions such as acne vulgaris, dandruff, tinea pedis or other fungal or skin infections. The selected strains are generally referred to herein as "YUN" strains and can compete with the skin pathogens and thereby restore a healthy skin microbiota. This restoration of a healthy microflora falls under the term probiotherapy, defined as the use of useful microorganisms or probiotics that restore a healthy microflora at the place where the microbial dysbiosis takes place.
    Accordingly, in a first aspect, the present invention provides a topical dermatological formulation containing one or more living Lactobacillus species; wherein at least one of the Lactobacillus species is L. plantarum; more particularly, a L. plantarum strain with at least 97% sequence identity with SEQ ID NO: 4 in the 16S rRNA gene.
    The composition of the present invention may further contain Lactobacillus species as selected, for example, from the non-limiting list: L. pentosus, L. gasseri, L. crispatus, L. acidophilus, L. jensenii, L. fermentum, L. rhamnosus.
    In the context of the present invention, the term "topical" is understood to mean: local delivery to a particular location of the body, especially administration to a specified location on or within the body. In particular, it covers the applications via non-solid formulations such as creams, foams, gels, lotions or ointments. The term "topical" is not intended to include dosage forms of solid preparations such as capsules, tablets, ...
    Accordingly, the following is understood by the term "topical dermatologically"; the local delivery of non-solid formulations directly on the skin. Preferably, the compositions of the present invention are applied over a large surface of the skin to be most effective.
    In the context of the present invention, the term "living Lactobacillus species" means viable Lactobacillus species, and non-fragments, culture supernatans, or killed forms thereof.
    In a further aspect, the present invention provides a living Lactobacillus species for use as skin probiotherapy, by topical route, wherein the Lactobacillus species is L. plantarum; more particularly, a L. plantarum strain with at least 97% sequence identity with SEQ ID NO: 4 in the 16S rRNA gene. As already defined above, wherein probiotherapy is intended for the recovery and / or maintenance of a healthy skin microbiota in a patient in need thereof.
    Persons who may benefit from this probiotherapy are, for example, persons with skin disorders linked to a disturbed skin microbiota possibly due to bacterial or fungal infections and / or dysbioses due to overgrowth of certain pathogenic microorganisms, such as acne vulgaris, tinea pedis, rose, rosaceae, impétigo. ..
    Accordingly, in a further aspect, the present invention provides the use of one or more living Lactobacillus species, in the preparation of a topical skin formulation for restoring and / or maintaining a healthy skin microbiota; wherein at least one of the Lactobacillus species is L. plantarum; more particularly, a L. plantarum strain with at least 97% sequence identity with SEQ ID NO: 4 in the 16S rRNA gene.
    The present invention also provides a method for repairing and / or maintaining a healthy skin microbiota; with at least one step by topically administering an effective amount of one or more living Lactobacillus species to an individual; wherein at least one of the Lactobacillus species is L. plantarum; more particularly, a L. plantarum strain with at least 97% sequence identity with SEQ ID NO: 4 in the 16S rRNA gene.
    In yet another aspect, the present invention provides a formulation containing one or more living Lactobacillus species for use in restoring and / or maintaining a healthy skin microbiota, via topical administration, wherein Lactobacillus species are selected from the list consisting of L. plantarum L. pentosus and L. rhamnosus; more in particular a L. plantarum strain with at least 97% sequence identity with SEQ ID NO: 4 in the 16S rRNA gene; an L. pentosus strain having at least 97% sequence identity with SEQ ID N ° 1 in the 16S rRNA gene and an L. rhamnosus strain having at least 97% sequence identity with SEQ ID N ° 5 in the 16S rRNA gene.
    The invention further provides a Lactobacillus strain selected from the list consisting of L. pentosus YUN-V1.0 deposited under deposit number LMG P-29455 (deposited at BCCM on March 9, 2016); L. plantarum YUN-V2.0 deposited under deposit number LMG P-29456 (deposited at BCCM on March 9, 2016); and L. rhamnosus YUN-S1.0 deposited under deposit number LMG P-29611 (deposited at BCCM on May 12, 2016).
    The microbiological 'deposits' mentioned here are made at BCCM / LMG or the Belgian coordinated collections of micro-organisms with correspondence address: Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium.
    Lactobacillus pentosus YUN-V1.0 is a single colony isolate obtained in our lab after subcultivation of a strain, originally a vaginal isolate of a healthy woman. The 16S rRNA gene sequence (SEQ ID N ° 1) for strain L. pentosus YUN-V1.0 was determined by PCR with primers 8F (5T - AGAGTTTGATCCTGGCTCAG-3 SEQ ID N 0 2) and 1525R (51 -AAGGAGGTGATCCAGCCGCA-3 SEQ ID N ° 3). YUN-V2.0 and YUN-V3.0 are single colony isolates obtained in our lab after subcultivation of Lactobacillus plantarum strains originally isolated from human saliva and a corn silo, respectively. The 16S rRNA gene sequence (SEQ ID N ° 4) for L. plantarum strain YUN-V2.0 was determined by PCR with primers 8F (51 - AGAGTTTGATCCTGGCTCAG-3 '- SEQ ID N ° 2) and 1525R (51 -AAGGAGGTGATCCAGCCGCA-3 SEQ ID NO 3). YUN-S1.0 is a single colony isolate obtained in our lab after subcultivation of a Lactobacillus rhamnosus strain that was originally isolated from a healthy person. The 16S rRNA gene sequence (SEQ ID NO: 5) for strain L. rhamnosus YUN-S1.0 was determined by PCR with primers 8F (5 '-AGAGTTTGATCCTGGCTCAG-3 SEQ ID NO 2) and 1525R (5' - AAGGAGGTGATCCAGCCGCA-3 SEQ ID N ° 3).
    These specific "YUN" strains can be used as such or preferably formulated in products with these strains. Said products are dermatological administration forms in particular in the form of non-solid formulations such as creams, foams, gels, lotions or ointments.
    In particular, the present invention provides the "YUN" strains defined above for use in probiotherapy, i.e., for repairing and / or maintaining a healthy skin microbiota.
    In yet another aspect, the present invention provides a topical application of one or more living Lactobacillus species as skin probiotherapy; wherein said Lactobacillus species are selected from the list consisting of L. plantarum, L. pentosus and L. rhamnosus; more in particular, said probiotherapy comprises restoring and / or maintaining a healthy skin microbiota in a patient in need thereof.
    In a specific application, the Lactobacillus species in the topical applications, methods and compositions as described herein are Lactobacillus strains selected from the list consisting of L. plantarum YUN-V2.0 deposited under deposit number LMG P-29456 (deposited with BCCM on March 9, 2016); L. pentosus YUN-V1.0 deposited under deposit number LMG P-29455 (deposited with BCCM on March 9, 2016); and L. rhamnosus YUN-S1.0 deposited under deposit number LMG P-29611 (deposited at BCCM on May 12, 2016).
    EXAMPLES
    MATERIAL AND METHODS
    Bacterial strains and growing condltles
    Lactobacillus strains (Table 1) were grown at 37 ° C in the Man, Rogosa and Sharpe (MRS) medium (Carl Roth). All bacteria were grown non-shaking and inoculated from glycerol stocks (-80 ° C). Solid media contain 1.5% (w / v) agar.
    Table 1: The bacterial strains used in this study
    Preparation of spent culture supernatant (SCS) from the selected strains
    To obtain culture supernatant (SCS) with the secreted active antimicrobial components,
    growth medium specifically for each species inoculated from a preculture and incubated for 24 hours. SCS was obtained by centrifugation for 30 minutes at 6797 g (8000 rpm) at 4 ° C. The SCS was then filtered sterilized (0.20 µm cellulose acetate, VWR).
    Antimicrobial activity of live lactobacilli in co-cultures with Malassezia furfur, Trichophyton rubrum, Propionibacterium acnes and Candida albicans.
    The antimicrobial activity of the selected bacteria was investigated by standard antimicrobial tests with some minor changes. The antimicrobial activity of the selected bacteria was investigated by a "spot assay" (Schillinger and Lücke 1989). Briefly, 1-3 μΐ of each culture was spotted on an agar plate. These plates were incubated for 24 to 72 hours depending on the strain. An overnight culture of the pathogen was then diluted in 7 ml of 'soft agar' of the medium of the pathogen and poured onto the plates with the 'spots' of the selected strains. The plates were incubated overnight at 30-37 ° C, after which the inhibition zones were measured. A spot of miconazole (for fungi) and / or 0.1% hexetidine and / or tetracycline (for Propionibacterium acnes) was added to the plate as a positive control before the 'soft agar' was poured. 'Radial diffusion' test of SCS from lactobacilli
    In addition, the antimicrobial activity of the culture supernatant (SCS) was investigated with a protocol as previously described for competition testing between lactobacilli and gastrointestinal pathogens (Coconnier et al. 1997). Miconazole (for fungi) and tetracycline (for Propionibacterium acnes) were used as a positive control. Sterile growth medium was used as a negative control.
    Antlmicrobial activity of SCS of the selected strains against Candida, Propionibacterium acnes, Malassezia furfur and Trichophyton spp (further referred to as 'pathogens') using spectrophotometric determinations over time
    Spectrophotometric determinations over time were also performed as previously described (De Keersmaecker et al. 2006) with minor modifications. Briefly, an overnight culture of the bacterium was added to the wells of a microtiter plate filled with 50-80% of the appropriate medium and 50-5% SCS lactobacilli. MRS at pH 4.3 and antibiotics and antimycotics in the correct concentration were used as a negative and as a positive control, respectively. Bacteria or fungi were grown and the optical density (OD) was measured at 590 nm every 30 minutes for 3 days with a Synergy HTX multi-mode reader (Biotek). Each test was measured at least in triplicate and the average OD was calculated. The antimicrobial activity was expressed as the relative optical density achieved after 24 hours (stationary phase) compared to the negative controls.
    Antibiogram using Kirby-Bauer test
    Sensitivity to antibiotics was evaluated using the Kirby-Bauer diffusion test. Briefly, antibiotics were spotted on paper slices and the bacterial inhibition zone was measured on agar plates. The antibiotics tested were erythromycin, normocin, tetracycline, ampicillin and clindamycin at relevant concentrations.
    Clinical study ('proof-of-concept') he patients with acne vulgaris
    A proof-of-concept clinical study was conducted on 20 patients with acne vulgaris. The patients were men between 12-25 years of age with mild inflammatory acne. The purpose of this proof-of-concept study was to assess the effect of a topical probiotic cream (with + -10-8 colony forming units (CFU) of L. pentosus YUN-V1.0, + -10-8 CFU L plantarum YUN-V2.0 and + -10-8 CFU L. rhamnosus YUN-S1.0 per application of 1 g of the topical cream ACN) to be assessed on the skin microbiota and on the acne. Patients were asked to apply the cream twice a day for 56 days (8 weeks). The patients were seen by a dermatologist at the start (before therapy) and at week 4, week 8 and week 10. A skin smear was taken with each visit. Bacterial DNA was isolated from these samples by the commercial Mobio Powersoil kit (see 'Human Microbiome Project'). Isolated DNA was analyzed via 16S rRNA amplicon sequencing with MiSeq Illumina and a bioinformatic analysis was performed. In addition, a clinical scoring was performed and a photo was taken with each visit.
    Clinical study ('proof-of-concept') he patients with tinea pedis ('swimmer's eczema')
    A proof-of-concept clinical study was conducted on 20 patients with tinea pedis. The patients were between 18-65 years old with tinea pedis. The purpose of this proof-of-concept study was to assess the effect of a topical probiotic cream (with + -10-8 colony forming units (CFU) of L. pentosus YUN-V1.0, + -10-8 CFU L plantarum YUN-V2.0 and + -10-8 CFU L. rhamnosus YUN-S1.0 per application of 1 g of the topical cream FNG) to be assessed on the skin microbiota and the Trichophyton infection. Patients were asked to request the cream twice a day for 56 days (8 weeks). The patients were seen by a dermatologist at the start (before therapy) and at week 4, week 8 and week 10. A skin smear was taken with each visit. Bacterial DNA was isolated from these samples by the commercial Mobio Powersoil kit (see 'Human Microbiome Project'). Isolated DNA was analyzed via 16S rRNA amplicon sequencing with MiSeq Illumina and a bioinformatic analysis was performed. For analysis of the presence of the fungi, the smears were also plated on Trichophyton-specific medium (medium represented by BCCM). Colony PCR was performed using universal ITS ('internai transcribed région') primers ITS1 (SEQ ID NO 6) (5 '-TCCGTAGGTGAACCTGCGG-3') and ITS4 (SEQ ID NO 7) (5 '-TCCTCCGCTTATTGATATGC-3') , followed by sequencing to identify the fungi. In addition, a clinical scoring was performed and a photo was taken with each visit.
    RESULTS
    Growth characteristics and lactate production
    Potentially useful or probiotic strains were characterized in terms of growth characteristics, lactate production and the possibility of lowering the pH of the medium. These properties are expected to be important for antipathogenic activity. These data show that Lactobacillus pentosus YUN-V1.0, L. plantarum YUN-V2.0 and L. rhamnosus YUN-S1.0 produce the highest amount of lactic acid (Fig 1).
    Antipathogenic activity against Propionibacterium acnes
    Kinetic experiments were performed for the analysis of the antimicrobial activity of culture supernatans (SCS) of the selected strains against Propionibacterium acnes. SCS of all lactobacilli tested inhibited the growth of Propionibacterium acnes while SCS of other bacterial species such as Streptococcus thermophilus and S. salivarius, both also lactic acid bacteria, and Staphyloccus epidermidis did not inhibit the growth of P. acnes. This suggests that the species and strain specific properties of the selected lactobacilli are important for the antipathogenic activity against P. acnes (Fig 2).
    Antipathogenic activity against Malassezia, Trichophyton and Candida
    In the next phase, the beneficial or probiotic bacteria were screened for the antipathogenic effect against specific skin pathogens. The results of the spot assays against Malassezia furfur, Trichophyton rubrum and Candida albicans are shown in Tables 2, 3 and 4 respectively.
    Table 2: "Spot assay" of the selected lactobacilli against Malassezia furfur.
    * three independent repetitions
    Table 3: "Spot assay" of the selected lactobacilli against Trichophyton rubrum.
    * three independent repetitions
    Table 4: "Radial diffusion assay" of the selected lactobacilli against Candida albicans.
    * three independent repetitions
    Culture supernatant from L. pentosus YUN-V1.0 and L. plantarum YTJN-V2.0 was also tested in radial diffusion assays and proved to be effective in inhibiting the growth of Malassezia, Trichophyton and Candida. L. rhamnosus YUN-S1.0 was not as effective in inhibiting the growth of Malassizia but of Trichophyton and Candida.
    Antiblogram using Kirby-Bauer test
    The selected bacteria were also tested for their sensitivity to antibiotics to prevent the spread of antibiotic resistance genes. All lactobacilli were sensitive to erythromycin, normocin, tetracycline, ampicillin and clindamycin, except L. plantarum 5057, which was found to be not sensitive to tetracycline. Therefore, strain L. plantarum 5057 was not considered suitable as a strain for probiotherapy.
    REFERENCES
    Beylot, C. et al., 2014. Propionibacterium acnes: an update on its role in the pathogenesis of acne. Journal of the European Academy of Dermatology and Venereology: JEADV, 28 (3), pp. 271-8.
    Chan, R.C. et al., 1985. Compétitive exclusion of uropathogens from human uroepithelial cells by
    Lactobacillus whole cells and cell wall fragments. Infection and immunity, 47 (1), pp.84-9.
    Chan, R. C., Bruce, A.W. & Reid, G., 1984. Adherence of cervical, vaginal and distal urethral normal microbial flora to human uroepithelial cells and the inhibition of adherence or gram-negative uropathogens by competitive exclusion. The Journal of urology, 131 (3), pp. 596-601.
    Grice, E.A. & Segre, J.A., 2011. The skin microbiome.
    Nature reviews. Microbiology, 9 (4), pp. 244-53.
    Reid, G., 1999. The Scientific Basis for Probiotic Strains or Lactobacillus. Appl. Envir. Microbiol., 65 (9), pp.37 63-37 66.
    Reid, G. & Bruce, A. W., 2001. Selection of lactobacillus strains for urogenital probiotic applications. The Journal of infectious diseases, 183 Suppl, pp. 77-80.
    Sugita, T. et al., 2015. Temporal changes in the skin Malassezia microbiota or members of the Japanese Antarctic Research Expedition (JARE): A case study in Antarctica as a pseudo-space environment. Medical mycology, 53 (7), pp. 717-24.
    Wang, L. et al., 2015. Characterization of the major bacterial-fungal populations colonizing dandruff scalps in Shanghai, China, shows microbial disequilibrium. Experimental dermatology, 24 (5), pp. 398-400.
    LIST OF SEQUENCIES
    <110> YUN NV
    <120> DERMATOLOGICAL PREPARATIONS FOR MAINTENANCE ΕΝ / OR REPAIR OF HEALTHY SKIN MICROBIOTA <130> AXC-007 <160> 7 <170> BiSSAP 1.3
    <210> 1 <211> 1406 <212> RNA <213> Lactobacillus pentosus <220> <223> 16S rRNA sequence <400> 1 cttaggcggc tggttcctaa aaggttaccc caccgacttt gggtgttaca aactctcatg 60 gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg ctgatccgcg 120 attactagcg attccgactt catgtaggcg agttgcagcc tacaatccga actgagaatg 180 gctttaagag attagcttac tctcgcgagt tcgcaactcg ttgtaccatc cattgtagca 240 cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac cttcctccgg 300 tttgtcaccg gcagtctcac cagagtgccc aacttaatgc tggcaactga taataagggt 360 tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac aaccatgcac 420 cacctgtatc catgtccccg aagggaacgt ctaatctctt agatttgcat agtatgtcaa 480 gacctggtaa ggttcttcgc gtagcttcga attaaaccac atgctccacc gcttgtgcgg 540 gcccccgtca attcctttga gtttcagcct tgcggccgta ctccccaggc ggaatgctta 600 atgcgttagc tgcagcactg aagggcggaa accctccaac acttagcatt catcgtttac 660 ggtatggact accagggtat ctaatcctgt ttgctaccca tactttcgag cctcagcgtc 720 agttacagac cagacagccg ccttcgccct cgttctctctctctctctctctctc a 780 ccgctacaca tggagttcca ctgtcctctt ctgcactcaa gtttcccagt ttccgatgca 840 cttcttcggt tgagccgaag gctttcacat cagacttaaa aaaccgcctg cgctcgcttt 900 acgcccaata aatccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta 960 gttagccgtg gctttctggt taaataccgt caatacctga acagttactc tcagatatgt 1020 tcttctttaa caacagagtt ttacgagccg aaacccttct tcactcacgc ggcgttgctc 1080 catcagactt tcgtccattg tggaagattc cctactgctg cctcccgtag gagtttgggc 1140 cgtgtctcag tcccaatgtg gccgattacc ctctcaggtc ggctacgtat cattgccatg 1200 gtgagccgtt accccaccat ctagctaata cgccgcggga ccatccagaa gtgatagccg 1260 aagccatctt tcaaactcgg accatgcggt ccaagttgtt atgcggtatt agcatctgtt 1320 tccaggtgtt gcc tgggggcgtcgggccgtcgtcggcggggcggcggcggcggggcggcggcggcgggcgggccg
    <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <22 0> <223> Primer 8F <400> 2 agagtttgat cctggctcag 20
    <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <22 0> <223> Primer 1525R <400> 3 aaggaggtga tccagccgca 20
    <210> 4 <211> 1425 <212> RNA <213> Lactobacillus plantarum <22 0> <223> 16S rRNA <400> 4 ggttcctaaa aggttacccc accgactttg ggtgttacaa actctcatgg tgtgacgggc 60 ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga ttactagcga 120 ttccgacttc atgtaggcga gttgcagcct acaatccgaa ctgagaatgg ctttaagaga 180 ttagcttact ctcgcgagtt cgcaactcgt tgtaccatcc attgtagcac gtgtgtagcc 240 caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt ttgtcaccgg 300 cagtctcacc agagtgccca acttaatgct ggcaactgat aataagggtt gcgctcgttg 360 cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc acctgtatcc 420 atgtccccga agggaacgtc taatctctta gatttgcata gtatgtcaag acctggtaag 480 gttcttcgcg tagcttcgaa ttaaaccaca tgctccaccg cttgtgcggg cccccgtcaa 540 ttcctttgag tttcagcctt gcggccgtac tccccaggcg gaatgcttaa tgcgttagct 600 gcagcactga agggcggaaa ccctccaaca cttagcattc atcgtttacg gtatggacta 660 ccagggtatc taatcctgtt tgctacccat actttcgagc ctcagcgtca gttacagacc 720 agacagccgc cttcgccact ggtgattactcg cctcgcctcg agttccac tgtcctcttc tgcactcaag tttcccagtt tccgatgcac ttcttcggtt 840 gagccgaagg ctttcacatc agacttaaaa aaccgcctgc gctcgcttta cgcccaataa 900 atccggacaa cgcttgccac ctacgtatta ccgcggctgc tggcacgtag ttagccgtgg 960 ctttctggtt aaataccgtc aatacctgaa cagttactct cagatatgtt cttctttaac 1020 aacagagttt tacgagccga aacccttctt cactcacgcg gcgttgctcc atcagacttt 1080 cgtccattgt ggaagattcc ctactgctgc ctcccgtagg agtttgggcc gtgtctcagt 1140 cccaatgtgg ccgattaccc tctcaggtcg gctacgtatc attgccatgg tgagccgtta 1200 ccccaccatc tagctaatac gccgcgggac catccaaaag tgatagccga agccatcttt 1260 caagctcgga ccatgcggtc caagttgtta tgcggtatta gcatctgttt ccaggtgtta 1320 tcccccgctt ctgggcaggt ttcccacgtg ttactcacca cagcggcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgccgcgcgcgccggcggcggcggcggccgccgc
    <210> 5 <211> 1403 <212> RNA <213> Lactobacillus rhamnosus <22 0> <223> 16S rRNA <400> 5 gtcgacgagt tctgattatt gaaaggtgct tgcatcttga tttaattttg aacgagtggc 60 ggacgggtga gtaacacgtg ggtaacctgc ccttaagtgg gggataacat ttggaaacag 120 atgctaatac cgcataaatc caagaaccgc atggttcttg gctgaaagat ggcgtaagct 180 atcgcttttg gatggacccg cggcgtatta gctagttggt gaggtaacgg ctcaccaagg 240 caatgatacg tagccgaact gagaggttga tcggccacat tgggactgag acacggccca 300 aactcctacg ggaggcagca gtagggaatc ttccacaatg gacgcaagtc tgatggagca 360 acgccgcgtg agtgaagaag gctttcgggt cgtaaaactc tgttgttgga gaagaatggt 420 cggcagagta actgttgtcg gcgtgacggt atccaaccag aaagccacgg ctaactacgt 480 gccagcagcc gcggtaatac gtaggtggca agcgttatcc ggatttattg ggcgtaaagc 540 gagcgcaggc ggttttttaa gtctgatgtg aaagccctcg gcttaaccga ggaagtgcat 600 cggaaactgg gaaacttgag tgcagaagag gacagtggaa ctccatgtgt agcggtgaaa 660 tgcgtagata tatggaagaa caccagtggc gaaggcggct gtctggtctg taactgacgc 720 tgaggctcga aagcatgggt caggagggggggggggggggggg atgaatgc taggtgttgg agggtttccg cccttcagtg ccgcagctaa cgcattaagc 840 attccgcctg gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca 900 caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac 960 atcttttgat cacctgagag atcaggtttc cccttcgggg gcaaaatgac aggtggtgca 1020 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080 tatgactagt tgccagcatt tagttgggca ctctagtaag actgccggtg acaaaccgga 1140 ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta 1200 caatggatgg tacaacgagt tgcgagaccg cgaggtcaag ctaatctctt aaagccattc 1260 tcagttcgga ctgtaggctg caactcgcct acacgaagtc ggatecgcta gtaatcgcgg 1320 atcagcacgc cgcgggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggg
    <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <22 0> <223> Primer Sequence <400> 6 tccgtaggtg aacctgcgg 19
    <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer Sequence <400> 7 tcctccgctt attgatatgc 20
    权利要求:
    Claims (12)
    [1]

    A topical dermatological formulation containing one or more living Lactobacillus species; wherein at least one of the Lactobacillus species is a L. plantarum strain with at least 97% sequence identity with SEQ ID N ° 4 in the 16S rRNA gene.
    [2]
    2. A living Lactobacillus species for use in restoring and / or maintaining healthy skin microbiota, by topical administration, wherein the Lactobacillus species is a L. plantarum strain with at least 97% sequence identity with SEQ ID N ° 4 in the 16S rRNA gene.
    [3]
    Use of one or more living Lactobacillus species, in the preparation of a topical dermatological formulation for the repair and / or maintenance of a healthy skin microbiota; wherein at least one of the Lactobacillus species is a L. plantarum strain with at least 97% sequence identity with SEQ ID N ° 4 in the 16S rRNA gene.
    [4]
    4. Method for repairing and / or maintaining a healthy skin microbiota; with at least one step by topically administering an effective amount of one or more living Lactobacillus species to an individual; wherein at least one of the Lactobacillus species is a L. plantarum strain with at least 97% sequence identity with SEQ ID N ° 4 in the 16S rRNA gene.
    [5]
    A formulation containing one or more live Lactobacillus species for use in restoring and / or maintaining a healthy skin microbiota, by topical administration, wherein a Lactobacillus species is selected from the list consisting of L. plantarum, L. pentosus and L rhamnosus; wherein the L. plantarum is an L. plantarum strain with at least 97% sequence identity with SEQ ID N ° 4 in the 16S rRNA gene, wherein the L. pentosus is an L. pentosus strain with at least 97% sequence identity with SEQ ID N 0 1 in the 16S rRNA gene and the L. rhamnosus is an L. rhamnosus strain with at least 97% sequence identity with SEQ ID NO: 5 in the 16S rRNA gene.
    [6]
    6. A Lactobacillus strain that is L. rhamnosus YUN-S1.0 that was deposited under deposit number LMG P-29611 (deposited at BCCM on 12 May 2016).
    [7]
    7. A formulation containing a Lactobacillus strain as defined in claim 6. 8 · The formulation according to claim 7; wherein the formulation is a topical dermatological formulation.
    [9]
    The use according to claim 3 or the formulation according to any of claims 1, 5, 7 or 8 wherein the formulation is a topical dermatological formulation in the form of a gel, cream, ovule, suppository, foam, lotion or ointment.
    [10]
    10. A Lactobacillus strain according to claim 6 or a formula rhinu vols.s one dor claims 7 nf 8: for use in repairing and / or maintaining a healthy skin microbiota, by topical administration.
    [11]
    11. Topical application of one or more living Lactobacillus species in skin probiotherapy; wherein said Lactobacillus species are selected from the list consisting of L. plantarum, L. pentosus and L. rhamnosus.
    [12]
    12. Topical application according to claim 11; wherein probiotherapy consists of restoring and / or maintaining a healthy skin microbiota in a patient in need thereof.
    [13]
    Topical use according to any of claims 11 or 12, the use according to claim 3, with the method as defined in claim 4, the living Lactobacillus species as defined in claim 2, or the formulation according to claim 5; said Lactobacillus species being a Lactobacillus strain selected from the list consisting of L. plantarum YUN-V2.0 deposited under deposit number LMG P-29456; L. pentosus YUN-V1.0 with deposit number LMG P-29455; and L. rhamnosus YUN-S1.0 with deposit number LMG P-29611.
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    同族专利:
    公开号 | 公开日
    US20190344100A1|2019-11-14|
    WO2017220525A1|2017-12-28|
    BE1024425A1|2018-02-14|
    CA3027458A1|2017-12-28|
    US20210361978A1|2021-11-25|
    BE1024425A9|2018-03-20|
    CN110121353A|2019-08-13|
    BE1024425B9|2018-03-26|
    EP3471836A1|2019-04-24|
    JP2019522649A|2019-08-15|
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    法律状态:
    2018-03-07| FG| Patent granted|Effective date: 20180220 |
    优先权:
    申请号 | 申请日 | 专利标题
    BE20165454A|BE1024425B9|2016-06-21|2016-06-21|DERMATOLOGICAL PREPARATIONS FOR MAINTENANCE AND / OR REPAIR OF HEALTHY SKIN MICROBIOTA|BE20165454A| BE1024425B9|2016-06-21|2016-06-21|DERMATOLOGICAL PREPARATIONS FOR MAINTENANCE AND / OR REPAIR OF HEALTHY SKIN MICROBIOTA|
    CA3027458A| CA3027458A1|2016-06-21|2017-06-20|Dermatological preparations for maintaining and/or restoring healthy skin microbiota|
    PCT/EP2017/065006| WO2017220525A1|2016-06-21|2017-06-20|Dermatological preparations for maintaining and/or restoring healthy skin microbiota|
    CN201780050648.1A| CN110121353A|2016-06-21|2017-06-20|Dermatological preparations for the skin microbial group that maintains and/or get well|
    US16/311,321| US20190344100A1|2016-06-21|2017-06-20|Dermatological preparations for maintaining and/or restoring healthy skin microbiota|
    JP2018566402A| JP2019522649A|2016-06-21|2017-06-20|Dermatological preparations for the maintenance and / or recovery of healthy skin microflora|
    EP17732379.7A| EP3471836A1|2016-06-21|2017-06-20|Dermatological preparations for maintaining and/or restoring healthy skin microbiota|
    US17/392,895| US20210361978A1|2016-06-21|2021-08-03|Dermatological preparations for maintaining and/or restoring healthy skin microbiota|
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