New composition for Anti-age treatment of skin

Abstract

The present invention relates to the treatment and / or prevention of skin aging. More specifically, the invention relates to the administration of live microorganisms to improve skin appearance and decrease sights of skin aging.

Description

[0001] New composition for anti-age treatment of skin

[0002] Technical field of the invention

[0003] The present invention is related to compositions and method for treatment of skin aging, in particular, to topical compositions and methods of use. The present invention is related to topical compositions that are comprising viable microorganisms. In particular, the present invention relates to compositions, and methods of topical use of compositions, comprising live probiotic microorganisms for improving skin appearance and decrease sights of skin aging.

[0004] In particular, the present invention relates to novel compositions comprising viable microorganisms with active metabolism on skin.

[0005] Background of the invention

[0006] This invention relates to a composition, a use of said composition and a method for use to prevent or treat skin aging or photodamage. The composition comprises live probiotic microorganisms being able to reverse the signs of aging skin.

[0007] During aging, the skin undergoes major clinical and morphological changes. The skin physiologically ages due to both intrinsic and extrinsic factors, known as chronological aging and photoaging, respectively. Intrinsic factors are driven by telomer shortening and cell senescence, while extrinsic factors such as ultraviolent light and pollution cause reactive oxygen species (ROS) and free radicals.

[0008] Ageing, the process of gradual decline whilst growing older affect everyone. One of the most visually prominent results of ageing can be seen on the skin. The skin loses its firmness and elasticity, resulting in sagging, more apparent pores, and wrinkles.

[0009] Additionally, the turnover number of the cells in the skin lowers with the skin seeming less radiant, rougher in appearance and it becomes drier as a result from reduced sebocyte activity, with an increasing number of pigment spots.

[0010] Ageing of the skin can be considered on several levels such as phenotypically (as described above), histologically and / or molecularly. Histologically, the junction between the dermis and epidermis becomes flattened and melanocytes density decreases. The dermis thickness decreases, accompanied by decrease in both vascularization and number of mast cells and fibroblasts. The reduced elasticity and firmness of the skin are a result of changes in the extracellular matrix (ECM) of the dermis, such as altered production and location of the fibres collagen and elastane. The glandular activity also decreases, resulting in less sebum and subsequently more dry skin. Cellu larly, ageing occurs due to accumulation of senescent cells, change in cell cycles and accumulation of free radicals / oxidative stress and changes in glycation patterns, specifically in that of collagen. Signs of aging, such as dryness, roughness, pigmented spots, and wrinkles, as well as decreased skin barrier and altered biomechanical functions are the main histological changes.

[0011] With the increasing age of the westernized population, treatments that can prevent and rejuvenate signs of skin ageing are receiving increased interest due cosmetic and economic potential.

[0012] The above-mentioned age related changes of the skin occur due to intrinsic and extrinsic factors, where intrinsic factors include chronological and genetical factors, whereas extrinsic factors are attributed to environmental effects, such as sun (termed photoaging), pollution exposure and smoking. Especially sun exposure can result in generation of reactive oxygen species (ROS) and free radicals ending with oxidative stress and subsequent cellular damage. Additionally, a recent study by Howard et al. (2022) found that the diversity of the skin microbiome increased in all sites sampled with age, but the abundance of Lactobacillus decreased, indicating that the skin microbiome may also play a role in skin ageing.

[0013] Ageing occurs due to a wide variety of factors, which intricately interact and affect each other. These changes are not only cosmetically displeasing to some but can result in protective barrier issues and subsequent medical issues. With the increasing age of the population, especially in the westernized world, there is a huge cosmetic and economic interest in reversing / preventing the appearance of aging-signs.

[0014] With the increasing age of the westernized population, treatments that can prevent and rejuvenate signs of skin ageing are receiving increased interest due cosmetic and economic potential. Anti-ageing efficacy on facial skin can be evaluated by assessing subepidermal low-echogenic band (SLEB) thickness, dermal density, skin firmness and elasticity, skin hydration, TEWL, crow's feet wrinkles, spots, smoothness and complexion radiance and comparing to baseline measurements. SLEB has previously shown to quantify cutaneous senescence, with the thickness expressing the degree of cutaneous ageing (Gniadecka et al 1994). Therefore, SLEB has shown to work well in quantifying ageing, especially in photo-exposed areas, such as the malar region. Skin lipid / bacterial genera correlations may be important for the age-dependent change of the skin microbiome. In the present invention, viable lactic acid bacteria producing antimicrobial metabolites and organic acids, are applied topical in a lipid formulation to modulate the skin microbiome and reverse aging. Applying viable bacteriocin producing microorganisms has surprisingly shown that these can improve skin appearance and decrease the sign of aging. The age-associated decrease in Lactobacillus is thus anticipated to result also in a decrease of bacteriocin producing Lactobacillus and thereby allowing for an increase in diversity of the skin microbiome. Applying viable Lactobacillus to aging skin will thus increase the abundance of Lactobacillus in the microbiome and by metabolism on skin, these Lactobacillus produced metabolites will decrease the diversity of the microbiome to reflect a younger skin microbiome.

[0015] Summary of the invention

[0016] The present invention relates to compositions for topical use comprising live isolated microorganisms or the methods of use of live isolated microorganisms for treatment or prevention of skin aging.

[0017] In one aspect of the invention the composition is for treatment or prevention of skin aging measurable as reduction in at least one of the following parameters: wrinkles, pigmentation, sub-epidermal low-echogenic band (SLEB) thickness or trans epidermal water loss (TEWL).

[0018] Or as increase in at least one of the following parameters: dermal density, skin hydration, skin elasticity, complexion radiance or smoothness.

[0019] In another aspect of the invention is a method of a skin treatment for treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging.

[0020] This invention is based upon multiple mechanisms of action caused by viable species of lactic acid bacteria which can decrease sign of aging.

[0021] An aspect of the present invention relates to a composition for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging where the composition comprising at least one live isolated microorganism.

[0022] Another aspect of the present invention relates to a composition for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the composition comprising at least one live isolated microorganism, by providing : (a) a reduction in wrinkles of at least 5 percent;

[0023] (b) a reduction in pigmentary spots density of at least 5 percent; and / or

[0024] (c) an increase in firmness measured as a reduction in suction distance of more than 5 percent.

[0025] A further aspect of the present invention relates to a composition for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the composition comprising at least 2 live lactic acid bacteria for use in the topical treatment of skin wherein an increase in dermal density of at least 5 percent is obtained after daily topical treatment for 28 days.

[0026] An even further aspect of the present invention relates to a composition for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the composition comprising at least one live isolated microorganism, by providing :

[0027] (a) a reduction in sub-epidermal low-echogenic band (SLEB) thickness of at least 2 percent; and / or

[0028] (b) a reduction in TEWL of at least 2 percent.

[0029] Yet an aspect of the present invention relates to a composition for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the composition comprising at least one live isolated microorganism, by providing :

[0030] (a) an increase in complexion radiance of more than 10 percent; and / or

[0031] (b) an increase in smoothness of more than 10 percent.

[0032] A further aspect of the present invention relates to a method of a skin treatment comprising administering for at least 28 days a live topical composition comprising at least one isolated lactic acid bacteria for treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the method provides:

[0033] (a) a reduction in wrinkles of at least 5 percent;

[0034] (b) a reduction in pigmentary spots density of at least 5 percent; and / or

[0035] (c) an increase in firmness measured as a reduction in suction distance of more than 5 percent.

[0036] A further aspect of the present invention relates to a method of a skin treatment comprising administering for at least 28 days a live topical composition comprising at least one isolated lactic acid bacteria for treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the method provides:

[0037] (a) an increase in dermal density of at least 5 percent;

[0038] (b) an increase in skin hydration of more than 10 percent; and / or (c) an increase in skin elasticity of more than 2 percent.

[0039] A further aspect of the present invention relates to a method of a skin treatment comprising administering for at least 28 days a live topical composition comprising at least one isolated lactic acid bacteria for treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the method provides:

[0040] (a) a reduction in sub-epidermal low-echogenic band (SLEB) thickness of at least 2 percent; and / or

[0041] (b) a reduction in TEWL of at least 2 percent.

[0042] A further aspect of the present invention relates to a method of a skin treatment comprising administering for at least 28 days a live topical composition comprising at least one isolated lactic acid bacteria for treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the method provides:

[0043] (a) an increase in complexion radiance of more than 10 percent; and / or

[0044] (b) an increase in smoothness of more than 10 percent.

[0045] A further aspect of the present invention relates to live lactic acid bacteria for use in the topical treatment of skin wherein a reduction in sub-epidermal low-echogenic band (SLEB) thickness of at least 2 percent is obtained after daily topical treatment for 28 days.

[0046] Yet an aspect of the present invention relates to live lactic acid bacteria for use in the topical treatment of skin wherein the lactic acid bacteria after daily topical treatment for 28 days provides:

[0047] (a) an increase in dermal density of at least 5 percent; and / or

[0048] (b) an increase in skin hydration of more than 10 percent; and / or

[0049] (c) an increase in skin elasticity of more than 2 percent.

[0050] The present invention will now be described in more detail in the following.

[0051] Detailed description of the invention

[0052] Definitions

[0053] Prior to discussing the present invention in further details, the following terms and conventions will first be defined :

[0054] The term "Skin aging" refers to the measurable signs of aging of skin which can be both visible by clinical evaluation and / or measurable. The typical signs of skin aging includes wrinkles, sagging, pigmentation or enlarged pore size, the skin appears less radiant, rougher, and becomes dry.

[0055] Efficacy measured as an anti-age improvement of at least one of the following parameters; SLEB thickness (pm), Dermal Density (pixel / mm2), Skin firmness(RO) (pm), Skin elasticity (R2) (%), Skin hydration (AU), TEWL (g / h / m2), Crow's feet wrinkles score, Spots score, Smoothness of the skin score, or Complexion radiance score.

[0056] In a preferred embodiment of the invention the anti-age improvement is measured as reduction in SLEB, wherein the SLEB thickness is reduced at least 2%, at least 3%, at least 4%, at least 5% or at least 6% after 56 days.

[0057] In a preferred embodiment of the invention the anti-age improvement is measured as increase in Dermal Density (pixel / mm2), wherein the dermal density is increased at least 5%, at least 10% or at least 15% after 56 days.

[0058] In a preferred embodiment of the invention the anti-age improvement is measured as increase in Skin firmness as determined as a reduction in suction distance (RO) (pm) of at least 5%, at least 8%, at least 10% or at least 12% after 56 days.

[0059] In a preferred embodiment of the invention the anti-age improvement is measured as increase in Skin elasticity (R2)(%), wherein the skin elasticity is increased at least 2%, at least 4% or at least 6% after 56 days.

[0060] In a preferred embodiment of the invention the anti-age improvement is measured as increase in skin hydration (AU), wherein the skin hydration is increased at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% after 56 days.

[0061] In a preferred embodiment of the invention the anti-age improvement is measured as decrease in TEWL (g / h / m2), wherein the TEWL is decreased at least 2%, at least 3%, or at least 4% after 56 days.

[0062] In a preferred embodiment of the invention the anti-age improvement is measured as decrease in Crow's feet wrinkles score (%), wherein the wrinkles are reduced at least 10%, at least 15%, or at least 20% after 56 days. In a preferred embodiment of the invention the anti-age improvement is measured as decrease in pigmentary spots score (%), wherein the pigment spot density is reduced at least 5%, at least 10%, at least 15%, at least 20% or at least 25% aft er 56 days.

[0063] In a preferred embodiment of the invention the anti-age improvement is measured as increase in Smoothness of the skin score, wherein the increase in smoothness of the skin score is at least 10%, at least 15%, at least 20%, at least 25% or at least 30% after 56 days.

[0064] In a preferred embodiment of the invention the anti-age improvement is measured as increase in Complexion radiance score, wherein the increase in complexion radiance score is at least 10%, at least 20%, at least 25%, at least 30% or at least 40% after 56 days.

[0065] In a preferred embodiment of the invention an anti-age improvement of the skin is measured for at least two parameters selected from the following : SLEB thickness (pm), Dermal Density (pixel / mm2), Skin firmness(RO) (pm), Skin elasticity (R2) (%), Skin hydration (AU), TEWL (g / h / m2), Crow's feet wrinkles score, Spots score, Smoothness of the skin score, or Complexion radiance score.

[0066] In a preferred embodiment of the invention an anti-age improvement of the skin is measured for at least three parameters selected from the following: SLEB thickness (pm), Dermal Density (pixel / mm2), Skin firmness(RO) (pm), Skin elasticity (R2) (%), Skin hydration (AU), TEWL (g / h / m2), Crow's feet wrinkles score, Spots score, Smoothness of the skin score, or Complexion radiance score.

[0067] In a preferred embodiment of the invention an anti-age improvement of the skin is measured for at least four parameters selected from the following : SLEB thickness (pm), Dermal Density (pixel / mm2), Skin firmness(RO) (pm), Skin elasticity (R2) (%), Skin hydration (AU), TEWL (g / h / m2), Crow's feet wrinkles score, Spots score, Smoothness of the skin score, or Complexion radiance score.

[0068] In a preferred embodiment of the invention an anti-age improvement of the skin is measured for at least five parameters selected from the following: SLEB thickness (pm), Dermal Density (pixel / mm2), Skin firmness(RO) (pm), Skin elasticity (R2) (%), Skin hydration (AU), TEWL (g / h / m2), Crow's feet wrinkles score, Spots score, Smoothness of the skin score, or Complexion radiance score.

[0069] In a preferred embodiment of the invention an anti-age improvement of the skin is measured for at least six parameters selected from the following : SLEB thickness (pm), Dermal Density (pixel / mm2), Skin firmness(RO) (pm), Skin elasticity (R2) (%), Skin hydration (AU), TEWL (g / h / m2), Crow's feet wrinkles score, Spots score, Smoothness of the skin score, or Complexion radiance score.

[0070] In a preferred embodiment of the invention an anti-age improvement of the skin is measured for at least seven parameters selected from the following: SLEB thickness (pm), Dermal Density (pixel / mm2), Skin firmness(RO) (pm), Skin elasticity (R2) (%), Skin hydration (AU), TEWL (g / h / m2), Crow's feet wrinkles score, Spots score, Smoothness of the skin score, or Complexion radiance score.

[0071] In a preferred embodiment of the invention an anti-age improvement of the skin is measured for at least eight parameters selected from the following: SLEB thickness (pm), Dermal Density (pixel / mm2), Skin firmness(RO) (pm), Skin elasticity (R2) (%), Skin hydration (AU), TEWL (g / h / m2), Crow's feet wrinkles score, Spots score, Smoothness of the skin score, or Complexion radiance score.

[0072] In a preferred embodiment of the invention an anti-age improvement of the skin is measured for at least nine parameters selected from the following : SLEB thickness (pm), Dermal Density (pixel / mm2), Skin firmness(RO) (pm), Skin elasticity (R2) (%), Skin hydration (AU), TEWL (g / h / m2), Crow's feet wrinkles score, Spots score, Smoothness of the skin score, or Complexion radiance score.

[0073] In a preferred embodiment of the invention an anti-age improvement of the skin is measured for at all the following parameters: SLEB thickness (pm), Dermal Density (pixel / mm2), Skin firmness(RO) (pm), Skin elasticity (R2) (%), Skin hydration (AU), TEWL (g / h / m2), Crow's feet wrinkles score, Spots score, Smoothness of the skin score, and Complexion radiance score.

[0074] Mode of action (MoA) of anti-ageing in topically applied microorganisms are not understood yet, but several hypotheses are possible. These include, but are not limited to, local immune modulation, resulting in reduced amount senescence and systemic effects, direct cell (keratinocyte) stimulation resulting in ECM modulation and increased collagen and elastane, ending with proper ECM build and structure. This can be mediated via mTOR mammalian signalling, antioxidant effects resulting in alleviation of oxidative stress which accumulates with age, albeit other mode of actions could be entirely possible. Moisturizing effects resulting in less cellular stress and better barrier function. mTOR signalling is targeted by the bacterial metabolite rapamycin and is known to have anti-ageing effects. This signalling pathway is also intricately interconnected with immune signalling, meaning that for MoA, all of the above effects may be possible at once, indicating multiple mode of actions.

[0075] Additionally, it is possible that microbiome modulation may affect ageing parameters. The amount of Lactobacillus species is decreased with age. This maybe age-related changes in physiology which result in subsequent microbiome change, but perhaps the microbiome may be able to modulate host physiology, thereby providing a more hospitable environment for them, allowing for colonization. Therefore, applying live microorganisms topically to skin can play a regulatory role in aging, further reaffirming that microbiome modulation may affect ageing.

[0076] Increased inflammation has been associated with aging, termed "inflammageing". The applied microorganisms can by acting as an immune stimulant, alleviate this age associated inflammation, resulting in a skin phenotype more similar to that of younger skin.

[0077] Another MoA can be by restoring acidic pH of the skin, alleviate oxidative stress, attenuate, and even restore photoaged skin, improve barrier function of skin. Having both an effect on the skin cells as well as these effects can work as modulators of the skin microbiome, and thereby indirectly improve skin appearance.

[0078] The present invention relates to topical application of live microorganisms.

[0079] The preferred microorganisms are in particular bacteria. The probiotic bacteria is preferably selected from the group comprising Lactiplantibacillus plantarum, Lactococcus lactis, Lacticaseibacillus rhamnosus, Lactobacillus helveticus, Lactobacillus jensenii, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus amylovorus, Lactobacillus amylolyticus, Lactobacillus alimentarius, Lactobacillus aviaries, Lactobacillus delbrueckii, Lactobacillus diolivorans, Lactobacillus farciminis, Lactobacillus gallinarum, Lacticaseibacillus easel, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus hilgardii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus mucosae, Lactobacillus panis, Lactiplantibacillus paraplantarum, Lactobacillus pontis, Latilactobacillus sake!, Lactobacillus saliverius, Lactobacillus sanfraciscensis, Lacticaseibacillus paracasei, Lactobacillus pentosus, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus coryniformis, Lactobacillus curvatus, Levilactobacillus brevis, Lactobacillus buchneri, Lactobacillus fructivorans, Lactobacillus hilgardii, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus ingluviei, Weissella viridescens, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium animalis, Carnobacterium divergens, Corynebacterium glutamicum, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Oenococcus oeni, Pasteuria nishizawae, Pediococcus acidilactici, Pediococcus dextrin icus, Pediococcus parvulus, Pediococcus pentosaceus, Probionibacterium freudenreichii, Probionibacterium acidipropoinici, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus clausii, Bacillus coagulans, Bacillus flexus, Bacillus fusiformis, Bacillus lentus, Bacillus licheniformis, Bacillus mega-terium, Bacillus mojavensis, Bacillus pumilus, Bacillus smithii, Bacillus subtilis, Bacillus vallismortis, Geobacillus stearothermophilus or mutants thereof.

[0080] In another aspect of the invention the probiotic microorganism is selected from the genera related to the natural healthy skin microbiome including genera Lactobacillus, Lactiplantbacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, Lentilactobacillus, LeuconostocProbionibacterium, Cutibacterium, Staphylococcus, Corynebacterium, Malassezia, Aspergillus, Cryptococcus, Rhodotorula, and / or Epicoccum. In another preferred embodiment of the invention the probiotic strain is Staphylococcus epidermidis, Staphylococcus hominis, Cutibacterium acnes (Probionibacterium acnes) or any combinations thereof.

[0081] In a preferred embodiment of the invention the probiotic strain is a Gram-positive bacteria. In one preferred embodiment of the invention the composition comprises at least one strain selected from the group consisting of Lactiplantibacillus plantarum LB356R (DSM 33094), Lactiplantibacillus plantarum LB244R (DSM 32996), Weissella viridescens LB10G (DSM 32906), Lacticaseibacillus paracasei LB113R (DSM 32907), Lacticaseibacillus paracasei LB116R (DSM 32908), Levilactobacillus brevis LB152G (DSM 32995), Lacticaseibacillus paracasei LB28R (DSM 32994), Enterococcus faecium LB276R (DSM 32997), Leuconostoc mesenteriodes LB349R (DSM 33093), Lactiplantibacillus plantarum LB316R (DSM 33091), Lactiplantibacillus plantarum LB312R (DSM 33098), Pediococcus pentosaceus LB606R (DSM 33730), Lactiplantibacillus plantarum LB679R (DSM 33731), Lactobacillus crispatus LB714R (DSM 33732), Lactobacillus gasseri LB905R (DSM 34094), Lactobacillus crispatus LB912R (DSM 34095), Lactobacillus crispatus LB919R (DSM 34097), Lactobacillus jensenii LB918R (DSM 34096) and / or any mutant strains and / or any combinations hereof. A "live" microorganisms means "viable" organisms being able to grow or metabolise. "Viability" of microorganisms is measured as Colony Forming Units CFU. A "decrease” in viability of microorganisms may be determined as the difference in CFU / g as compared to the CFU / g at the time of formulating the composition.

[0082] Preferably, the live microorganism may be viable on skin for at least 4 hours after topical application, e.g. for at least 6 hours, such as for at least 8 hours, e.g. for at least 10 hours, such as for at least 12 hours after topical application.

[0083] The microorganisms according to the invention are preferably in isolated or purified form, where the term "isolated" means in particular that the microorganism is cultivated as a monoculture and is derived from the culture medium including their natural medium, for example. The term "purified" is not restricted to absolute purity.

[0084] The microorganisms may advantageously be present in viable dried and / or spray-dried and / or lyophilized and / or vacuum dried crystalized form.

[0085] In a preferred embodiment of the invention the probiotic strain is used as a live isolated microorganism in a crystalized dried form.

[0086] In a preferred embodiment of the invention the strain is used as a viable isolated strain dried into a crystal of cryoprotectant. Wherein the crystal in a preferred form comprises at least 10% cryoprotectant.

[0087] In one aspect of the invention the cryoprotectant is selected from salts, proteins or polysaccharides.

[0088] In one aspect of the invention the cryoprotectant is selected from maltodextrin, trehalose, saccharose, lactose, mannitol, sucrose, glycerol, sorbitol, dextran, inulin.

[0089] In one aspect of the invention the cryoprotectant composition comprises at least one of the following maltodextrin, trehalose, saccharose, lactose, mannitol, sucrose, glycerol, sorbitol, dextran and / or inulin.

[0090] In addition, it is preferable for the microorganism to be present in the composition in the amount by weight of 0.001 wt % to 20 wt %, preferably 0.005 wt % to 10 wt %, especially preferably 0.01 wt % to 5 wt %. A preferred embodiment of the present invention involves the administration of from approximately lxl03to lxlO14CFU of viable bacteria per gram, more preferably from approximately lxlO4to lxlO10CFU / g, and most preferably from approximately lxlO5to lxlO9CFU of viable bacteria per gram of composition.

[0091] In one preferred embodiment of the invention the dosage of live probiotic microorganisms in the composition is above approximately lxlO4CFU of viable bacteria per gram of the composition, preferably above approximately lxlO5CFU / g, preferably above approximately lxlO5CFU / g.

[0092] "Lactic acid bacteria" includes species from the families Lactobacillaceae, Aerococcaceae, Bifidobacteriaceae, Carnobacteriaceae, Enterococcaceae, Leuconostocaceae and Streptococcaceae. These are considered non-pathogenic and are used as probiotic bacteria in general to improve gastrointestinal flora and in the treatment of gastrointestinal symptoms.

[0093] In one embodiment of the invention the preferred microorganism is an isolated wild type lactic acid bacteria.

[0094] The term "bacteriocin" refers to an antimicrobial peptide or protein produced by a bacteria that is active against microorganisms but does not harm the producing bacteria. For purposes of the present invention, bacterocins or bacterocin sources generally include antimicrobial agents suitable for use in formulations as cosmetics or pharmaceuticals. Especially preferred antimicrobial agents include "lantibiotics" (i.e., polypeptides containing lanthionine and beta -methyl lanthionine). Non-limiting examples of such lantibiotics are nisin, such as nisin A or nisin Z, or nisin analogs or related lanthionine-containing peptides, such as pediocin, lactosin, lactacins (e.g., lacticin A, lacticin B, lactacin F), camocin, enterocin, plantaricin, subtilin, epidermin, cinnamycin, duramycin, ancovenin, Pep 5, and the like, individually or in any combination thereof. Other bacterocins that are useful in the present invention include, for example, lactococcins (e.g., lactococcin A, lactococcin B, lactococcin M), leucocoin, helvetican, acidophilucin, caseicin, and the like, individually or in any combination, bacteriocin that is a lantibiotic and / or a non-lantibiotic. According to another embodiment of the present teachings, the bacteriocin is selected from a group comprising nisin A, nisin Z, nisin Q, nisin F, nisin U, nisin U2, salivarcin X, lacticin J46, lacticin 481, lacticin 3147, salivarcin A, salivarcin A2, salivarcin A3, salivarcin A4, BHT-Aa, BHT Ab, salivarcin A5, salivarcin B, streptin, salivaricin Al, streptin, streptococcin A- FF22, mutacin BNY266, mutacin 1140, mutacin K8, mutacin II, smbAB, bovicin HJ50, bovicin HC5, macedocin, leucocin C, sakacin 5X, enterocin CRL35 / mundticin, avicin A, mundticin I, enterocin HF, bavaricin A, ubericin A, leucocin A, mesentericin Y105, sakacin G, curvacin A / sakacin A, lactocin 5, cyctolysin, enterocin A, divercin V41, divercin M35, bavaricin, coagulin, pediocin PA-1, mundticin, piscicocin CS526, piscicocin 126 / Vla, sakacin, Pcarnobacteriocin BM1, enterocin P, piscicoin Vlb, penocin A, bacteriocin 31, bacteriocin RC714, hiracin JM79, bacteriocin T8, enterocin SE-K4, carnobacteriocin B2 and Plantaricins.

[0095] The term "plantaricin" refers to bacteriocins from Lactiplantibacillus plantarum, the major types of plantaricins includes Plantaricin A, Plantaricin E, Plantaricin F, Plantaricin J, Plantaricin K, Plantaricin C, Plantaricin D, Plantaricin W, Plantaricin T and Plantaricin S. As well as other plantaricins e.g. Plantaricin35d, Plantaricin MG, Plantaricin 423, Plantaricin 154, Plantaricin 149, Plantaricin 163, Plantaricin LC74, Plantaricin K25, Plantaricin ST31, Plantaricin SA6. In particular broad spectrum Plantaricins e.g. Plantaricin F, Plantaricin DL3, Plantaricin ZJ008, Plantaricin MG, Plantaricin Q7, Plantaricin KL-1Y, Plantaricin 163, Plantaricin 154.

[0096] In one preferred embodiment of the present invention the bacteriocin is a plantaricin. Plantaricin is produced by Lactiplantibacillus plantarum LB244R (DSM32996), Lactiplantibacillus plantarum LB356R (DSM33094), Lactiplantibacillus plantarum LB312R (DSM33098), and Lactiplantibacillus plantarum LB316R (DSM33091).

[0097] In one preferred embodiment the anti-age strain is an isolated Lactiplantibacillus plantarum.

[0098] In one preferred embodiment the anti-age strain is Lactiplantibacillus plantarum LB244R (DSM32996).

[0099] In one preferred embodiment the anti-age strain is a lactic acid bacteria able to produce at least two bacteriocins, more preferred at least three bacteriocins, more preferred at least four bacteriocins.

[0100] A "antimicrobial metabolite" may include at least one antimicrobial lactic acid producing bacteria metabolite chosen from a group comprising phenyllactic acid, 3-hydroxy phenyllactic acid, 4-hydroxy phenylactic acid, 3- hydroxy propan aldehyde, 1,2 propandiol, 1,3 propandiol, hydrogen peroxide, ethanol, carbon dioxide, carbonic acid, propanoic acid, butyric acid, cyclic dipeptides, cyclo(L-Phe- L-Pro), cyclo(L P-Traps-4-OH-L-Pro), 3-(R)- hydroxydecanoic acid, 3-hydroxy-5-cic dodecanoic acid, 3-(R)-hydroxy dodecanoic acid, and 3-(R)-hyroxytetradecanoic acid. In one aspect of the invention a lipid is included in the topical formulation, preferable the lipid is in form of a natural oil or fat. More preferably the lipid is from a vegetable oil or fat. The vegetal oil or fat may be selected from least one of the following sources: acai, acai berry, almond sweet, aloes vera, andiroba, apricot kernel, arnica, argan, avocado, babassu, boabab, black berry seed, black cumin, black currant seed, blueberry, borage, brazil nut, brocoli seed, buriti, calendula, camellia seed, cannabis oil including CBD and THC, canola, copaiba balsam, cape chestnut (yangu), carrot (daucus carrota), castor, Chardonnay grape, chaulmoogra, cherry Kernel, chia seed, chickweed, coconut, coconut fractionated, cotton seed, comfrey, corn, crambe seed, cranberry seed, cucumber seed, echium seed, evening primrose, emu, flax seed, grape seed, hazelnut, hemp seed, horsechest nut seed, jojoba, karanj seed, kiwi seed, kukuinut, macadamia nut, marula, marshmallow, manketti, meadowfoam, milk thistle seed, moringa, mullein, mustard seed, neem, olive, palm, papaya seed, passionflower seed, peach kernel, peanut, perilla, pomegranate, Pentaclethra macroloba, pumpkin seed, raspberry seed, rice bran, rosehip, St. John's Wort oil, safflower, sea buckthorn pulp, sheabutter oil, sesame roast-ed, sesame seed, soya been, sunflower, tamanu (Calophyllum In-ophyllum), thistle, tomato, turkey red, sangre de drago, walnut, watermelon seed, wheatgerm, Abyssinian, Colza, bees wax, lanolin, linseed, mortierella oil, ongokea, paraffinum liquid, peacan, Pegui, Poppy seed, Pracaxi, rapeseed, soybean, tall, tung, veronica, Wheat germ, yangu seed and any combination thereof.

[0101] In a preferred embodiment of the invention the lipids are from vegetable oils, fats or wax selected from almond oil, sunflower oil, hemp oil, almond sweet oil, shea butter, cocoa butter, Rose Hip oil, jojoba oil, Jojoba Golden oil, rapeseed oil, Camomile oil, Calendula oil, evening prim rose, borage oil, acai oil, Sea buck-thorn oil, Jafflower oil, castor oil, olive oil, linseed oil, apricot kernel oil, argan oil, camelina oil, comfrey oil, grape seed oil, kiwi seed oil, mullein oil, peach kernel oil, thistle oil and sesame oil.

[0102] A "therapeutic effective amount" of a compound with respect to the subject method of treatment refers to an amount of the compounds in a preparation which, when administered as part of a desired dosage regimen (to a mammal, preferably a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit risk ratio applicable to any medical treatment.

[0103] As used herein, the term "treating" or "treatment" includes reversing, reducing, or arresting the symptoms, signs, and underlying pathology of aging in a manner to improve or stabilize a subject's condition. Preferably, a change from a baseline may be observed within 20 days, such as within 28 days, e.g. within 35 days, e.g. within 46, such as within 50 days, e.g. within 56 days, from application of the first dose of the composition according to the present invention.

[0104] It will be clear to those skilled in the art that here, as well as in all the statements of range given in the present invention, characterized by such terms as "about" or "approximately," that the precise numerical range need not be indicated with expressions such as "about" or "approx." or "approximately," but instead even minor deviations up or down with regard to the number indicated are still within the scope of the present invention.

[0105] The composition according to the present invention may preferably comprise a pharmaceutically or cosmetically acceptable vehicle or excipient. In an embodiment of the present invention the composition may be provided in solid form, liquid form, viscous form, emulsion or as a dried form.

[0106] The composition according to the present invention may preferably be for topical application (a topical composition). Preferably the composition may be formulated into emulsion, a mist, a paste; a talc; a powder; a lotion; a custard; a foam; a cream; an oil; a gel; a serum; an ointment; a spray or semi-solid formulation.

[0107] The preferred pH of the composition will be pH 2.5 to pH 7, more preferred from pH 3 to pH 6.5, and even more preferred from pH 4 to pH 6.

[0108] A preferred embodiment according to of the present invention relates to a composition for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging where the composition comprising at least one live isolated microorganism.

[0109] A further preferred embodiment according to the present invention relates to a composition for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the composition comprising at least one live isolated microorganism, by providing :

[0110] (a) a reduction in wrinkles of at least 5 percent;

[0111] (b) a reduction in pigmentary spots density of at least 5 percent; and / or

[0112] (c) an increase in firmness measured as a reduction in suction distance of more than 5 percent.

[0113] Yet a preferred embodiment according to the present invention relates to a composition for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the composition comprising at least 2 live lactic acid bacteria for use in the topical treatment of skin wherein an increase in dermal density of at least 5 percent is obtained after daily topical treatment for 28 days.

[0114] An even further preferred embodiment according to the present invention relates to a composition for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the composition comprising at least one live isolated microorganism, by providing :

[0115] (a) a reduction in sub-epidermal low-echogenic band (SLEB) thickness of at least 2 percent; and / or

[0116] (b) a reduction in TEWL of at least 2 percent.

[0117] A further preferred embodiment according to the present invention relates to a composition for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the composition comprising at least one live isolated microorganism, by providing :

[0118] (a) an increase in complexion radiance of more than 10 percent; and / or

[0119] (b) an increase in smoothness of more than 10 percent.

[0120] A further preferred embodiment of the present invention relates to a method of a skin treatment comprising administering for at least 28 days a live topical composition comprising at least one isolated lactic acid bacteria for treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the method provides:

[0121] (a) a reduction in wrinkles of at least 5 percent;

[0122] (b) a reduction in pigmentary spots density of at least 5 percent; and / or

[0123] (c) an increase in firmness measured as a reduction in suction distance of more than 5 percent.

[0124] Yet a preferred embodiment of the present invention relates to a method of a skin treatment comprising administering for at least 28 days a live topical composition comprising at least one isolated lactic acid bacteria for treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the method provides:

[0125] (a) an increase in dermal density of at least 5 percent;

[0126] (b) an increase in skin hydration of more than 10 percent; and / or

[0127] (c) an increase in skin elasticity of more than 2 percent.

[0128] Another preferred embodiment of the present invention relates to a method of a skin treatment comprising administering for at least 28 days a live topical composition comprising at least one isolated lactic acid bacteria for treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the method provides: (a) a reduction in sub-epidermal low-echogenic band (SLEB) thickness of at least 2 percent; and / or

[0129] (b) a reduction in TEWL of at least 2 percent.

[0130] A further preferred embodiment of the present invention relates to a method of a skin treatment comprising administering for at least 28 days a live topical composition comprising at least one isolated lactic acid bacteria for treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the method provides:

[0131] (a) an increase in complexion radiance of more than 10 percent; and / or

[0132] (b) an increase in smoothness of more than 10 percent.

[0133] An even further preferred embodiment of the present invention relates to live lactic acid bacteria for use in the topical treatment of skin wherein a reduction in sub-epidermal low- echogenic band (SLEB) thickness of at least 2 percent is obtained after daily topical treatment for 28 days.

[0134] A preferred embodiment of the present invention relates to a composition for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging where the composition comprising at least one live isolated microorganism wherein the composition after daily topical treatment for 28 days provides:

[0135] (a) an increase in dermal density of at least 5 percent; and / or

[0136] (b) an increase in skin hydration of more than 10 percent; and / or

[0137] (c) an increase in skin elasticity of more than 2 percent.

[0138] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0139] The invention will now be described in further details in the following non-limiting examples.

[0140] Examples

[0141] Example 1. Clinical trial

[0142] Materials and methods

[0143] Test product

[0144] The investigational product, a probiotic serum (PS), was an oil-based serum including natural vegetable lipids; Butyrospermum parkii butter, Brassica campestris seed oil, Simmondsia chinensis seed oil, Hydrogenated olive oil, Helianthus annuus hybrid oil and live Lactiplantibacillus plantarum LB244R (1 x 109CFU / g).

[0145] Clinical trial

[0146] The trial was designed as a single-center study of a topical cream. The Characteristics of the study population is shown in Table 1. The trial was conducted in accordance with the Declaration of Helsinki principles and subsequent amendments and carried out in the spirit of Good Clinical Practice, reviewed by the internal review board (opinion no 8397 / 2022) and approved by the Independent Ethics Committee for human application. Informed consent was obtained prior to the study.

[0147] Table 1 Trial demographics

[0148] Demographic Data Skin Subjects

[0149] Number 23 (100 %) Skin reactivity Included 23

[0150] (100 %)

[0151] Sensitive 14 Completed 21

[0152] (60.9 %) (91.3 %)

[0153] Mean age 56.6 Normal 9 Dropouts 2

[0154] (39.1 %) (8.7 %)

[0155] Age min 49

[0156] Age max 62 Skin condition:

[0157] Normal 9

[0158] (39.1 %)

[0159] Phototype II 4 (17.4 %) Mixed / Dry 10

[0160] (43.5 %)

[0161] Phototype III 16 (69.6 %) Dry 4

[0162] (17.4 %)

[0163] Phototype IV 3 (13.0 %)

[0164] 23 Subjects were included based on being postmenopausal females, between 40-65 years old, with skin type normal, mixed / dry to dry skin with Fitzpatrick skin Phototype II to IV and signs of skin aging. The skin aging was determined by wrinkles, fine lines and pigmented spots (at least grade 2 and up to grade 5 on the Bazin scale). Subjects were excluded if they had cutaneous marks interfering with assessment, history of known allergies to the product, history of skin cancer, changed hormonal treatment during study, prior vitamin A or anti-aging treatments, or forecast of intensive sun exposure during the test period. Subjects were instructed to apply the PS twice daily and evaluated by a dermatologist three times over the trial; at treatment initiation (DO), after 28±2 and 56±3 days of use (D28, D56). All evaluations were performed after an acclimatization process of at least 15 minutes in a fully controlled and acclimatized room (temperature 21 ± 2 °C, Relative humidity 55 ± 10%).

[0165] Product compatibility and acceptability assessment

[0166] Compatibility was assessed by the sensation of discomfort described by the subjects and the visible reactions of irritation determined by dermatologists trained technicians.

[0167] Skin ultrasonography

[0168] A Dermascan C ultrasound system (Cortex technology, Denmark) with a modified 20 Mhz ultrasound probe was used to detect dense tissue and structures in the dermis in the malar area. The SLEB (sub-epidermal low echogenic Band) thickness and skin density were calculated.

[0169] Skin biomechanical evaluation

[0170] Skin firmness and elasticity evaluation was performed by a Cutometer® dual MPA 580 using a 2mm probe utilizing a negative pressure of 450 mbars for 2 seconds followed by a 2 second release. Measurements were obtained in the malar area and suction height in mm was used to interpret firmness (R0), while visco-elasticity was measured by recovery distance as a percentage of suction height(R2).

[0171] Efficacy evaluation by Scores

[0172] Dermatologists or qualified technicians assessed pigmentary spot density score and Crow's feet wrinkles clinical scores according to Bazin (2011) Skin Res Technol. May;17(2) : 135- 40. doi: 10.1111 / j.1600-0846.2010.00481.x. As well as smooth aspect and complexion radiance on a scale of 0 to 9 with 9 being the highest.

[0173] Skin barrier function assessment

[0174] Trans Epidermal Water Loss (TEWL) was used to measure the skin barrier function. Measurements were assessed by Tewameter® TM 300 (Courage - Khazaka Electronic GmbH, koln, Germany) in the malar area of the face.

[0175] Assessment for skin hydration

[0176] Skin hydration was measured indirectly through measurement of electrical capacitance which depends on the water content of the stratum corneum by corneometer CM825 probe connected to a cutometer dual MPA 580 (Courage & Khazaka, Germany). Measurements were conducted in the malar area of the face. Measurements are obtained in arbitrary units as a reference to a factory standard. Statistical analysis

[0177] Students-t or Wilcoxon signed ranks test was performed for all continuous data comparisons between day 0 and day 28 and 56 respectively. A level of significance of 95% was adopted. To evaluate relative change each subject served as her own reference. Results

[0178] Tolerability of PS

[0179] A total of 23 subjects were enrolled in the trial with 21 completions of treatment and 2 dropouts (Table 1). All subjects tolerated the treatment with PS with no adverse reactions ascribable to the product, emphasizing the safety of topical application of cream formulation(s) with live lactic acid bacteria. In essence, the product showed very good dermatological compatibility and acceptability.

[0180] Table 2 Anti-Aging efficacy (n = 21) All parameters

[0181] Parameters Time point

[0182] DO D28 D56

[0183] SLEB thickness (pm) 261 ± 69 245 ± 53* 237 ± 55*

[0184] Dermal Density (pixel / mm2) 325 ± 58 324 ± 87* 368 ± 76*

[0185] Skin firmness(RO) (pm) 264 ± 38 235 ± 37* 228 ± 37*

[0186] Skin elasticity (R2) (%) 57.8 ± 4.5 60.3 ± 4.7* 61.8 ± 4.4*

[0187] Skin hydration (AU) 34.1 ± 6.9 40.3 ± 10.3** 51.3 ± 10**

[0188] TEWL (g / h / m2) 9.1 ± 2 9.1 ± 1.4 8.5 ± 1.3**

[0189] Crow's feet wrinkles score 3.0 ± 0.8 2.5 ± 0.8* 2.3 ± 0.8*

[0190] Spots score 3.5 ± 0.8 2.9 ± 0.8* 2.5 ± 0.8*

[0191] Smoothness of the skin score 4.2 ± 0.7 5.0 ± 0.7* 5.5 ± 0.7*

[0192] Complexion radiance score 4.1 ± 0.6 5.2 ± 0.6* 5.9 ± 0.5*

[0193] Values in table are given as the mean ± SD

[0194] * Significance different at p < 0.05 in comparison to DO baseline values by Wilcoxon signed- rank test for paired data.

[0195] ** Significance different at p < 0.05 in comparison to DO baseline values by students t-test.

[0196] Skin ultrasonography The mean SLEB thickness was significantly reduced from 261±69 pm to 245±53 pm after 28 days of use (-3.6% reduction, p < 0.05) and to 237±55 pm after 56 days (-6.4% reduction, p < 0.05). Dermal density was increased from 325±58 pixel / mm2to 368±76 pixel / mm2after 56 days. Thus, PS had a positive anti-age effect on both SLEB thickness and dermal density in subjects (Figure 1 & Table 2).

[0197] Skin biomechanical evaluation

[0198] Skin firmness (R0) was reduced with 10.8% (p < 0.05) after 28 days and 13.1% (p < 0.05) after 56 days of application (Table 2). In addition, the skin elasticity (R2) was increased with 4.70% and 7.40% (p < 0.05) after 28 and 56 days, respectively (Figure 2 and Table 2).

[0199] Clinical Scores

[0200] Probiotic Serum had a positive effect on Crow's feet wrinkles, pigmentary spots density, smoothness, and complexion radiance. Crow's feet wrinkles were significantly reduced after 28 days ( -17.2 %; p > 0.05) and 56 days (-22.4 %; p > 0.05) of use (Table 2). The pigmentary spots density was significantly reduced after 28 days ( -16.3 %; p > 0.05) and 56 days (-27.5 %; p > 0.05) of use (Table 2). The skin smoothness was significantly increased after 28 days (19.4 %; p > 0.05) and 56 days (33.5 %; p > 0.05) of use (Table 2). The complexion radiance was also significantly increased after 28 days (26.4 %; p > 0.05) and 56 days (44.4 %; p > 0.05) of use (Table 2).

[0201] Clinically, PS helped reverse the investigated signs of aging skin. The ultrasonographic results show a significant reduction of the SLEB thickness indicating an antiaging effect as SLEB thickness increases proportionally to age. While the SLEB thickness decreases the dermal density increases. By image analysis, the dermal density is increased in both the Papillary dermis and the Reticular dermis indicating an increase in collagen bundles, as it is an echogenic marker protein. This suggests a reversal from aging skin to matured skin and increase of collagen bundle thickness. This is further underlined by the biomechanical evaluation of the trial where both skin firmness and elasticity were increased. The skin firmness increases, with the suction distance being reduced (Table 2, Figure 2A). The firmness is a combination of the collagen and elastin fibers elasticity, the viscoelasticity as well as the skins plasticity. This is supported by an increase in elasticity (Table 2, Figure 2B), suggesting an improved strengthening of the collagen and elastin fibers. As the measurements are done with a 2 mm probe these changes are observed in the stratum corneum and papillary dermis.

[0202] Wrinkle formation is associated with saggy and un-elastic skin caused by damage to the dermal extracellular matrix of collagen and elastin. As the dermis ages, the collagen fibers are disorganized, which results in increase of wrinkles, roughness, and radiance. Topical treatment with PS significantly reduced crow's feed wrinkles, along with a decrease in pigmentary spot density, improved complexion radiance, and improved smoothness. These results are a physical manifestation of the ultrasonographic and biomechanical improvements, underlying the anti-aging benefits of PS (Table 2).

[0203] Additionally, the increased smoothness is a direct reflection of PS restorative effect on the skin barrier after 56 days of treatment as shown by the TEWL. The increased barrier function is followed by improved skin hydration was also significantly increased (Table 2).

[0204] In conclusion the clinical study of PS included 21 individuals and showed both dermatological compatibility and acceptability. PS significantly reduced SLEB thickness and increased dermal density as well as firmness and elasticity of the skin, improving the clinical signs of skin ageing. The product also improved crow's feet wrinkles, pigment spot density and complexion radiance. These results suggest that PS would be a useful antiaging product.

[0205] Example 2:

[0206] Determination of viability of applied microorganisms on skin.

[0207] L. plantarum LB316R, L. plantarum LB244R, L. plantarum LB312R were formulated as viable strain into a topical oil formulations of almond oil (50% w / w) and jojoba oil (49% w / w) and approximately 1% of a freeze dried microrganism. The concentration of live bacteria were determined in each formulation by plate counting on MRS media.

[0208] Each topical formulation was applied on human skin and the skin surface was divided into areas of 4x4 cm, one area was swapped only once. The number of viable lactic acid bacteria (LAB) was evaluation by swapping with a sterile cotton swap and drawn out on MRS agar plates. The number of viable LAB was followed by swapping each hour from a 4x4 area, survival of LAB on human skin was determined relative to the number of cells at time of application of the topical formulation.

[0209] The number of CFU / g of topical formulation was determined in each batch to be approximately 108CFU / g.

[0210] Formula A (LB316R) : 8xl08CFU / g

[0211] Formula B (LB244R) : 7xl08CFU / g

[0212] Formula C (LB312R) : 5xl08CFU / g During the test the skin was not covered, exposed to sun, rinsed, or washed.

[0213] When applied to human skin, all 3 strains were fully viable on the skin for about 8-10 hours, thereafter the viability starts to decline.

[0214] Example 3:

[0215] Pigmentary spots.

[0216] Formula A from example 2, comprising the strain L. plantarum LB316R was applied to photo-aged skin two times daily, after 2 weeks, a significant visible reduction in both pigmentary spots and wrinkles were observed. Figure 4A is the skin before treatment and figure 4B is same area of skin after 2 weeks of application.

[0217] Skin hydration, firmness and smoothness was evaluated by test persons, and all parameters were evaluated as being improved after 2 weeks of treatment.

[0218] Example 4:

[0219] Placebo controlled double-blinded clinical trial

[0220] The second clinical trial was designed as a single-center, double-blinded, vehicle-ointment placebo-controlled study. All subjects were randomly assigned either the topical ointment without active (placebo ointment) or with active (live L. plantarum LB244R).

[0221] Inclusion criteria included : Postmenopausal females aged 40-65 with normal, mixed / dry or dry skin with visible signs of aging, such as fine lines and pigmented spots on a minimum grade 2 on the Bazin scale. Subjects were instructed to apply their assigned topical ointment to the face and periocular area twice daily, morning and evening, on clean skin and massage until complete absorption for 56 consecutive days.

[0222] All parameters were assessed at day 0, day 26 and day 56 for included subjects by trained technicians or dermatologists. All assessments were performed after a minimal acclimatization process of 15 minutes in a fully controlled and acclimatized room with temperatures T=21°C ± 2°C and a relative controlled humidity of RH = 55% ± 10%.

[0223] Trial demographics. All subjects included were female.

[0224] Demographic data Skin type Subjects Age: Skin reactivity: Included: 23 (100%)

[0225] Mean age: 58.7 Sensitive: 13 (56.5%) Analyzed : 21 (91.3%) Minimum age: 47 Normal: 10 (43.5%) Dropouts: 2 (8.7%) Maximum age: 65

[0226] Skin condition:

[0227] Skin phototypes: Normal: 10 (43.5%) Phototype II: 10 Mixed / dry: 7 (30.4%) (43.5%) Dry: 6 (26.1%)

[0228] Phototype III: 12 (52.2%)

[0229] Phototype IV: 1 (4.3%) Age: Skin reactivity: Included: 23 (100%)

[0230] Mean age: 58.7 Sensitive: 15 (65.2%) Analyzed: 23 (100%) Minimum age: 47 Normal: 8 (34.8%) Dropouts: 0 (0%) Maximum age: 65

[0231] Skin condition:

[0232] Skin phototypes: Normal: 14 (60.9%) Phototype II: 10 Mixed / dry: 2 (8.7%) (43.5%) Dry: 7 (30.4%)

[0233] Phototype III: 12 (52.2%)

[0234] Phototype IV: 1 (4.3%)

[0235] Skin ultrasonography

[0236] Skin ultrasonography was measured as in previous study example 1. Tissue density and dermis structures were assessed in the malar region, randomly assigned either left or right side, using a dermascan C ultrasound system (Cortex technology, Denmark) with a modified 20 Mhz probe. Measurements were used to calculate the Sub-epidermal low echogenic band (SLEB) thickness as well as skin density. Skin biomechanical evaluation

[0237] Skin biomechanical parameters were measured as in example 1, using a Dual-cutometer MPA 580® (Courage & Khazaka Electronic GmbH, Germany). Measurements were performed using a 2 mm probe, where negative pressure is used to deform the skin mechanically. Negative pressure is created in the device (450 mBar) and after 2 seconds the skin is released. Penetration depth is determined using a non-optical measuring system. The penetration depth is used as a measurement for skin firmness and the skin's ability to return to its original position as a measurement for skin elasticity. Skin hydration

[0238] Skin hydration assessments were measured using a Corneometer CM825 probe connected to a Cutometer dual MPA 580 (Courage & Khazaka, Germany). Measurements were performed in the malar area of the face, with measurements randomly assigned to either the left or right side. The hydration is assessed by measuring the capacitance of the skin expressed in arbitrary units (AU) to a factory standard.

[0239] Skin structure visualization

[0240] Skin conditions were visualized and characterized using Confocal microscopy with Vivascope® 1500 (Mavig, Germany). Using in vivo confocal laser scanning microscopy (CLSM) or Line-field confocal optical coherence tomography (LC-OCT)(Damae, France) the epidermis and reticular dermis were imaged and inspected. Images are generated using a 830 or 800 nm laser hitting defined spots at specific skin depths. Images can be used to create a 3D object by scanning several optical planes and stacking them using suitable microscopy software.

[0241] Clinical evaluations

[0242] Efficacy of the ointments were assessed by a dermatologist at baseline and after application for 28 and 56 days. The following parameters were assessed :

[0243] Crow's feet wrinkle clinical score was assessed using the Bazin scale, Spot clinical score according to the Bazin scale, Smoothness of the skin score on a scale from 0 to 9 (with 9 meaning very smooth skin and 0 meaning not smooth skin), and complexion radiance score on a scale from 0 to 9 (9 meaning very radiant complexion and 0 meaning not radiant complexion).

[0244] These observations were supported by self-assessment questionnaires.

[0245] Statistical analysis

[0246] Statistical analysis was done using either Students T test or Wilcoxon's signed ranks test for all the continuous data comparisons; between DO, D28 and D56 as well as both products). For subjective data binomial tests were used. A significance level of 95% was adopted for all tests.

[0247] For all measured parameters, the active works better than the placebo vehicle ointment, showing statistically significant effect of the live probiotic applied topical. Time point

[0248] Parameter

[0249] GROUP A (placebo) SLEB thickness (mm) 0.291 ± 0.096 0.287 ± 0.105 0.291 ± 0.098 Dermal Density 296.635 ± 300.026 ± 54.073 309.346 ± (pixels / mm2) 65.945 57.804* Skin hydration (AU) 43.1 ± 8.8 47.9 ± 9.3 51.8 ± 9.0 SLEB thickness (mm) 0.284 ± 0.068 0.256 ± 0.054* 0.243 ± 0.055* Skin elasticity (%) 0.595 ± 0.119 0.673 ± 0.094* 0.646 ± 0.088* Skin hydration (AU) 37.7 ± 8.5 51.6 ± 8.2* 59.7 ± 10.3*

[0250] *Significant difference with p < 0.05 between active ointment and placebo.

[0251] Figure 1 : Ultrasonographic assessment shows reduced SLEB thickness and increased dermal density Fig 1A) Bars showing mean SLEB % change with subjects' day 0 serving as a reference. Fig. IB) Bars showing mean Dermal Density % change with subjects' day 0 serving as a reference.

[0252] Figure 2: Biomechanical properties of the skin measured by a cutometer improved in terms of firmness (R0) and elasticity (R2). Fig 2A) Bars showing mean skin firmness % change with subjects' day 0 serving as a reference. Fig 2B) Bars showing mean skin elasticity % change with subjects' day 0 serving as a reference. Significance levels in comparison to baseline values were tested by Wilcoxon signed-rank test for paired data. * indicates p < 0.05.

[0253] Figure 3: Clinical assessment of Crow's feet wrinkles, pigmentary spots density, complexion radiance, and smoothness. A) Bars showing mean Crow's feet wrinkles % change with subjects' day 0 serving as a reference. B) Bars showing mean pigmentary spots density % change with subjects' day 0 serving as a reference. C) Bars showing mean complexion radiance % change with subjects' day 0 serving as a reference. D) Bars showing mean smoothness % change with subjects' day 0 serving as a reference. Significance levels in comparison to baseline values were tested by Wilcoxon signed-rank test for paired data. * indicates p < 0.05. Figure 4: Visual assessment of wrinkles and pigmentation A) Before treatment. B) After 2 weeks of treatment with 2 applications per day.

Claims

Claims1. A composition for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging where the composition comprising at least one live isolated microorganism.

2. The composition according to claim 1 for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the composition comprising at least one live isolated microorganism, by providing :(a) a reduction in wrinkles of at least 5 percent;(b) a reduction in pigmentary spots density of at least 5 percent; and / or(c) an increase in firmness measured as a reduction in suction distance of more than 5 percent.

3. The composition according to claim 1 for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the composition comprising at least 2 live lactic acid bacteria for use in the topical treatment of skin wherein an increase in dermal density of at least 5 percent is obtained after daily topical treatment for 28 days.

4. The composition according to claim 1 for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the composition comprising at least one live isolated microorganism, by providing:(a) a reduction in sub-epidermal low-echogenic band (SLEB) thickness of at least 2 percent; and / or(b) a reduction in TEWL of at least 2 percent.

5. The composition according to claim 1 for use in the treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the composition comprising at least one live isolated microorganism, by providing:(a) an increase in complexion radiance of more than 10 percent; and / or(b) an increase in smoothness of more than 10 percent.

6. The composition according to anyone of the preceding claims, wherein the microorganism is a lactic acid bacteria.

7. The composition according to claim 6, wherein the lactic acid bacteria produce antimicrobial metabolites and / or bacteriocins.

8. The composition according to anyone of claims 1 to 7, wherein the change from baseline is observed within 20 days, such as within 28 days, e.g. within 35 days, e.g. within 46, such as within 50 days, e.g. within 56 days, from application of the first dose of the composition.

9. The composition according to anyone of the preceding claims, wherein the composition is a topical composition.

10. The composition according to anyone of the preceding claims, wherein the topical composition is a cream, a lotion, a spray, a solution, a gel, a serum, an ointment, a fat, an oil, a powder, a paste, a foam, a plaster, a paint, an adhesive, a suspension or an emulsion.

11. The composition according to anyone of the preceding claims, wherein the composition, e.g. the topical composition, comprising a live microorganism in a concentration of at least 104CFU / g, such as at least 105CFU / g, such as at least 105CFU / g, such as at least 107CFU / g, such as at least 108CFU / g.

12. The composition according to anyone of the preceding claims, wherein the composition further comprises at least one natural lipid.

13. A method for of a skin treatment comprising administering for at least 28 days a live topical composition comprising at least one isolated lactic acid bacteria for treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging.

14. The method according to claim 13 of a skin treatment comprising administering for at least 28 days a live topical composition comprising at least one isolated lactic acid bacteria for treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the method provides:(a) a reduction in wrinkles of at least 5 percent;(b) a reduction in pigmentary spots density of at least 5 percent; and / or(c) an increase in firmness measured as a reduction in suction distance of more than 5 percent.

15. The method according to claim 13 of a skin treatment comprising administering for at least 28 days a live topical composition comprising at least one isolated lactic acid bacteria for treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the method provides:(a) an increase in dermal density of at least 5 percent;(b) an increase in skin hydration of more than 10 percent; and / or(c) an increase in skin elasticity of more than 2 percent.

16. The method according to claim 13 of a skin treatment comprising administering for at least 28 days a live topical composition comprising at least one isolated lactic acid bacteria for treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the method provides:(a) a reduction in sub-epidermal low-echogenic band (SLEB) thickness of at least 2 percent; and / or(b) a reduction in TEWL of at least 2 percent.

17. The method according to claim 13 of a skin treatment comprising administering for at least 28 days a live topical composition comprising at least one isolated lactic acid bacteria for treatment, alleviation, suppression, prophylaxis and / or prevention of skin aging, wherein the method provides:(a) an increase in complexion radiance of more than 10 percent; and / or(b) an increase in smoothness of more than 10 percent.

18. The method according to claim 13 to 17 wherein the live microorganism is applied to skin in a concentration of at least 104CFU / cm2, such as at least 105CFU / cm2, such as at least 105CFU / cm2, such as at least 107CFU / cm2, such as at least 108CFU / cm2.

19. The method according to any of claims 13 to 18 wherein the live microorganism is applied to skin at least once daily.

20. The method according to any of claims 13 to 19 wherein the live microorganism is viable on skin for at least 8 hours after topical application.

21. Live lactic acid bacteria for use in the topical treatment of skin wherein a reduction in sub-epidermal low-echogenic band (SLEB) thickness of at least 2 percent is obtained after daily topical treatment for 28 days.

22. Live lactic acid bacteria for use in the topical treatment of skin wherein the lactic acid bacteria after daily topical treatment for 28 days provides:(a) an increase in dermal density of at least 5 percent; and / or(b) an increase in skin hydration of more than 10 percent; and / or(c) an increase in skin elasticity of more than 2 percent.

23. Live lactic acid bacteria according to claim 19 or 20 wherein the lactic acid bacteria is isolated and applied to skin in a concentration of at least 104CFU / cm2, such as at least 105CFU / cm2, such as at least 105CFU / cm2, such as at least 107CFU / cm2, such as at least 108CFU / cm2.

24. Live lactic acid bacteria according to claim 19 to 21 wherein the lactic acid bacteria belong to the family Lactobacillaceae.

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