Synbiotic compositions for skin care

Abstract

At the heart of the invention is a biological gene delivery system allowing a coordinated delivery of bioactives with proven benefits to the skin and hair. To that end, the invention proposes the use of genetically manipulated live probiotics as a manufacturing platform that remains dormant (spores) before application to the skin and becomes activated (viable germinating spores) after the topical application and exposure thereof to the specific conditions on skin surface. The products of the invention are topical formulations that can be exceptionally useful in dermatological, veterinary, and cosmetic contexts due to improved properties of topical delivery, efficacy, and shelf-life and surprising reparative and curative effects on the skin and hair.

Description

[0001] SYNBIOTIC COMPOSITIONS FOR SKIN CARE

[0002] TECHNOLOGICAL FIELD

[0003] The invention pertains to the field of dermatological, veterinary, and cosmetic formulations containing probiotic and prebiotic components, wherein the probiotic component was genetically modified to confer improved reparative and curative effects onto the skin, hair follicles and hair.

[0004] BACKGROUND

[0005] The skin microbiome has become the focus of much attention in recent years, which in turn has led to two main realizations: (1) that a “healthy” skin microbiome is closely associated with skin health and healthy skin appearance; and (2) that imbalance or anomaly in the diversity of microbial species making the skin microbiome (dysbiosis) is one of the factors underlying various skin conditions. Known examples are repeated findings of association between the composition of microbial flora constructing the skin microbiome and the occurrence of various skin disorders such as atopic dermatitis, acne vulgaris, seborrheic dermatitis, pityriasis versicolor, psoriasis, eczema, and many others, as well as much more ubiquitous phenomena commonly referred to as unhealthy or uneven skin appearance, skin wrinkling and drying, and other signs of skin aging. Thus, making “a balanced and healthy skin microbiome” has become the focal point of many pharma and cosmetic companies.

[0006] Several effective probiotics have been developed so far, predominantly for the gut and intestinal microbiomes. In the cometic industry, due to safety concerns, cosmetic products are expected to have relatively low content of contaminating microorganisms (<500 colony forming units (CFU) / g for eye products and 1000 CFU / g for the rest). Therefore, the prevailing belief in the cosmetic industry has been that “a true probiotics” is to be avoided, and replaced instead with components of non-living probiotic bacteria, such as bacterial lysates, ferments, and filtrates deprived from live bacterial cells.

[0007] Probiotic compositions have gained more experience in dermatological uses. Topical creams with live lactobacilli were used to reduce acne and in the treatment of other skin infections. However, one of the problems with formulating live probiotics is to be able to achieve sufficient stability and shelf-life of the probiotics, without resorting to strong antiseptics and preservatives that would jeopardize its beneficial properties. GENERAL DESCRIPTION

[0008] This invention stems from the general notion that certain types of probiotics, and especially Bacillus strains, can contribute to “a healthy and balanced” skin microbiome, which in turn would lead to a more healthy and rejuvenated skin appearance, and more generally, improve the integrity and functionality of the epidermis and skin barrier.

[0009] Intensive R&D in biotech yielded a number of technologies using Bacillus as a cell factory for the production of self and foreign proteins, peptides, vitamins, hormones, enzymes, and other substances of interest for pharma, cosmetics, agriculture and food industries. By the current estimates, about half of all biotech produced enzymes are derived from Bacillus strains, and specifically B. subtilis. Moreover, B. subtilis has been known for its positive effects on skin barrier and skin microbiome and is generally considered as GRAS (Generally Recognized as Safe). Therefore, B. subtilis, and Bacillus in general, offers an advantageous and accessible tool for producing important bioactive substances on a sufficiently large scale and in nature mimicking conditions.

[0010] In the most general terms, the invention proposes to use B. subtilis and other taxonomically related bacterial strains as a resource of various bioactive substances with proven benefits to the skin and to skin integrity and intactness. As the perception of skin well-being, integrity and intactness is a direct outcome of the integrity and functionality of the skin barrier and skin microbiome, the bioactives of the invention can be thither articulated in terms of substances that contribute to the functionality and integrity of skin barrier and / or the functionality and balance in the diversity of skin microbiome, and as a result to an improved skin appearance and the general perception of “a healthy skin”.

[0011] An additional advantage of using Bacillus resides in its ability to survive or preserve viability in vegetative and non-vegetative (spore) states. Bacillus spores can remain in a “dormant” non-vegetative state for years, and only upon exposure to certain external triggers, they start to germinate or enter a vegetative cycle. Therefore, Bacillus further provides an attractive manufacturing platform for controlled production of bioactives that can be downregulated or activated by specific environmental triggers.

[0012] A refinement of this platform is proposed by combining genetic engineering and bacterial culturing methods to produce specifically tailored Bacillus spores that have the potential to produce a variety of skin and hair beneficial bioactives. In the original state, before application to the skin, the genetically modified probiotic bacteria remain dormant state, whereas after their topical application and exposure to the specific triggers on the skin surface (e.g., skin temperature, acidity, lipid content, moisture, pressure, etc.), they start to germinate and produce / express / secrete the cloned bioactives.

[0013] Another improvement to this platform is provided by supplementing the probiotic component with a prebiotic component (e.g., a fructan polysaccharide, inulin) that supports germination and preservation of the probiotics on the skin surface. To further improve sustainability and viability of the probiotic bacteria on the skin, the invention further contemplates the use of a mix of genetically engineered spores from various Bacillus strains or a mix of genetically engineered and non-engineered spores from the same strain, and various types of prebiotic components.

[0014] From a broader point of view, the invention provides an improved solution to the problem of delivery and preservation of dermatologically important and cosmetic actives by incorporating them into genetically engineered probiotics in the form of spores and combining them with a prebiotic component to produce sustainable and controllable constructs that become effective and functional only upon topical application thereof onto the skin. As such, the presently proposed approach can lay the foundation for the design and development of a new family of dermatological, veterinary, and cosmetic products.

[0015] The effectiveness of skin probiotics is highly dependent on the ability to preserve viability of the probiotics before application (in a stored product) and after its application onto the skin, this is especially true for leave-on cosmetic products. In a previous study, the inventors and colleagues found that a preparation comprising spores from a specific mix of Bacillus strains and inulin (i.e., synbiotics) yielded detectable and viable bacteria more than 10 days after topical application on the skin. In a more recent study, they found that topical application of this synbiotics on 3D human skin model can have profound effects on the expression of mammalian genes and specific molecular pathways, including genes belonging to cell cycle signaling, pluripotency and keratinocytes differentiation pathways and genes partaking in the biosynthesis and metabolism of important skin bioactives such as retinol, collagen, and mediators of radical scavenging. Furthermore, a clinical study using the same synbiotics found that it is beneficial for skin microbiome, evident by the enrichment of commensal microbiota and the reduction of pathologic microbiota within a relatively short period of topical use (within the period of 4 weeks). In addition, most of the study participants reported that topical application of the synbiotics had led to significant reduction of acne, skin oiliness and redness, reduction of skin pores, and to improvement in skin texture and generally healthy appearance. All the above formed the basis for the presently proposed inventive concept, suggesting that (1) cosmetic or dermatological topical formulations combining probiotic and prebiotic components can provide proactive as well as protective means to support the functionality, integrity and intactness of the mammalian skin barrier and the mammalian skin microbiome; (2) providing the probiotic component in the form of spores can improve storability and efficacy of the formulation per se; and further, (3) all these properties can be further enhanced by introducing specific genetic alterations into the probiotic component to drive it towards production of various structural or secretable bioactive compounds that are beneficial to mammalian skin barrier and / or microbiome.

[0016] Some examples of potentially interesting genetic alterations are listed below: a) Sequences encoding bioactive compounds or enzymes associated with attenuation of skin aging, e.g., Hyaluronic acid, Super Oxide Dismutase, or Coenzyme Q10. These would be especially useful for cosmetic or dermatological leave-on products or products for long-term or continuous use, optionally in the formulation context allowing slow or controlled release of the bioactives. b) Sequences encoding weak therapeutic compounds, e.g., retinoids for acne treatment, or weak steroids to treat inflammation and eczema. These would be applicable for dermatological products for limited or short-term use, optionally in the formulation context allowing immediate release of the bioactives. c) Sequences encoding proteins, peptides, or small interference (siRNA) acting as regulators of native skin enzymes and assisting in the maintenance of skin homeostasis, e.g., Cytochromes P450 (CYPs), Aldehyde Dehydrogenases (ALDHs), Flavin-containing Monooxygenases (FMOs), Cyclooxygenases (COX) and others. d) Sequences encoding foreign enzymes with proven dermatological, veterinary, and cosmetic benefits. Examples of those are certain types of plant-derived proteins, such as Bromelain with benefits of scars and burns healing, or Papain and Chymopapain and other bio-peeling enzymes.

[0017] Specific examples of substances produced / expressed / secreted by genetically modified B. subtilis strains are:

[0018] • S.O.D as an example of radical scavenging enzyme (antioxidant)

[0019] • Vitamin C / Ascorbic acid producing enzyme (collagen production, skin whitening).

[0020] • P-galactosidase (P GAL) as a general example of excreted enzymes • Hyaluronic acid (HA) producing enzyme (enhances skin moisturizing)

[0021] • Tranexamic acid producing enzyme (skin whitening agent)

[0022] • Kojic acid producing enzyme (skin whitening agent)

[0023] • Papain (enzymatic peeling effect)

[0024] • Bromelain (enzymatic peeling effect)

[0025] • Vitamin D producing enzyme (improves skin barrier functioning)

[0026] • Vitamin B3 producing enzyme (improves skin barrier functioning)

[0027] • Melatonin producing enzyme (circadian day cycle effector)

[0028] • Human copper binding GHK-Cu tripeptide (increases collagen and elastin synthesis, supports dermal fibroblasts growth, and increases hair growth)

[0029] • Benzoyl peroxide producing enzyme (anti acne drug)

[0030] • Lidocaine producing enzyme (analgesic)

[0031] • Lutein producing enzyme (antioxidant effector)

[0032] • Caffeine producing enzyme (for treating skin cellulite and hair loss)

[0033] In summary, the invention offers a new line of dermatological, veterinary, and cosmetic formulations, the common feature of which is incorporation of genetically altered probiotic bacteria that have the potential to produce dermatologically or cosmetically relevant bioactives. An important feature of these formulations is in providing said probiotics in the form of spores together with a prebiotic compound, so that upon application onto the skin the probiotic bacteria are triggered to germinate and produce / express / secrete the bioactives of interest. The formulations of the invention are essentially base-formulations that can be incorporated into a variety of leave-on and rinse- off products in the forms of sprays, gels, lotions, creams, masks, ointments, and powders. They can be used for curative as well as preventive purposes in a wide range of clinical, subclinical, and non-clinical conditions, all stemming from disruption of functionality and integrity of skin barrier and / or skin microbiome, or skin homeostasis in general. In the upper clinical end, such conditions can include atopic dermatitis, acne vulgaris, rosacea, seborrheic dermatitis, pityriasis versicolor, psoriasis, actinic keratosis, eczema, and other skin disorders. In the lower sub- or non-clinical end, they can include dandruff, sunburn, skin irritations, discolorations, unhealthy skin appearance and skin aging. Ultimately, the formulations of the invention can have exceptionally wide applicability in dermatology, veterinary and cosmetics, especially in highly demanded cosmetic products for restoring a healthy and rejuvenated complexion of the skin.

[0034] BRIEF DECSRIPTION OF DRAWINGS

[0035] Figs 1A-1C illustrate some examples of recombinant constructs of the invention, using pNNBT20 / 21 vectors to include the Hyaluronic acid (HA) expressing cassettes incorporated into the B. subtilis amyE locus (1A-1B), including a heterologous hasA gene (e.g., Streptococcal hasA) and B. subtilis derived tuaD, gtaB, and / or gcaD genes, repositioned within about 50 bp intervals to maximize expression efficiency (1C), in conjunction with a strong promoter (mutated B. amyloliquefaciens derived amyQ promoter), a strong ribosome binding site and a strong transcription terminator (both derived from the B. clausii aprL gene) to further enhance expression efficiency.

[0036] Figs 2A-2B illustrate the main steps in the spores’ enrichment procedure of the invention when applied to B. subtilis 168 and KO7 A strains (2A) to produce the respective sporulation rates (2B). Figures show that one of the determining factors of sporulation efficiency is exposure to oxygen during the starvation step (2A-2B left, DSM 24-72 h), with 168 responding to low oxygen (stress) and K07A to high oxygen to yield sporulation rates over 80% and up to 100%, respectively (2B right, histogram). Visual analysis of spores under the phase-contrast microscope is also shown (2B right, 168 after 48 h).

[0037] Fig, 3 illustrates additional examples of recombinant constructs of the invention, using expression cassettes of a heterologous superoxide dismutase (e.g., the human SOD) or a 0-galactosidase (e.g., E. coli P-gal) in conjunction with the pSigH-p43 dual promoters (growth-dependent and constitutive promoters) spaced with a 100 bp linker, and B. clausii aprL derived terminator, a structure previously related to the production of a secretable enzymes., all incorporated into the B. subtilis AmyE locus.

[0038] Figs 4-5 illustrate example assays for verification of successful integration of the cloned genes into the AmyE locus of B. subtilis, by Amylase assay and PCR, respectively.

[0039] Figs 6A-6B illustrate applications these methods for isolation of specific genetically modified B. subtilis stains, with the example of PCR method for the detection of candidate 168 and K07A containing heterologous SOD expression cassettes (6A), and specific antibodies for the detection of the respective expression of SOD enzyme (6B). Antibodies for the effective detection of the P-gal gene product are also available (6B). DESCRIPTION OF SPECIFIC EMBODIMENTS

[0040] In the broadest sense, the present invention can be articulated in terms of a topical delivery system of bioactive that modulates functionality and integrity of mammalian skin barrier and / or functionality and diversity of mammalian skin microbiome.

[0041] The term “ mammalian skin barrier" relates herein to the multiple functionalities of epidermis as a barrier to external environmental insults and a protector against water loss from the skin. It further implies functionality and integrity of the epidermal barrier on physical, chemical, neuronal, immune and microbial levels, including its functions as a provider of physical protection by keratinization and formation of stratum corneum, an architect and a preserver of skin homeostasis via various types of epidermal cell junctions, a protector against UV damage due to the presence of various skin chromophores, a provider of immune barrier due to a variety of resident immune cells populating epidermis and dermis, a sensory organ that comprises a large range of sensory receptors associated with non-neuronal components, and further, a keeper of the skin’s natural microbiome.

[0042] The term “mammalian skin microbiome” predominantly relates herein to the functionalities and diversity of microbial communities constructing the skin microbiome, or the microbiome colonizing the epidermal surface. The normal or “healthy” skin microbiome is composed of bacteria, fungi, and viruses, with relatively stable representation of members of these groups. Unlike the gut microbiome, the skin microbiota is dominated by Actinobacleria with prevalence of Gram-positive bacteria, such as Staphylococcus, Propionibacterium, and Corynebacterium species. The terms “a dysbalanced microbiome ” or ’dysbiosis" imply herein a deviation from the normal composition of skin microbiome, usually revealed by relatively increasing representation bacterial and fungal taxa associated with moist, dry, and sebum-rich microenvironments, for example lipophilic Propionibacterium species, or humidity favoring Staphylococcus and Corynebacterium species. Dysbiosis of skin microbiome can be further prompted by various skin and hair disorders and subclinical and non-clinical skin and hair conditions.

[0043] The term “bioactive“ encompasses herein any substance that has a biological effect on functionalities of mammalian skin barrier and / or skin microbiome. It encompasses bioactives which are direct gene products such as proteins or peptides, enzymes or amine hormones acting on mammalian skin barrier or skin microbiome, as well as their downstream effectors such as lipids, metabolites, steroid hormones, and vitamins. Examples of such useful bioactives are provided in EXAMPLE 4. The construct “ modulating functionality” encompasses herein any change in any measurable parameter or indicator of functionalities of mammalian skin barrier or skin microbiome, including up- and down-regulation of any mediators of said functionalities. This “modulating functionality” is mostly applied herein in the sense of restoring the normal functionality and integrity of skin barrier or the normal functionality and diversity of skin microbiome, or in the sense of repairing previous or current dysfunctionality of skin barrier or dysbiosis of skin microbiome.

[0044] In other words, in some embodiments the bioactive of interest is meant to repair or reverse disfunctions or disruptions of functionality and integrity of mammalian skin barrier.

[0045] In some embodiments the bioactive of interest is meant to repair or reverse disfunctions or disruptions of functionality and diversity of mammalian skin microbiome.

[0046] More broadly, the bioactive of Interest is meant to repair or reverse disfunctions or disruptions of mammalian skin homeostasis.

[0047] In some embodiments the bioactive can modulate or restore functionality and integrity of the skin barrier by modulating one or more mediators of immunologic, antiallergic, anti-inflammatory, sensory responses of cutaneous tissue and cells.

[0048] In some embodiments the bioactive can modulate or restore functionality and integrity of the skin barrier by modulating one or more mediators of proliferation or differentiation of cutaneous cells.

[0049] In some embodiments the bioactive can modulate or restore functionality and integrity of the skin barrier by modulating processes underlying moisturizing, tightening, anti- wrinkling, wound healing as well as skin color and smell, or rejuvenation of the skin, and / or more uniform and healthy skin and healthy hair appearance in general.

[0050] The term “skin” is perceived herein as a part of the integumentary system that encompasses the epidermal, dermal and hypodermal skin tissues, and related structures such hair follicles, hair, nails and glands. The functionality and integrity of these structures, especially the condition of skin scalp and nail bed, affect hair and nail growth, shape and appearance.

[0051] In some embodiments the bioactive can modulate or restore functionality and diversity of the skin microbiome by modulating or inhibiting persistent growth or quorum sensing of harmful microorganisms. In some embodiments the bioactive can modulate or restore functionality and diversity of the skin microbiome by modulating or inhibiting persistent dysbiosis of skin microbiome.

[0052] In some embodiments the bioactive can modulate or restore functionalities of the skin barrier and skin microbiome by modulating or inhibiting one or more manifestations of a persistent skin disorder or a subclinical or non-clinical skin condition.

[0053] A key feature of this delivery system is a preparation combining live probiotic microorganisms and one or more prebiotic compounds (often referred to in the text as a synbiotic preparation or synbiotics). The term “probiotic microorganisms"" encompasses herein any microscopic organism which is friendly or not harmful to the skin, including archaea, bacteria, fungi and viruses. The term “probiotic compound" encompasses any type of compound which supports or maintains the viability of probiotic microorganisms, including sugars, polysaccharides, and others.

[0054] In some embodiments the probiotic microorganisms can be or can comprise live bacteria or live yeasts.

[0055] In some embodiments the prebiotic compounds can be a sugar or a sugar alcohol

[0056] A specific feature of this synbiotic preparation is that in its original state, i.e., before its application onto the skin, the live probiotic microorganisms are maintained in in a non- vegetative state (spores) and transition to a vegetative state (germinating spores) only after its application onto the skin.

[0057] Another specific feature of this preparation is that the probiotic microorganisms are genetically engineered to include at least one bioactive genetic alteration relative to the respective wild-type microorganism. The term “bioactive genetic alteration"" implies herein any genetic change relative to the wild-type genome of the same microorganism, which either directly or indirectly facilitates or promotes the expression and / or section of the bioactive of interest.

[0058] Still another important feature of the preparation, owing to the combination of live non-vegetative probiotics and prebiotics, is that before it is applied onto the skin, the genetic alteration remains dormant in the probiotic microorganisms or is not expressed but is expressed exclusively after they transition to the vegetative state upon application thereof onto the skin surface. In other words, in some embodiments the probiotic microorganisms do not express the bioactive genetic alteration(s) prior to topical application of the delivery system onto mammalian skin.

[0059] In some embodiments upon topical application of the delivery system onto mammalian skin, the probiotic microorganisms transition from a non-vegetative to a vegetative state.

[0060] In some embodiments the probiotic microorganisms start expressing the bioactive genetic alteration(s) after topical application of the delivery system onto mammalian skin.

[0061] All these aspects can be further articulated in terms of topical formulations, i.e.:

[0062] In some embodiments the invention provides topical formulations comprising live probiotic microorganisms in a non-vegetative state and one or more prebiotic compounds, wherein the probiotic microorganisms are genetically modified to comprise at least one bioactive genetic alteration relative to the respective wild-type microorganism, and said genetic alteration is selected to modulate functionality and integrity of mammalian skin barrier and / or functionality and diversity of mammalian skin microbiome.

[0063] Alternatively, that the invention provides topical formulations for modulating functionality and integrity of mammalian skin barrier and / or functionality and diversity of mammalian skin microbiome, the main features of which are that the formulations comprise at least one prebiotic compound and dormant live probiotic microorganisms comprising at least one bioactive genetic alteration relative to the respective wild type, and that the probiotic microorganisms are configured to transition from a non-vegetative to a vegetative state upon application of the formulations onto the mammalian skin.

[0064] In other words, the inventive strategy behind the present formulations is to exert control on the expression of the bioactive genetic alteration(s) by controlling the transition of the prebiotic microorganisms from non-vegetative to vegetative states.

[0065] More specifically, in some embodiments the formulations of the invention can comprise probiotic microorganisms which are live dormant probiotic bacteria.

[0066] In some embodiments the formulations can comprise probiotic bacteria in the form of viable spores.

[0067] In some embodiments the viable spores can constitute at least about 50-100% of the probiotic bacteria in the formulation, or they can constitute at least about 50-60%, 60- 70%, 70-80%, 80-90% and 90-100%, or at least about 50%, 60%, 70%, 80%, 90%, 100% of the probiotic bacteria in the formulation. In some embodiments the spores can remain viable and dormant during storage of the formulation for at least about 1-12 months at RT or 4°C, or storage of the formulation for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or 1 year or more at RT or 4°C.

[0068] In some embodiments the spores can start to germinate, or they enter a vegetative cycle, after topical application of the formulation onto the mammalian skin.

[0069] In some embodiments the germinating spores, or the resulting bacteria, can remain viable for at least about 1 day after topical application of the formulation onto the skin, or they remain viable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days on the mammalian skin.

[0070] Sporulation and germination rates can be measured by one of the known methods, e.g., CFU count or under the phase-contrast microscope (see present EXAMPLES).

[0071] In other words, in some embodiments the strategy would be to exert control on the expression of the bioactive genetic alteration(s) by controlling the sporulation and germination rates of the prebiotic bacteria.

[0072] A specific aspect of this invention deals with a method of inducing and enriching sporulation in genetically altered probiotic bacteria. The ability to induce high sporulation rates in genetically altered B. subtilis was demonstrated in the present EXAMPLES.

[0073] The rate determining steps in the methods of the invention are:

[0074] (i) culturing the genetically altered probiotic bacteria in a minimal medium while exposing them to predetermined oxygen conditions, and optionally

[0075] (ii) subjecting the probiotic bacteria to a heat shock.

[0076] The predetermined oxygen conditions can be high or low oxygen conditions, depending on the type of probiotic bacteria, and can be induced by monitoring the degree of shaking during the culturing step. As has been presently demonstrated, different strains of bacteria respond differently to exposure to oxygen during growth in minimal media, and thus require different oxygen exposures to confer optimal sporulation rates. Some require low oxygen, or oxygen stress (e.g., B. subtilis 168 strain) and others high oxygen (e.g., B. subtilis K07A strain)

[0077] In some embodiments the culturing step or the growing in a minimal medium can continue for at least about 24 h, 48 h or 72 h, or more.

[0078] In some embodiments the probiotic bacteria can be exposed to low or high oxygen for the same duration of time.

[0079] In some embodiments the heat shock can involve exposure of the probiotic bacteria to at least about 80°C for at least about 15, 30, 45, or 60 min. The ability to control germination time and site, i.e., to avoid early germination before application of the formulation onto the skin and induce germination upon contact of the formulation with the skin surface, is dependent on various factors which are intrinsic and extrinsic to the formulation, including (but not limited to) water and mineral content, the presence of water binding molecules (e.g., glycerol, sucrose, propylene glycol and others), the content of bacterial nutrients (e.g., prebiotics, skin lipids) vs. preservatives as well as pH, temperature and pressure on the skin (e.g., massaging the skin). All these are carefully calibrated to control germination rate of the probiotic component in the formulations per se and upon their application onto the skin.

[0080] One parameter that seems to be especially important is the water activity (WA), a physical measure defined as the ratio of vapor pressure of water in a substance to vapor pressure of pure water at the same temperature. In other words, WA is indicative of the amount of free water that is available for reactions or interactions with other substances. Substances with high WA generally support microbial growth and are therefore more vulnerable to microbial contamination. WA of less than 1, 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.91, 0.9, 0.89, 0.88, 0.86, 0.87, 0.85, 0.84, 0.83, 0.82, 0.81, 0.80, 0.79, 0.78, 0.77, 0.76, 0.75, 0.74, 0.73, 0.72, 0.71, 0.70, 0.69, 0.68, 0.67, 0.66, 0.65, 0.64, 0.63, 0.62, 0.61, 0.60 is generally considered to be inhibiting for bacterial growth.

[0081] WA of less than 1, 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.91, 0.9, 0.89, 0.88, 0.86, 0.87, 0.85, 0.84, 0.83, 0.82, 0.81, 0.80, 0.79, 0.78, 0.77, 0.76, 0.75, 0.74, 0.73, 0.72, 0.71, 0.70, 0.69, 0.68, 0.67, 0.66, 0.65, 0.64, 0.63, 0.62, 0.61, 0.60, 0.59, 0.58, 0.57, 0.56, 0.55, 0.54, 0.53, 0.52, 0.51, 0.50 is considered to be inhibiting for fungal growth.

[0082] To further enhance the ability to control germination time of the probiotic bacteria, in some embodiments the formulations can comprise mixes of genetically engineered spores from various bacterial strains or mixes of genetically engineered and nonengineered spores from the same strain, and various types of prebiotic components.

[0083] In some embodiments the formulations can comprise at least one strain of Bacillus species (Bacillus spp), or several members of Bacillus spp or mixes thereof. Bacillus predominantly refers herein to a genus of Gram-positive rod-shaped bacteria with a chromosome of more than 4M bp, and potentially additional extrachromosomal DNAs. Importantly, in the context of skin, Bacillus is a colonizer and is not part of the species constructing the native human skin microbiome. In some embodiments the Bacillus spp can be selected from B. subtilis, B. licheniformis, B. megaterium and B. amyloliquefaciens or taxonomically related bacteria or mixes thereof.

[0084] In some embodiments the Bacillus spp can be selected from B. coagulans, B. subtilis, B. laterosporus and B. laevolacticus or taxonomically related bacteria or mixes thereof.

[0085] Regarding the prebiotic component in these formulations, in some embodiments the prebiotics can belong to the group of sugars, sugar alcohols, oligosaccharides, polysaccharides, or any combination thereof.

[0086] In some embodiments the sugars can be mono-, di- or tri-saccharide, or any combination thereof.

[0087] In some embodiments the oligosaccharides can be fructooligosaccharides (FOS), galactooligosaccharides (GOS), human milk oligosaccharides (HMDS), or any combination thereof.

[0088] In some embodiments the sugar alcohol can be Xylitol or a derivative thereof.

[0089] In some embodiments the sugar can be Rhamnose or a derivative thereof.

[0090] In some embodiments the polysaccharide can be Inulin or a derivative thereof.

[0091] In some embodiments the formulations can further comprise one or more postbiotic compounds, or substances that are beneficial to mammalian microbiome.

[0092] In some embodiments the postbiotic compounds can be nutrients, vitamins, amino acids, fatty acids, proteins, peptides, or any combination thereof.

[0093] In many embodiments the formulations of the invention can be applied to humans.

[0094] In some embodiments the formulations can be applied to non-human mammals, including livestock animals and pets.

[0095] Regarding genetic modifications of the probiotic component, as of now, there is a plethora of methods for introducing and expressing foreign DNA or RNA into the bacterial genome. The classical method is by cloning the foreign DNA / RNA into a vector such as a bacteriophage or a plasmid. Both types of vehicles are commercially available, with various improvements of cloning sites, linkers and signal sequences controlling the expression of cloned genes or proteins.

[0096] Another approach is by introducing naturally occurring or modified small single stranded RNA or DNA. It was extensively demonstrated that small single stranded (ss) RNA or DNA (e.g., siRNA, shRNA, microRNA, aptamers, morpholinos, and sense and antisense oligos with a general length in the range of <20 bp, 20-50 bp and 50-70 bp), provide an efficient tool for inhibiting or interfering with gene expression in a more precise quantitative manner. One way of introducing this type of molecules into probiotic bacteria is via gene editing.

[0097] Gene editing technology has developed to provide the first-generation gene editing using Zinc Finger Nucleases (ZFNs), the second-generation gene editing using Transcription Activator-like Effector Nucleases (TALEN), and in recent years, the more popular third-generation gene editing using Clustered Regularly Spaced Short Palindromic Repeats CRISPR (CRISPR-Cas). Specifically:

[0098] 1. ZFN based gene editing uses ZFNs consisting of an engineered nuclease (Fokl) fused to zinc finger DNA-binding domains. Additionally, ZFNs function as dimers, increasing the length of the DNA recognition site and consequently increasing specificity.

[0099] 2. TALEN based gene editing is similar in terms of requirements of Fokl nuclease and dimerization to function. The DNA binding domains further employ an effector array of 33-35 amino acid repeats (TALEs) enabling single-nucleotide recognition, thereby improving targeting capabilities and specificity of ZFNs.

[0100] 3. CRISPR-Cas-mediated genome editing is a 2-component system consisting of a guide RNA (sgRNA) and a Cas9 nuclease, wherein the former targets the DNA sequence and the latter cuts the DNA defined by the sgRNA. The main advantage is that sgRNA can be carefully customized to improve targeting and specificity, which makes CRISPR-Cas the most handy, adaptable, and cost-effective gene editing method today.

[0101] 4. Base editing is derived from CRISPR-Cas9 and uses inactive Cas9 (dCas9) fused to bacterial DNA Cytidine or Adenine Deaminases (CBE or ABE) to allow single nucleotide substitutions, i.e., C>T or A>G, which is the main advantage of this method.

[0102] 5. Prime editing is used for introducing transition mutations and small deletions / insertions (50-80 bp) without double- stranded DNA breaks. It uses Cas9-nickase fused to Reverse Transcriptase (RT) and a modified guide RNA (pegRNA) consisting of the primer binding site (PBS) and a sequence containing the desired edit.

[0103] 6. Programmable Addition via Site-specific Targeting Elements (PASTE) is the most recent development in gene editing that allows to introduce larger insertions (about xlOK bp) without double-stranded DNA breaks due to the use of bacteriophage serine integrase enzymes. It incorporates certain aspects of Prime editing. In short, recent advances in gene editing technologies, and especially CRISPR, provide multiple accessible tools for the design and development of genetically modified probiotics. So far, CRISPR and CRISPR derived technologies have been successfully used for screening and engineering genomes for loss of function using CRISPR knock out (CRISPRKO) or CRISPR interference or inhibition (CRISPRi) cassettes and gain of function using CRISPR activation (CRISPRa) cassettes.

[0104] Regarding specific genetic alterations, in some embodiments the genetic change can comprise or consist of one or more deletion, insertion, duplication, rearrangement and / or single nucleotide variation in the genome of the probiotic microorganism relative to the wild type.

[0105] In some embodiments the genetic change can comprise or consist of one or more chimeric fusions of heterologous DNA or RNA with the DNA or RNA of the probiotic microorganism.

[0106] In some embodiments the genetic alterations can change can comprise or consist of extrachromosomal or intrachromosomal additions of DNA or RNA to the genome of the probiotic microorganism relative to the wild type

[0107] In some embodiments the intrachromosomal or extrachromosomal addition can be an addition of DNA or RNA sequence contained in a plasmid or a bacteriophage.

[0108] In some embodiments the genetic alterations can further comprise or consist of increased or decreased expression of one or more genes, proteins, or peptides in the probiotic microorganisms relative to the respective wild type.

[0109] In some embodiments the genetic alterations can further comprise or consist of increased or decreased expression of one or more genes, protein, or peptides in a mammalian host upon application of the formulation onto the mammalian skin.

[0110] In some embodiments said proteins or peptides can be structural proteins or peptides, or enzymes.

[0111] In some embodiments said proteins or peptides can be secreted proteins or peptides, or secreted enzymes.

[0112] In some embodiments the formulations of the invention can be sophisticated to provide controlled release of the bioactive(s), including controlled, modified, sustained, prolonged, immediate release of the bioactive.

[0113] In some embodiments the formulations can include additional active ingredients, such as antibiotics, anti-inflammatory agents, whitening agents, etc. In some embodiments the formulations can include non-active ingredients to obtain the desired properties of skin moisture and integrity and protectivity against environmental hazards.

[0114] Altogether, the formulations of the invention can serve as a basis for new lines of products for dermatological, veterinary and cosmetic uses.

[0115] This aspect can be further articulated in terms of dermatological, veterinary or cosmetic formulations comprising any of the above-mentioned probiotic formulations, and optionally the respective acceptable carriers or excipients.

[0116] In some embodiments they can be provided in the form of leave-on formulations, such as moisturizing masks and nourishing creams, to provide long-term effects of the cloned bioactive(s).

[0117] In some embodiments they can be provided in the form of rinse-off formulations, such as soaps, shampoos, skin cleansers and hair conditioners, to provide short-term effects of the bioactive(s) and avoid side effects.

[0118] The dermatological, veterinary, or cosmetic formulations of the invention can be further incorporated into a wide range of advantageous topical products in the form of topical gels, ointments, creams, lotions, masks, shampoos, sprays, powders, etc.

[0119] In terms of intended uses, it is projected that the formulations of the invention can serve for the prevention, alleviation and treatment of many clinical, sub-clinical and non- clinical conditions in humans, animals and pets, and mammals in general.

[0120] In some embodiments the dermatological formulations of the invention can be used in the prevention, alleviation or treatment of many types of mammalian skin disorders or skin conditions, and especially in humans.

[0121] Some examples of relevant human skin disorders that can be treated by the dermatological formulations of the invention include (but are not limited to) atopic dermatitis, acne vulgaris, rosacea, seborrheic dermatitis, pityriasis versicolor, psoriasis, lupus erythematosus, diabetic foot ulcer, actinic keratosis, eczema or xerosis.

[0122] Examples of relevant sub-clinical and non-clinical human skin conditions that can be treated by the cosmetic formulations of the invention include (but are not limited to) dandruff, sunburns, mild skin irritations, skin discolorations, unhealthy skin, hair and nail appearance and / or skin aging. More broadly, the cosmetic formulations of the invention can be used for skin whitening or brightening and / or for restoring a healthy complexion and rejuvenated appearance of the skin.

[0123] This aspect can be further articulated in terms of cosmetic or dermatological or veterinary methods applied to mammalian, animals or human subjects, the main feature of which is topical administering the formulations to the mammalian subject.

[0124] Ultimately, the invention can be further articulated in terms of uses of any of the formulations as above in the manufacture of dermatological, veterinary or cosmetic products serving for the prevention, alleviation, or treatment of various skin, hair and nail abnormalities, or clinical, subclinical or non-clinical conditions affecting the skin, hair or nail in mammals, and especially humans.

[0125] The terms “about” or “approximately” are meant to encompass herein a deviation of up to ±15% from the specified values or ranges, and more specifically up to a deviation of about ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±11%, ±12%, ±13%, ±14% or ±15% therefrom.

[0126] EXAMPLES

[0127] The figures and examples provided herein serve only illustrative purposes and are not intended to be limiting since the scope of invention will be decided by the scope of appended claims.

[0128] EXAMPLE 1: Strategies for inducing bacterial sporulation and germination

[0129] Bacterial sporulation

[0130] Sporulation is a natural bacterial survival mechanism that comes in response to environmental stress. Under stress, the bacterial genome is sequestered in a dormant (spore) non-vegetative state, and upon stress removal, is triggered to germination or reversal to the vegetative state. Sporulation is driven by phosphorylation and activation of certain bacterial transcription factors, leading to compartmentalization of the bacterial ribosomes, enzymes and Ca2+-dipicolinic acid complex (CaDPA) in the core or cortex of mature spores. As a result, the mature spores are partially dehydrated and metabolically dormant, which makes them relatively resistant to UV, a range of chemicals (e.g., peroxide and hypochlorite), extreme heat and other stressors. Bacterial germination

[0131] Bacillus spores constantly monitor the environment by various germination receptors (GR) that are responsive to nutrient (e.g., amino acids, sugars, purine nucleosides) or non-nutrient (e.g., salts (KBr, K2HPO4), pressure (100-600 MPa) and temperature (37°C) triggers of germination. Exposure to such triggers initiates a series of reactions, involving changes in GR conformation, conductivity of transmembrane channels, membrane permeability and eventually in the release of CaDPA and monovalent cations (e.g., H+, K+, Na+) and increase spores’ water content (up to 80% wt). The CaDPA release and spores’ hydration are the hallmarks of transition from the non- vegetative (dormant) to the vegetative (germination) state.

[0132] Strategy for controlled transition from sporulation to germination

[0133] The ability to control bacterial transition from a non-vegetative to vegetative states is key to success of a probiotic -based product. Premature germination often leads to reduced bacterial viability and bacterial death, reflected in reduced number of CFU (Colony Forming Units). Premature germination can also affect a product’s stability and its ultimate efficacy in situ.

[0134] In the context of skin, there are apparent advantages in being able to preserve probiotic bacteria in a non-vegetative state prior to application of the product for product’ s storability. On the other hand, after application of the product onto the skin, one would allow transition to a vegetative state for complete manifestation of its cosmetic or dermal benefits. In case of topical probiotics, a transition from non-vegetative to vegetative states can be triggered by a series of specific environmental conditions on the skin surface, including (but not limited to) skin temperature, acidity, lipid content, and moisture, and pressure exerted by applying the probiotics onto the skin.

[0135] EXAMPLE 2: Strategies for inducing genetic alterations in Bacillus

[0136] The Bacillus genus

[0137] The Bacillaceae family are predominantly Gram-positive bacteria, which include anaerobic spore-forming bacilli, and aerobic or facultatively anaerobic endospore forming bacteria. Bacillaceae encompass at least 226 species which can be distinguished by 16SrRNA gene sequencing and other molecular methods. One of the distinguishing features of Bacillaceae is the ability to form endospores which are highly resistant to adverse environmental insults such as heat, radiation, chemicals, and drought. The choice of B. subtilis

[0138] B. subtilis is rod-shaped Gram-positive spore forming bacteria that can survive with minimal supplementation of salts, carbon, nitrogen, and phosphorus. When exposed to nutrient limitations, B. subtilis activates several pro-surviving mechanisms, such as synthesis of antibiotics and extracellular enzymes, motility, DNA uptake, formation of biofilm and sporulation. Upon exposure to certain growth stimulating triggers, the spores undergo germination and reverse to a vegetative state.

[0139] In nature, B. subtilis is widely prevalent in soil, plant roots and aquatic environments. It is regarded as GRAS and owing to its affordable and easy growing conditions in vitro, it has become an integral part of the biotech industry. It is therefore widely used in the production of fermented foods, various food additives, household detergents, antibiotics, vitamins, and vaccines.

[0140] Genetic engineering of B. subtilis

[0141] An increased understanding of B. subtilis biochemistry, genetics, and physiology, created an abundance and versatility of engineered B. subtilis strains. B. subtilis 168, one of the most utilized strains, is a tryptophan auxotroph (trpC2). BSB 1, a more recent tryptophan-prototrophic (trp+) 168 variant, has become available. The Bacillus Genetic Stock Center and Japanese National BioResource Project provide comprehensive collections of B. subtilis variants.

[0142] B. subtilis has been known for its natural competence for DNA uptake and high transformation efficiency. These properties can be further enhanced and manipulated by known in the art methods of genetic engineering. Examples of some current approaches to gene manipulation include: 1. traditional methods via genetic competence and homologous recombination, 2. deletion library containing erythromycin and kanamycin cassette interruptions of non-essential genes, 3. CRISPR-interference (CRISPRi), targeting essential genes, 4. pMiniMad for marker-less allelic exchange, and 5. CRISPR / Cas9 gene editing.

[0143] 1. The genes responsible for DNA uptake in B. subtilis are activated by ComK - a global competence transcription regulator that acts during the stationary phase (minimal medium). One example of ComK dependent genes is Rec A, promoting pairing and strand exchange via homologous recombination. Homologous recombination between the B. subtilis chromosome and an exogenous DNA can be enhanced by the incorporation of specific genetic modifications, using PCR, plasmids or genomic DNA. The minimal requirement for optimal recombination is about ~ 500 bp homology with the host chromosome.

[0144] 2 / 3. Various deletion libraries can be obtained from Bacillus Genetic Stock Center, e.g., strains with erythromycin / kanamycin cassettes interruptions of non-essential genes and strains enabling CRISPR-interference (CRISPRi) of essential genes, the latter allows more precise control on gene transcription level. These resources can be used to produce engineered strains by integration of knockout alleles (gene elimination) or knockdown (reduced gene expression).

[0145] 4. The pMiniMad approach facilitates marker- less integration of point mutations, insertions, or deletions. It involves integration and looping out of the pMiniMad plasmid carrying the genetic alteration of interest embedded in temperaturesensitive origin of replication and antibiotic sensitive cassette. The transformed cells are first grown in a non-permissive temperature to induce a crossover event that confers antibiotic resistance, and then switched to a permissive temperature to induce plasmid excision and antibiotic sensitivity.

[0146] 5. The CRISPR / Cas9 method enables to achieve high precision genetic alterations with remarkable efficiency, including introduction of substitutions, deletions, and insertions on diverse genetic background (up to 20 kb). It may require specific materials compared to other methods, it is a highly versatile and accessible tool for introducing multiple genetic modifications.

[0147] Figs 2A-2C and Fig. 5 show certain examples of recombinant constructs for introducing and expressing heterologous genes of interest in B. subtilis, the methods of implementation thereof are shown in EXAMPLE 5.

[0148] EXAMPLE 3: Producing genetically altered B. subtilis spores

[0149] Main steps in the methods of producing genetically altered B. subtilis

[0150] (i) Selecting B. subtilis strains, e.g., strains 168 or K07A (BGSC 1 A1605).

[0151] (ii) Designing a recombinant construct containing the gene of interest, promoter(s) and termination sequences, ribosomal binding site(s) and marker region(s).

[0152] (iii) Introducing the recombinant construct into the B. subtilis genome and verifying the presence of the acquired genotype / phenotype.

[0153] (iv) Verifying functionality of the inserted gene by respective RNA- or protein-based or activity assays in cell extracts and growth medium. (v) Quantifying the expression of the respective gene product or its downstream effector(s).

[0154] Example of methods for inducing B. subtilis sporulation

[0155] Bacterial cells were grown in LB over night at 37°C at 200 rpm, diluted to ODeoo = 0.05 in LB and grown at 37°C until OD600 = 0.7-0.8. Cells were diluted to ODeoo = 0.01 in fresh DSM (per IL: 8 gr Bacto nutrient broth, 10 mL 10% w / v KC1, 10 mL 1.2% (w / v) MgSO4-7H2O, pH adjusted to 7.6 by I M NaOH, volume adjusted to 1 L with water and autoclave, ImL IM Ca(NO3)2-4H2O, ImL lOmM MnCh-4H2O and 1 mL ImM FeSO4-7H2O added before use), grown for 24 and 48 h. At each time point, ODeoo was measured, cells were washed 3 times with cold water, serial dilutions were performed and samples were plated on LB agar before and after healing (30 min at 80°C). Sporulation efficiency was calculated as CFU after heating divided by CFU before heating.

[0156] Example of methods for inducing B. subtilis spores’ germination

[0157] For activity assays, spores were diluted to 1-OD final in 10ml LB medium (100ml v), incubated at 37°C overnight. ODeoo is measured every 5 min for verification of OD increase.

[0158] Germinated spores were subjected to qualitative and quantitative analyses of expression of elected genes or proteins as per respective genetic modifications and analyses pertaining to the regulatory requirements of safety and efficacy.

[0159] Figs 3A-3C illustrate the main steps in the spores’ enrichment and germination procedures, and their evaluation under the phase-contrast microscope.

[0160] EXAMPLE 4: Strategy for selecting candidate genes

[0161] Candidate bioactives were selected based on prior literature analysis exploring relationships between specific genes / gene products and specific manifestations of skin damage and aging, such as excessive pigmentation, inflammation, skin wrinkling and dryness, UV damage and symptoms of various skin conditions. The bioactives of interest we grouped into the following functional groups:

[0162] I. Skin moisturizing and skin conditioning agents

[0163] II. Skin whitening and brightening agents

[0164] III. Enzymes and enzymes' co-factors

[0165] IV. Vitamins

[0166] V. Factors of healing / attenuating of symptoms of skin diseases VI. Factors of UVA / UVB absorption / defense, and

[0167] VII. Other factors acting on the skin health and healthy skin appearance.

[0168] Each group was further dissected into subgroups and specific examples of elected agents. Preliminary results are shown in Table 1 below.

[0169] Table 1. Selection of candidate bioactives to be expressed in B. subtilis

[0170] Certain candidates were elected for the initial proof of concept:

[0171] • Hyaluronic acid

[0172] • Vitamin C [Ascorbic acid]

[0173] • Botox-like peptide

[0174] • Facial firming peptide

[0175] • Bromelain proteolytic enzyme

[0176] • SOD radical scavenging enzyme

[0177] • Melatonin hormone-like

[0178] • Tranexamic acid

[0179] • GHK-Cu tripeptide

[0180] • P-galactosidase

[0181] • Caffein

[0182] Figs 1A-1C and Fig. 3 show some example constructs with heterologous hasA (Hyaluronic acid), P-Gal (P-galactosidase), hSOD (Superoxide dismutase), the methods of implementation thereof in B. subtilis are shown in EXAMPLE 5 and Figs 4-6. EXAMPLE 5: Examples of genetically altered B. subtilis strains

[0183] I. Hyaluronic acid (HA) producing strains

[0184] HA is synthesized by polymerization of monosaccharides from two nucleotide sugars, UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP- GlcNAc), by hasA encoded HA synthase (HAS). hasA, and as a result HA production, is attributed to only group A and C Streptococci and Pasteurella multocida. In Streptococci, hasA makes part of a multiple gene operon encoding enzymes of the HA biosynthetic pathway to produce UDP-GlcUA and UDP-GlcNAc from glucose as a carbon source. The wild type B. subtilis lacks hasA, as such, but only hasB, hasC, and hasD homologues (tuaD, gtaB, and gcaD, respectively). The adopted strategy used recombinant methods to construct an artificial has-operon including Streptococcal hasA in conjunction with B. subtilis derived has-homologues in combination with carefully oriented transcription promoters and terminators and marker genes.

[0185] Figs 1A-1C show some example expression constructs with heterologous hasA. These constructs are now being expressed and tested in B. subtilis strains 168, and K07A (BGSC 1 Al 605). HA production is detected in the medium of growing bacteria by known quantitative assays using specific biotinylated HA binding proteins (e.g., Corgenix, Westminster, CO). Genetically modified B. subtilis are further subjected to specific conditions encouraging sporulation (see EXAMPLE 3).

[0186] II. Introducing HA by CRISPR Cas9 method

[0187] An alternative approach for producing HA in B. subtilis can use CRISPR Cas9 method to insert the Streptococcal hasA and the B. subtilis tuaD, gtaB, and gcaD in the same operon in conjunction with 6-HisTag at the C-terminus of the hasA gene to allow detection and quantification of HA production by HPLC MS (Widner B et al 2021, Anal Chem 93(11):4809-4817).

[0188] III. Vitamin C producing strains

[0189] L-ascorbic acid (L-AA, also vitamin C) in its native form is highly susceptible to oxidation and degradation due to the reactive 2-OH group. The glycosylated L-AA, 2-O-a-D-glucopyranosyl-L-ascorbic acid (AA-2G), is more stable in aqueous solutions, and is further capable of efficiently releasing L-AA via hydrolysis. AA-2G is functionally similar to L-AA but exhibits much less cytotoxicity. AA-2G is produced from L-AA via one-step glycosylation process by sucrose phosphorylase (SPase) and sucrose substrate. A previous study showed that SPase genes from heterologous bacterial sources can be successfully incorporated into B. subtilis using recombinant cloning methods. It was further shown that a secretable recombinant SPase did not require the presence of a signal peptide but only the presence of a strong promoter (Gan T et al 2023, Enzyme Microbiol Technol 169:110267). The adopted strategy uses a heterologous SPase gene, e.g., B. breve BbrSPase, (GenBank Acc. No. POO08785B) or B. longum BloSPase (GenBank Acc. No. AAO84039) in conjunction with dual promoters, growth-dependent and constitutive promoters (e.g., pSigH-p43) and a terminator sequence, which are introduced into the AmyE gene in B. subtilis (e.g., 168, and K07A (BGSC 1 A 1605) via homologous recombination.

[0190] Fig. 3 shows analogous constructs using heterologous P-Gal and hSOD in conjunction with the pSigH-p43 dual promoters, a linker and B. clausii aprL derived terminator integrated into the B. subtilis AmyE locus.

[0191] IV. Secreted enzymes producing strains

[0192] Two recombinant constructs were engineered for the expression / secretion of two additional enzymes in B. subtilis - the human Cu, Zn superoxide dismutase (Protein Acc. No. CR541742.1) and the E. coli 0-galactosidase (Protein Acc. No. WP_000177906.1)]. In both cases, the coding regions were cloned into the AmyE locus (integration homology arms from pDRl l l plasmid) of 168 and KO7 A (BGSC 1 Al 605) strains, including the spectinomycin resistance cassette (spec sequence from pDRl l l), the dual pSigH-p43 promoter and B. clausii aprL transcription terminator (see also Fig. 3).

[0193] Current studies are using these constructs to produce genetically altered B. subtilis strains. The expression / secretion of the respective enzymes is tested by functional colorimetric assays such as WST-8 xanthine and xanthine oxidase method for SOD activity and ONPG assay for P-Gal, including respective controls. The genetically modified B. subtilis are further subjected to specific conditions encouraging sporulation (see EXAMPLE 3). The main steps in the spores’ enrichment and germination procedures are shown Figs 2A-2B. Figures show that one of the determining factors of sporulation efficiency is exposure to oxygen during the starvation step, with 168 responding to low oxygen (stress) and KO7A to high oxygen to yield sporulation rates over 80% and up to 100%, respectively. Alternative methods are using chimeric constructs wherein the candidate gene is fused with a sequence encoding a bacterial secretion signal peptide. Sequences encoding effective bacterial signal peptides can be retrieved from multiple sources (e.g., Simonen and Palva 1993, Microbiol Rev 57:109-137 and others).

[0194] Figs 4-6 show certain methods for the verification of successful integration of the cloned genes into the AmyE locus of B. subtilis, and further methods for the detection of candidate genetically altered B. subtilis strains and their respective expression products.

Claims

CLAIMS:

1. A topical delivery system of a bioactive that modulates functionality and integrity of mammalian skin barrier and / or functionality and diversity of mammalian skin microbiome, the delivery system comprising live probiotic microorganisms and at least one prebiotic compound, wherein the probiotic microorganisms are in a non-vegetative state and comprise at least one genetic alteration, relative to the respective wild-type microorganism, that facilitates the expression and / or secretion of said bioactive upon application of the delivery system on the mammal’s skin.

2. A topical formulation comprising live probiotic microorganisms in a non- vegetative state and at least one prebiotic compound, wherein the probiotic microorganisms are genetically modified to comprise at least one bioactive genetic alteration, relative to the respective wild-type microorganism, said genetic alteration being selected to modulate functionality and integrity of mammalian skin barrier and / or functionality and diversity of mammalian skin microbiome.

3. A topical formulation for modulating functionality and integrity of mammalian skin barrier and / or functionality and diversity of mammalian skin microbiome, the formulation comprising at least one prebiotic compound, and live probiotic microorganisms, comprising at least one bioactive genetic alteration relative to the respective wild-type microorganism, and are configured to transition from a non- vegetative to a vegetative state upon application of the formulation onto the mammalian skin.

4. The formulation of claim 2, wherein the probiotic microorganisms do not express the at least bioactive genetic alteration, prior to topical application of the formulation onto the mammalian skin.

5. The formulation of claim 2, wherein the probiotic microorganisms express the at least bioactive genetic alteration, after topical application of the formulation onto the mammalian skin.

6. The formulation of claim 2, wherein upon topical application of the formulation to the mammalian skin, the probiotic microorganisms transition from the non-vegetative to the vegetative state.

7. The topical formulation of any one of claims 2 to 6, wherein said probiotic microorganisms are probiotic bacteria.

8. The topical formulation of claim 7, wherein the formulation comprises probiotic bacteria in the form of dormant viable spores.

9. The topical formulation of claim 8, wherein the dormant viable spores constitute at least about 50-100% of the probiotic bacteria.

10. The topical formulation of claim 8, wherein the spores remain viable in the formulation for at least about 1-12 months.

11. The topical formulation of claim 8, wherein the spores start to germinate after topical application of the formulation onto the mammalian skin.

12. The topical formulation of claim 11, wherein the germinating spores remain viable for at least about 1 day after topical application of the formulation onto the mammalian skin.

13. The topical formulation of claim 7, wherein said probiotic bacteria comprise at least one strain of Bacillus species (Bacillus spp), or a mix thereof.

14. The topical formulation of claim 13, wherein the Bacillus spp is selected from B. subtilis, B. licheniformis, B. megaterium and B. amyloliquefaciens or taxonomically related bacteria, or a mix thereof.

15. The topical formulation of claim 13, wherein the Bacillus spp is selected from B. coagulans, B. subtilis, B. laterosporus and B. laevolacticus or taxonomically related bacteria, or a mix thereof.

16. The topical formulation of any one of claims 2 to 6, wherein the at least one prebiotic compound is selected from a sugar, a sugar alcohol, an oligosaccharide, a polysaccharide, or a combination thereof.

17. The topical formulation of claim 16, wherein the at least one sugar is selected from a mono-, a di- or a tri- saccharide, or a combination thereof.

18. The topical formulation of claim 16, wherein the at least one oligosaccharide is a fructooligosaccharide (FOS), a galactooligosaccharide (GOS), a human milk oligosaccharide (HMDS), or a combination thereof.

19. The topical formulation of claim 16, wherein the at least one sugar alcohol is Xylitol or a derivative thereof.

20. The topical formulation of claim 16, wherein the at least one sugar is Rhamnose or a derivative thereof.

21. The topical formulation of claim 16, wherein the at least polysaccharide is Inulin or a derivative thereof.

22. The topical formulation of any one of claims 2 to 6, wherein said mammal is human.

23. The topical formulation of any one of claims 2 to 6, further comprising at least one postbio tic compound.

24. The topical formulation of claim 23, wherein the at least one postbiotic compound is a nutrient, a vitamin, an amino acid, a fatty acid, a protein, a peptide, or a combination thereof.

25. The topical formulation of any one of claims 2 to 6, wherein the at least one bioactive genetic alteration is selected from at least one of a deletion, an insertion, an addition, a duplication, a rearrangement, a single nucleotide variation in at least one gene of the probiotic microorganisms, relative to the respective wild type.

26. The topical formulation of any one of claims 2 to 6, wherein the at least one bioactive genetic alteration comprises a chimeric fusion of a heterologous DNA or RNA sequence with an autologous DNA or RNA sequence of the probiotic microorganisms.

27. The topical formulation of any one of claims 2 to 6, wherein the at least one bioactive genetic alteration comprises an extrachromosomal or an intrachromosomal addition of at least one DNA or RNA sequence to the genome of the probiotic microorganisms, relative to the respective wild type.

28. The topical formulation of claim 27, wherein said extrachromosomal or intrachromosomal addition of at least one DNA or RNA sequence is contained in a plasmid or a bacteriophage.

29. The topical formulation of any one of claims 2 to 6, wherein said at least bioactive genetic alteration comprises increased or decreased expression of at least one gene, protein, or peptide in the probiotic microorganisms, relative to the respective wild type.

30. The topical formulation of any one of claims 2 to 6, wherein said at least one bioactive genetic alteration comprises increased or decreased expression of at least one gene, protein, or peptide in a mammalian host upon application of the formulation onto the mammalian skin.

31. The topical formulation of claims 29 or 30, said protein or peptide being a structural protein or peptide, or an enzyme.

32. The topical formulation of claims 29 or 30, said protein being a secreted protein or peptide, or a secreted enzyme.

33. The topical formulation of any one of claims 2 to 6, wherein said modulating functionality and integrity of mammalian skin barrier and / or functionality and diversity of mammalian skin microbiome comprises modulating at least one of: an immunologic, allergic, inflammatory reaction, proliferation and / or differentiation of cutaneous tissue and / or cells in a mammalian host.

34. The topical formulation of any one of claims 2 to 6, wherein said modulating functionality and integrity of mammalian skin barrier and / or functionality and diversity of mammalian skin microbiome comprises modulating at least one of: moisturizing, tightening, antiwrinkle, wound healing, brightening, whitening of the skin and / or enhancing rejuvenated or uniform or healthy skin appearance in a mammalian host.

35. The topical formulation of any one of claims 2 to 6, wherein said modulating functionality and diversity of mammalian skin microbiome comprises inhibition of growth of at least one harmful microorganism on the skin of a mammalian host.

36. The topical formulation of any one of claims 2 to 6, wherein said modulating functionality and diversity of mammalian skin microbiome comprises inhibition of dysbiosis of skin microbiome in a mammalian host.

37. The topical formulation of any one of claims 2 to 6, wherein the functionality and integrity of mammalian skin barrier and / or the functionality and diversity of mammalian skin microbiome further comprises inhibition or reduction of at least one symptom of at least one skin disorder or skin condition, or a condition involving abnormalities of hair or nails.

38. The formulation of any one of claims 1 to claim 37, further formulated to provide at least one of a controlled, a modified, a sustained, a prolonged, an immediate release of said bioactive.

39. A cosmetic formulation comprising the formulation of any one of claims 1 to 38, and optionally further comprising a cosmetically acceptable carrier or excipient.

40. The cosmetic formulation of claim 39, which is a leave-on cosmetic formulation.

41. The cosmetic formulation of claim 39, which is a rinse-off cosmetic formulation.

42. A dermatological formulation comprising the formulation of any one of claims 1 to 38, and optionally further, comprising a dermatologically acceptable carrier or excipient.

43. The dermatological formulation of claim 42 for use in the prevention, alleviation or treatment of at least one dermatologic disorder or condition.

44. The dermatological formulation for use according to claim 43, wherein said at least one dermatologic disorder or condition is selected from atopic dermatitis, acne vulgaris, rosacea, seborrheic dermatitis, pityriasis versicolor, psoriasis, lupus erythematosus, diabetic foot ulcer, actinic keratosis, eczema, xerosis.

45. The cosmetic formulation of any one of claims 39 to 41 for use in the prevention or alleviation of at least one cosmetic skin condition, or a condition involving abnormalities of hair or nails.

46. The cosmetic formulation for use according to claim 45, wherein said at least one cosmetic condition comprises is selected from dandruff, sunburn, a mild skin irritation, a skin discoloration, an unhealthy skin appearance, skin aging.

47. The cosmetic formulation of any one of claims 39 to 41 for use in restoring a healthy complexion and rejuvenated appearance of the skin, or a healthy appearance of hair or nails.

48. A method of preventing, alleviating, or treating at least one dermatologic disorder or condition in a mammal, the method comprises topical administering the dermatological formulation of claim 42 onto the mammals’ skin.

49. A method of preventing, alleviating, or treating at least one cosmetic skin condition in a mammal, the method comprises topical administering the cosmetic formulation of any one of claims 39 to 41 onto the mammals’ skin, hair or nails.

50. Use of the formulation of any one of claims 2 to 47 in the manufacture of a dermatological, veterinary or cosmetic product for the prevention, alleviation, or treatment of at least one dermatologic disorder or clinical, sub-clinical or non-clinical skin condition, or a condition involving abnormalities of hair and nails.

51. The topical formulation of any one of claims 2 to 6, wherein said mammal is a livestock animal or a pet.

52. The formulation of any one of claims 1 to 38 for use in veterinary medicine.

53. A method of inducing sporulation in genetically altered probiotic bacteria, the method comprises(i) culturing the genetically altered probiotic bacteria in a minimal medium while exposing them to predetermined oxygen conditions, and optionally(ii) subjecting the probiotic bacteria to a heat shock.

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