New container, method and delivery system stabilizing topical formulations

Abstract

The present invention relates to a closed container for storage of live microorganisms in a formulation for topical application comprising a three-phase system a) microcrystals comprising live microorganism; b) an anhydrous carrier; and c) an air phase in contact with a desiccant.

Description

[0001] NEW CONTAINER, METHOD AND DELIVERY SYSTEM STABILIZING TOPICAL FORMULATIONS

[0002] Technical field of the invention

[0003] The present invention is related to a container, a new three-phase topical delivery system of stable live microorganisms, a method for suspending water soluble crystals in an anhydrous carrier and with moisture free air phase in a closed container. Products based thereon are stable over prolonged periods of time and are well suited for topical administration. The method of the invention has particular advantage when the water soluble crystals of live microorganism is applied topical as stabilized water soluble crystals in an anhydrous phase,

[0004] The present invention relates to water free three-phase systems comprising viable microorganisms. In particular the present invention relates to water free three-phase systems for topical use on skin or mucous membranes comprising at least one viable probiotic microorganism.

[0005] In particular the present invention relates to three-phase system for formulation of a product for topical application having low water activity in the range of about 0.1 to about 0.75.

[0006] Background of the invention

[0007] There is considerable interest in the use of probiotic bacteria. Probiotics are live microorganisms that confer health benefits to the host when administered at adequate levels (FAO WHO, 2006). However, to exert these benefits, the microorganisms must remain viable during the processing and storage of the product containing live probiotics, considerable amount of research have been done to stabilize probiotics for oral consumption and ensure resistance to gastrointestinal fluids. Because probiotics are sensitive to a number of factors, including the presence of oxygen and acidic media, microencapsulation has been studied as a method of increasing the viability of probiotic cells. Microencapsulation of probiotics is a process that surrounds probiotic microorganisms in a polymeric membrane, protecting them and, in certain cases, allowing their release under specific conditions. The techniques commonly applied to encapsulate probiotics are extrusion, atomization or spray drying, emulsion, coacervation and immobilization in starch granules (Favaro-Trindade et al., 2011). Polysaccharides, such as alginate, gellan, K-carrageenan, and starch are the most commonly used materials in the microencapsulation of bifidobacteria and lactobacilli. In order to meet the demand of skin care products comprising live microorganisms it is necessary to develop stable compositions for topical use which can maintain viability of the microorganisms as well as secure activation of the microorganism when applied on skin or mucous membranes.

[0008] Topical formulations and products for pharmaceutical or cosmetic purposes are developed to have a long shelf life and to be stable towards contamination and spoilage caused by microorganisms. The stability of viable probiotics in these topical formulations are thus very limited, however, the use of probiotics in topical formulations could have a huge potential if viability can be maintained in the formulation. Topical formulations like creams, lotions, gels, mists inherently contain a high degree of water, i.e. in order to be suitably formulated into a gel, cream, foam, lotion, ointment etc. Evidently, the presence of such high degrees of water in these formulations, poses a problem for the storage of probiotics in their metabolically inactive condition. A second problem occurring in such topical formulations, is that these generally contain agents, which are not compatible with the survival of microorganisms, such as preservatives, surfactants, emulsifiers and other ingredients in order to protect such formulations against the growth of unwanted microorganisms as well as for forming stable emulsions. These agents, preservatives, will naturally be a major problem in the formulation of beneficial viable microorganisms.

[0009] W018002248 disclose a concept of formulating microorganisms in a 2-compartment system, protecting the microorganisms of the inner core compartment from the ingredients in the outer compartment once the content of both compartments is combined, this microencapsulation is for topical use, however, still this encapsulation comprises microcapsules of a size touchable to the skin and which needs to be rubbed into the skin to break the capsules. The capsules not broken by friction will then not release the viable microorganisms to the surface of the skin. Another problem will be the survival or activation on the skin when the capsules are broken and the viable probiotics released to the skin with the ingredients in the other compartment which can include preservatives inactivating the probiotic strain.

[0010] The use of viable probiotics for topical application is very limited and most products are based on lysates (inactivated dead bacteria) of the probiotic strain to overcome the problems of maintaining viability of the microorganisms in the topical composition. The problems observed when formulating live probiotic strains in gels, emulsions, lotions and the like for topical application on the skin of mammals are lack of viability and stability. Hence, it was an object of the present invention to provide a system allowing for long-term storage of viable microorganisms, which does not substantially harm such microorganisms upon use thereof and which does release the viable microorganisms when applied on the skin or on mucous membranes

[0011] It was surprisingly found that formulating the microorganisms in a three-phase system of dried water-soluble crystals, an anhydrous carrier and a dried air phase will significantly improve stability and shelf life of the live microorganism. A further surprising benefit was that when releasing the dried water-soluble crystals on skin or mucous membranes then the crystals easily release the microorganisms in a viable form on the skin.

[0012] Summary of the invention

[0013] Thus, an object of the present invention relates to a three-phase delivery system, a container comprising a three-phase system and a method of preparing a three-phase formulation.

[0014] In particular an object of the present invention relates to a closed container for storage of live microorganisms in a formulation for topical application comprising a three-phase system, the three-phase system comprising; a) microcrystals comprising live microorganism; b) an anhydrous carrier; and c) an air phase in contact with a desiccant.

[0015] A further object of the present invention relates to a method of prolonging the stability of cosmetic products for topical application comprising a) adding microcrystals of live microorganisms to an anhydrous carrier to create a two-phase system; b) dispensing the two-phase composition to a container; and c) sealing with an air phase of at least 0.1 cm3air in direct contact with a desiccant.

[0016] In particular, it is an object of the present invention to maintain stability of live microorganisms.

[0017] In yet another aspect of the invention the crystals with lyophilized microorganisms are with an average diameter less than 2 mm, more preferably an average diameter of less than 1 mm, more preferably an average diameter less than 0.5 mm.

[0018] Yet an object of the present invention relates to a method for storing live microorganisms maintaining the viability of the microorganisms with less than about 2 log reduction per month at about 25 degrees C, wherein the method comprises storing the live microorganisms in a container with a three-phase composition with an internal relative humidity of about 30 percent or less.

[0019] Another object of the present invention relates to a method of preparing an anhydrous topical composition comprising a) providing viable microorganisms in water soluble crystals of at least one probiotic bacterial strain in an amount of at least 105CFU / g, b) providing a two-phase composition, c) dispensing the two phase system into a container, and d) closing the containing with an air phase in contact with a desiccant.

[0020] The present invention will now be described in more detail in the following.

[0021] Detailed description of the invention

[0022] Definitions

[0023] By "embedding" or "mixing" or "dispersing" or "enrobing" or "coating" it is meant that the crystals of probiotic microorganism is dispersed within and fully enveloped by the anhydrous carrier in a two-phase system characterized by a solid water soluble crystal and a liquid or partly liquid anhydrous carrier.

[0024] In a preferred embodiment the two-phase system of crystals and anhydrous carrier is liquid or partly liquid at temperatures below 37 degrees Celsius.

[0025] It is preferred that the probiotic culture products disclosed herein remain essentially dry, and that they contain no more than a trace of water. The use of substantial quantities of water in processing is typically incompatible with the coating anhydrous carrier and the product stability.

[0026] Structuring the anhydrous carrier with a water-free organogelator and be used to create a partly liquid two-phase system. An organogel, also called oleogel, is a class of gel made of a liquid organic phase immobilized by a three-dimensional network formed by an organogelator.

[0027] Although many types of organogelators have been developed, plant waxes and hydrogenated vegetable oils such as Rapeseed wax (hydrogenated rapeseed oil), candelilla wax (Euphorbia cerifera cera), rice bran wax (Oryza Sativa Bran Cera), berry wax (Rhus Verniciflua peel cera / Rhus Succedanea fruit cera), Oliwax (hydrogenated olive oil), Tea wax (camellia sinensis cera), Myrica fruit wax (myrica cerifera fruit wax), sunflower wax (Hydrolyzed sunflower seed wax), Sunflower seed wax (Helianthus Annuus Seed cera, ascorbyl palmitate, tocopherol), Castor wax (Hydrogenated castor oil), carnauba wax (Copernicia cerifera cera) or any other vegetable based wax (hydrogenated vegetable oil). When used as organogelators the waxes can be mixed to create a gel with particular physical properties.

[0028] Organogelator is preferable used in concentrations from 0.1 to 40% (w / w) of the anhydrous carrier. More preferable the concentration of the organogelator is 0.5 to 20% and even more preferable the concentration is 1 to 17%.

[0029] Polymers and synthetic waxes can also be used to gel the anhydrous carrier. The polymer suited for the invention are hydrogenated oils and polyurethane polymers and co-polymers being able to gel oils. Examples of such polyurethane polymers are disclosed in WO18185432. Only few polyurethane polymers are able to gel oils and examples of these are Oilkemia 5S polymer from Lubrisol and EstoGel M polymer from Polymerexpert. These polyurethane polymers comprise caprylic / capric triglycerides (castor oil) and are typically co-polymers of castor oil and polyurethane. The polymer of the invention is a polyurethane based on vegetable oils. Some vegetable oils used for production of polyurethane may need chemical modifications before polymerization.

[0030] In a preferred embodiment of the invention the polyurethane polymer is based on Castor oil.

[0031] In a preferred embodiment the polymer comprises more than 10% w / w castor oil, in a more preferred embodiment the polyurethane polymer comprises more than 20% w / w castor oil.

[0032] The invention is not limited to commercially available polyurethane products but to any polyurethane polymer / co-polymer product being able to gel anhydrous carriers.

[0033] The caprylic / capric triglyceride and polyurethane polymers are used in the oil in a concentration from 0.1% (w / w) to 20% (w / w). Preferable in the concentration from 0.3% (w / w) to 10% (w / w) and more preferable from 0.5% (w / w) to 6% (w / w).

[0034] Synthetic waxes include microcrystalline wax which is produced by de-oiling petrolatum as part of its refining process. Parafin wax is also derived from petroleum. Ozokerite, ceresin, and montan waxes are originally mineral waxes which are derived from coal and shale. Ozokerite for cosmetics are nowadays synthesized from petroleum, exactly like microcrystalline waxes. The present invention relates to live microorganisms including any bacteria, archaea, phages, viruses, yeast or fungi or any combinations thereof.

[0035] In an embodiment of the present invention the microorganism may be a bacteria or a mixture of bacterial strains.

[0036] Examples of suitable probiotic microorganisms include yeasts such as Saccharomyces, Debaromyces, Candida, Pichia and Torulopsis, moulds such as Aspergillus, Rhizopus, Mucor, and Penicillium and Torulopsis and bacteria such as the genera Bifidobacterium, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus, Cutibacterium, Lactobacillus, Lactiplantbacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, LacticaseibaciHus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, Lentilactobacillus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, and Weissella.

[0037] The most commonly used probiotics are strains of the lactic acid bacteria (LAB).

[0038] These are considered non-pathogenic and are used as probiotic bacteria in general to improve gastrointestinal flora and in the treatment of gastrointestinal symptoms. The present invention relates to stabilization of any viable microorgniam in a composition for application. The bacteria are preferably selected among the genera The microorganism is preferably selected among the genera Lactobacillus, Lactiplantbacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, LacticaseibaciHus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, Lentilactobacillus, Leuconostoc, Bifidobacterium, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, and Weissella. Lactobacillus, Leuconostoc, Bifidobacterium, Pediococcus, Lactococcus, Streptococcus Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, and Weissella.

[0039] The preferred microorganisms are in particular bacteria. The probiotic bacteria is preferably selected from the group comprising Lactococcus lactis, LacticaseibaciHus rhamnosus, Lactiplantibacillus plantarum, Lactobacillus helveticus, Lactobacillus jensenii, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus amylovorus, Lactobacillus amylolyticus, Lactobacillus alimentarius, Lactobacillus aviaries, Lactobacillus deibrueckii, Lactobacillus diolivorans, Lactobacillus farciminis, Lactobacillus gallinarum, Lacticaseibacillus easel, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus hiigardii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus mucosae, Lactobacillus panis, Lactiplantibacillus para planta rum, 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 hiigardii, 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 dextrinicus, Pediococcus parvulus, Pediococcus pentosaceus, Probionibacterium freudenreichii, Probionibacterium acidipropoinici, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus clausii, Bacillus coaguians, Bacillus fiexus, Bacillus fusiformis, Bacillus lentus, Bacillus licheniformis, Bacillus mega-terium, Bacillus mojavensis, Bacillus pumilus, Bacillus smithii, Bacillus subtilis, Bacillus vallismortis, Geobacillus stearothermophilus or mutants thereof.

[0040] 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.

[0041] 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.

[0042] In a preferred embodiment the anhydrous carrier embedded dried crystal microorganism is selected from the list but not restricted to: Bifidobacterium lactis DSM10140, B. lactis LKM512, B. lactis DSM 20451, Bifidobacterium bifidum BB-225, Bifidobacterium adolescentis BB-102, Bifidobacterium breve BB-308, Bifidobacterium longum BB-536 from Zaidanhojin Nihon Bifizusukin Senta (Japan Bifidus Bacteria Center), Bifidobacterium NCIMB 41675 described in EP2823822. Bifidobacterium bifidum BB-225, Bifidobacterium adolescentis BB- 102, Bifidobacterium breve BB-308, Bifidobacterium lactis HN019 (Howaru) available from DuPont Nutrition Biosciences ApS, Bifidobacterium lactis DN 173 010 available from Groupe Danone, Bifidobacterium lactis Bb-12 available from Chr. Hansen A / S, Bifidobacterium lactis 420 available from DuPont Nutrition Biosciences ApS, Bifidobacterium breve Bb-03, B. lactis BI-04, B. lactis Bi-07 available from DuPont Nutrition Biosciences ApS, Bifidobacterium bifidum Bb-02, Bifidobacterium bifidum Bb-06, Bifidobacterium longum KC-1 and Bifidobacterium longum 913 (DuPont Nutrition Biosciences ApS), Bifidobacterium breve M- 16V (Morinaga) and / or a Lactobacillus having a probiotic effect and may be any of the following strains; Lactobacillus rhamnosus LGG (Chr. Hansen), Lactobacillus acidophilus NCFM (DuPont Nutrition Biosciences ApS), Lactobacillus bulgaricus 1260 (DuPont Nutrition Biosciences ApS), Lactobacillus paracasei Lpc-37 (DuPont Nutrition Biosciences ApS), Lactobacillus rhamnosus HN001 (Howaru)available from DuPont Nutrition Biosciences ApS, Streptococcus thermophilus 715 and Streptococcus thermophilus ST21 available from DuPont Nutrition Biosciences ApS, Lactobacillus paracasei subsp. paracasei CRL431 (ATCC 55544), Lactobacillus paracasei strain F-19 from Medipharm, Inc. L. paracasei LAFTI L26 (DSM Food Specialties) and L. paracasei CRL 431 (Chr. Hansen), Lactobacillus acidophilus PTA-4797, L. salivarius Ls-33 and L. curvatus 853 (DuPont Nutrition Biosciences ApS). Lactobacillus casei ssp. rhamnosus LC705 is described in Fl Patent 92498, Valio Oy, Lactobacillus DSM15527 (Bifodan), Lactobacillus DSM15526 (Bifodan), Lactobacillus rhamnosus GG (LGG) (ATCC 53103) is described in US Patent 5,032,399 and Lactobacillus rhamnosus LC705 (DSM 7061), Propionic acid bacterium eg. Propionibacterium freudenreichii ssp. shermanii PJS (DSM 7067) described in greater details in FI Patent 92498, Valio Oy, Nitrosomonas eutropha D23 (ABIome), Staphylococcus hominis strains A9, C2, AMT2, AMT3, AMT4-C2, AMT4-GI, and / or AMT4-D12. (all from Matrisys Bioscience), L. rhamnosus PB01, L. gasseri EB01, L. curvatus EB10, L. acidophilus 5, Bifidobacterium animalis ssplactis 12, Bifidobacterium longum 536 all available from Bifodan A / S. Staphylococcus epidermidis strains M034, M038, All, AMT1, AMT5-C5, and / or AMT5-G6 (all from Matrisys Bioscience), L. plantarum YUN-V2.0 (BCCM LMG P-29456), L. pentosus YUN- VI.0 (BCCN LMG P-29455), L. rhamnosus YUN -S 1.0 (BCCM LMG P-2961), 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.

[0043] The use of viable probiotics for topical application is very limited and most products are based on lysates of the in-activated probiotic strain to overcome the problems of maintaining viability of the microorganisms in the topical composition. The problems observed when formulating live probiotic strains in gels, serums, emulsions, lotions and the like for topical application on the skin of mammals are lack of viability and stability.

[0044] Compositions for topical applications are typically to be stable for months at room temperature, this is a major problem for maintaining viability of live probiotic microorganisms in skin care products.

[0045] Another problem is activation of the probiotic strain when applied on the skin of a mammal. If the probiotic strain is microencapsulated following the procedures used for stabilization of probiotics for oral consumption then the microcapsules are designed to protect the live probiotic strain in the gastrointestinal fluids and will thus not dissolve on the skin surface. Therefore, the probiotic strain will not be released from the encapsulation and thereby not able to establish a binding, a metabolism or colonization of the probiotic strain on the skin surface or on mucous membranes.

[0046] The present invention solves the problem of stabilization of the live probiotic strain in a three-phase delivery system for topical use on skin or mucous membranes.

[0047] It will be understood that in the following, preferred embodiments referred to in relation to one broad aspect of the invention are equally applicable to each of the other broad aspects of the present invention described above. It will be further understood that, unless the context dictates otherwise, the preferred embodiments described below may be combined. When used herein, the term topical includes references to formulations that are adapted for application to body surfaces (e.g. the skin or mucous membranes). Mucous membranes that may be mentioned in this respect include the mucosa of the vagina, the penis, the urethra, the bladder, the anus, the mouth, the nose and the ear.

[0048] The present invention discloses new methodologies and delivery systems for stabilization of live probiotic strains in a composition for topical use to mucous membranes.

[0049] The utilization of these compositions comprising probiotic bacteria further facilitate the probiotic effects on skin of both humans and animals.

[0050] The present invention discloses methodologies for preparing a three-phase delivery system of an anhydrous carrier comprising viable microorganisms.

[0051] In an embodiment of the present invention the anhydrous carrier may be a vegetable oil.

[0052] The present invention further provides a topical therapeutic composition for the treatment or prevention of an skin disorder, comprising a therapeutically-effective concentration of one or more live species or strains or live biotherapeutic products within a pharmaceutically- acceptable anhydrous carrier suitable for topical administration on the skin or mucous membranes of a mammal, wherein said probiotic strain possesses the ability to maintain viable in the composition at room temperature and be released when applied to the skin surface.

[0053] In another aspect, the invention relates to a composition comprising a cosmetically acceptable anhydrous carrier. It is preferable for the composition to be present in liquid, or viscous form.

[0054] The three-phase delivery system is preferable essentially water free. More preferable the composition comprises less than 5% water, more preferable the composition comprises less than 1% water, more preferable the composition comprises less than 0.5% water, more preferable the composition comprises less than 0.1% water, more preferable the composition comprises less than 0.05% water.

[0055] The three-phase delivery system may advantageously further comprise other probiotics, prebiotics, or other active substances and / or may preferably also contain one or more of the following substances selected from antioxidants, vitamins, coenzymes, fatty acids, amino acids and cofactors. In a preferred embodiment the antioxidant is Vitamin E or a variant thereof (wherein the variants are selected from alfa, beta, gamma, delta tocoferols, tocotrienols and tocomonoenols).

[0056] In a preferred embodiment the antioxidant is tocopherol.

[0057] Preferably, the wherein anhydrous phase may comprise an antioxidant. Preferably, the anhydrous carrier may be an oil comprising tocopherol as an antioxidant.

[0058] In a further preferred embodiment the tocopherol is in the anhydrous phase, in a more preferred embodiment the tocopherol is present in a concentration below 5 wt%, even more preferred in a concentration below 4 wt%, or even more preferred in a concentration below 3 wt%.

[0059] Vegetable oils can contain natural antioxidants, in a preferred embodiment of the invention further antioxidants are incorporated into the composition. Antioxidants are preferred tocopherol (0.25 to 2.5 wt%) and / or Rosemary extract (0.1 to 0.75 wt%).

[0060] A "decrease" in viability may be "statistically significant" as compared to the viability determined at the time of formulating the composition. Decrease is measured as a log reduction and may include a log reduction of 0.1, 0.5, 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5 or 5.

[0061] "Viability" of microorganisms is measured as Colony Forming Units CFU / g. 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.

[0062] 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.

[0063] The microorganisms may advantageously be present in viable dried and / or spray-dried and / or lyophilized and / or vacuum dried crystalized form.

[0064] In a preferred embodiment of the invention the probiotic strain is used as a live isolated microorganism in a crystalized dried form. The microcrystals comprising microorganisms may be provided in a lyophilized form. Preferably, microcrystals comprising microorganisms may be provided in a lyophilized form in a water soluble cryoprotectant.

[0065] 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.

[0066] In one aspect of the invention the cryoprotectant is selected from maltodextrin, trehalose, saccharose, lactose, mannitol, sucrose, glycerol, sorbitol, dextran, inulin.

[0067] 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.

[0068] In addition, it is preferable for the crystals of microorganism to be present 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 %.

[0069] A preferred embodiment of the present invention involves the administration of from approximately lxlO3to 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.

[0070] 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.

[0071] Crystals comprising microorganisms (e.g. microcrystals) may be less than 2 mm in diameter, more preferable less than 1 mm in average diameter, even more preferably, less than 0.5 mm in average diameter, typically in the interval from 5 pm to 2 mm in average diameter, such as in the interval from 25 pm to 1 mm in average diameter, e.g. in the interval from 50 pm to 0.5 mm in average diameter.

[0072] In an embodiment of the present invention the average diameter may be determined by sieving.

[0073] Another surprising advantage of the preferred composition is that the microorganisms are able to activate on the skin and re-establish metabolic activity. 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.

[0074] A "mammal" include, but are not limited to, humans, primates, farm animals, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; piglets; sows; poultry; turkeys; broilers; minks; goats; cattle; horses; and non-human primates such as apes and monkeys.

[0075] Preferable the delivery system is for delivery of a composition for topical use on human skin or human mucous membranes.

[0076] An "effective amount" depends upon the context in which it is being applied. In the context of administering a composition comprising a viable microorganism topically on a skin or mucous membrane surface, an effective amount will be the number of viable microorganisms determined as CFU / gram which has a probiotic effect on skin or mucous membranes.

[0077] In one aspect of the invention the composition comprising the microorganism and a prebiotic. "Prebiotics" are components that increase the growth of specific microorganisms. "Synbiotics” are compositions comprising at least one probiotic and at least one prebiotic. Such compositions are understood to encourage the growth of beneficial microorganisms (e.g. the probiotic). Thus, powerful synbiotics are based on a combination of specific strains of probiotic microorganisms with carefully selected prebiotics. They can lead to an important health benefit to a mammal.

[0078] According to another aspect of the present invention there is provided a probiotic composition comprising the probiotic micro-organism and at least one more active ingredient. Prebiotics refer to chemical products that induce the growth and / or activity of commensal microorganisms of the microbiota (e.g., bacteria and fungi) that contribute to the well-being of their host. Prebiotics stimulate the growth and / or activity of advantageous bacteria that colonize the skin.

[0079] Some oligosaccharides that are used as prebiotics are fructooligosaccharides (FOS), xylooligosaccharides (XOS), polydextrose, pectins, galactooligosaccharides (GOS) or human milk oligo saccharides (HMO). Moreover disaccharides like lactulose, lactose or some monosaccharides such as or tagatose can also be used as prebiotics.

[0080] The other active ingredient (or other ingredients) is not limited in any way. In a preferred aspect, at least one prebiotic compound is comprised in the composition of the invention, i.e. as other ingredient. In a very broad concept, prebiotics are all those compounds which can be metabolized by probiotics. Prebiotics can thus serve as a food source for probiotics. Prebiotics are well known in the art and when used in the present invention there is no particular limitation of the prebiotic as such. In preferred embodiments at least one prebiotic product in the composition is selected from the following compounds and compositions: carbohydrates, glucans, alpha-glucans, beta-glucans, mannan-oligosaccharides, inulin, oligofructose, human milk oligosaccharides (HMO), galactooligosaccharides (GOS), lactulose, lactosucrose, galactotriose, fructooligosaccaride (FOS), cellobiose, cellodextrins, cylodextrins, maltitol, lactitol, glycosilsucrose, betaine, Vitamin E or a variant thereof (wherein the variants are selected from alfa, beta, gamma, delta tocoferols, tocotrienols and tocomonoenols). Optionally, mannanoligosaccharides and / or inulin may be preferred.

[0081] HMOs include lacto-N-tetraose, lacto-N-fucopentaose, lacto-N-triose, 3 '-sialyllactose, lacto- N-neofucopentaose, sialic acid, L-fucose, 2-fucosyllactose, 6 '-sialyllactose, lacto-N- neotetraose and 3-fucosyllactose.

[0082] In a preferred embodiment at least one of the following prebiotic compounds are used in the topical composition of the invention; lactose, beta-glucans, mannan-oligosaccharides, inulin, oli-gofructose, galactooligosaccharides (GOS), lactulose, lactose, lactosucrose, galactotriose, fructo-oligosaccaride (FOS), cellobiose, cellodextrins, cylodextrins, maltitol, lactitol, glycosilsucrose, betaine, lacto-N-tetraose, lacto-N-fucopentaose, lac-to-N-triose, 3 '-sialyllactose, lacto-N-neofucopentaose, sialic acid, 2-fucosyllactose, 6 '-sialyllactose, lacto-N-neotetraose and 3-fucosyllactose. Optionally, lactose and / or mannan- oligosaccharides and / or inulin may be preferred.

[0083] Fucose, in particular L-fucose is believed to strengthen natural defence of skin, stimulate epidermis immune defence and / or pre-vent and / or treat cutaneous autoimmune disease. In one preferred embodiment of the invention the composition comprises L-fucose and / or D- fucose.

[0084] In one preferred embodiment of the invention the composition further comprises L-fucose and / or D-fucose in a concentration in the composition of 10 mM to 500 mM. According to still further features in the described preferred embodiments the crystals comprising the microorganism further comprises at least one further probiotic microorganism selected from the group consisting of bacteria, archaea, phages, virus, yeasts or molds.

[0085] In a preferred embodiment the at least one further probiotic microorganism is a bacteria.

[0086] In one embodiment of the invention the anhydrous carrier is an oil.

[0087] An "oil" of the invention is an oil being liquid at storage temperature, thus the liquid oil has a freezing point below 25 degrees Celsius.

[0088] In a preferred embodiment the oil is a vegetable oil which can be absorbed by the skin or the mucous membrane.

[0089] In a preferred embodiment of the invention the oil is a vegetable oil selected from almond oil, sunflower oil, hemp oil, CBD oil, cannabis oil, Evening prim rose, Borage oil, baobab oil, acai oil, Almond sweet oil, Rose Hip oil, jojoba oil, Jojoba Golden oil, Camomile oil, shea oil, coconut oil, calendula oil, sea buck-thorn oil, Jafflower oil, castor oil, olive oil, corn oil, soya bean oil, cotton seed oil, wheat 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.

[0090] In one preferred embodiment of the invention, the oil is selected from sunflower oil, jojoba oil, olive oil and / or almond oil.

[0091] The vegetal oil may comprise at least one of: acai, acai berry, almond sweet, aloes vera, andiroba, apricot kernel, arnica, argan, avocado, babassu, baobab, 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.

[0092] The oil may be dried by using a desiccant, by vacuum tray drying, dehydrated air before forming a two-phase composition, or a combination hereof.

[0093] Anhydrous carrier material which may suitably be used in the delivery system according to the invention includes: waxes, such as beeswax, paraffin wax, animal stearates, glycol esters, mono and diglycerides and wax mixtures; oils, such as olive oil, sunflower oil, canola oil, coconut oil, corn oil, palm oil, sesame oil, peanut oil, along with medium chain triglycerides; an emulsifier, for example a phospholipid such as lecithin; a glyceride such as a mono- or di-glyceride or a combination thereof, for example a stearate, for example glycerol monostearate, such as an animal or vegetable stearate, and / or a palmitate such as glycerol monopalmitate, a dispersant or a surfactant such as polyethylene glycols.

[0094] Low water activity of the three-phase delivery system significantly increases its shelf-life. Water activity reflects the part of moisture content of a product which can be exchanged between the product and its environment. Water activity determines the lower limit of "available" water for microbial growth. Since bacteria, yeast, and molds require a certain amount of "available" water to support growth. Water may be present, even at high content levels, in a product, but if its energy level is sufficiently low the microorganisms cannot remove the water to support their growth. In an embodiment of the invention, the reactive amount of water is in the range of about 0.01 to about 0.80 aw, such as in the range of about 0.05 to about 0.75 aw, e.g. in the range of about 0.1 to about 0.70 aw.

[0095] In a further embodiment of the present invention the water activity (aw) in the air phase may be in the range of about 0.01 to about 0.80 aw, such as in the range of about 0.05 to about 0.75 aw, e.g. in the range of about 0.1 to about 0.70 aw.

[0096] Preferably, the anhydrous carrier may be an oil with a water content of 3% (w / w) or less, such as 2% (w / w) or less, e.g. 1% (w / w) or less, such as 0.5% (w / w) or less, e.g. 0.1% (w / w) or less.

[0097] By "air phase" it is meant the gas phase between the dessicant and the anhydrous carrier. The air phase can contain, but is not limited to be atmospheric air or a modified atmosphere packaging (MAP). Non-limiting examples of MAP are oxygen gas, carbon dioxide gas, nitrogen gas, helium gas, xenon gas, argon gas, nitrous oxide gas or any combinations thereof.

[0098] In a preferred embodiment the air phase is atmospheric air.

[0099] In another preferred embodiment the air phase comprises at least 5% oxygen.

[0100] The air phase according to the present invention may constitute at least 0.1 cm3air in direct contact with a desiccant, such as at least 0.5 cm3, e.g. at least 0.75 cm3, such as at least 1.0 cm3, e.g. at least 1.5 cm3, such as at least 2.0 cm3, e.g. in the range of 0.01-2.0 cm3, such as in the range of 0.025-1.5 cm3, e.g. in the range of 0.05-1.0 cm3, such as in the range of 0.075-0.5 cm3, e.g. in about 0.1 cm3.

[0101] One solution to maintain a particularly low level of moisture within a package is to incorporate sachets of desiccant material into the internal space of the package to remove the moisture from the headspace of the package. The desiccant material is generally known to reduce the moisture content within a package. Typical desiccant materials are "physical" desiccant materials, such as molecular sieves that bind water molecules within pore spaces of a material. Another type of desiccant material includes hydrate forming agents such as salts, such as ammonium chloride. Desiccant materials may also be used that form no hydrates, such as common salt (NaCI) or potassium bromide (KBr).

[0102] As used herein, the delivery system includes a desiccant in contact with the air phase. The desiccant may include, but is not limited to, sodium carboxymethylcellulose, calcium carboxymethylcellulose, silica oxide, colloidal silica dioxide, and combinations thereof.

[0103] In a preferred embodiment the desiccant is silica oxide.

[0104] In a preferred embodiment of the invention the crystal phase is about 0.01% (vol) to 10% (vol), the anhydrous phase is about 50% (vol) to 99% (vol) and the air phase is about 1% to 15% (vol) of the space in the container.

[0105] In a further preferred embodiment of the invention the crystal phase is about 0.1% (vol) to 5% (vol), the anhydrous phase is about 70% (vol) to 96% (vol) and the air phase is about 2% to 10% (vol) of the space in the container.

[0106] The present invention provides several advantages. In particular, viability of the microorganisms is kept in the composition even at storage at room temperature. The microorganisms activated by the temperature and moisture of the skin releasing the microorganisms from the anhydrous carrier as it is absorbed by the skin.

[0107] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0108] The invention will now be described in further details in the following non-limiting examples.

[0109] Examples

[0110] Example 1.

[0111] Manufacturing of a three-phase delivery system.

[0112] Cold pressed organic Jojoba oil (Simmondsia chinensis seed oil) was obtained from Hedenhus, Denmark. Organic refined sweet almond oil was obtained from Nardos Cosmeceuticals and organic sunflower oil (Helianthus annuus seed oil) was obtained from Hedenhus, Denmark.

[0113] Lyophilized Lactiplantibacillus plantarum LB244R (DSM 32996) was prepared by growing the bacteria in MRS broth at 30°C for 24 hours, harvested by centrifugation (1000 g, 10 min) and washed in phosphate / peptone buffer and the concentrated cells were freeze dried using trehalose, glycerol and maltodextrin as cryoprotectants.

[0114] Cell pellets are resuspended in a preservation medium solution, using 2.5 mL of preservation solution per gram of cell paste. The preservation medium solution contains 13 percent trehalose, 4 percent xylitol, 5 percent maltodextrin and 1 percent sodium ascorbate in a 10 mM sodium phosphate buffer (pH 7.4). The resulting batches of the preservation medium cell slurry is transferred to sterile trays and lyophilized in a Virtis Genesis freeze-drier. Viability of the cell slurry is determined prior to lyophilization by plate counting. The trays containing the cell cakes are placed in heat-sealed bags with desiccant, purged with nitrogen gas, and held at 2-8 degrees centigrade until milling. The lyophilized cell cakes using a cone mill to a crystal size below 1 mm.

[0115] Mixing of ingredients.

[0116] 1 : Mix the three dried oils 1 : 1 : 1 and adding 0.5% tocopherol.

[0117] 2: Adding the crystals with dried microorganisms in a concentration corresponding to approximately 108CFU / ml.

[0118] 3: Mix slowly until homogenous. 4: The suspension is discharged to containers of 25 ml leaving an air space of 0.5-1 ml

[0119] 5: sealing with a lid comprising silica oxide (Kalku 0675, Kisico GmbH)

[0120] Viability is followed by plate counting

[0121] Container is opened and 1 mL of the two-phase anhydrous carrier is transferred to tubes and centrifuged at 10.000 rpm for 4 minutes.

[0122] The oil is removed from pellet, and 100 pL Maximum Recovery Diluent (Sigma 07233) is added.

[0123] 900 pL MRS broth is added to each tube and mixed until pellet is dissolved. CFU / mL determined by plate counting.

[0124] Table 1 : Stability determined as viable strain at time = 0, and 1, 4, 12 and 24 months respectively. Shown for storage temperature 25 degrees Celsius. Cell counts are measured as CFU / ml. (Average of a triplet CFU determination)

[0125] Control is the two-phase system of anhydrous carrier with crystals comprising microorganisms, but without the additional closed air phase in contact with a desiccant.

[0126] Example 2:

[0127] Distribution and size of the embedded crystals of microorganism was determined for the compositions by contrast phase microscopy and image analysis using the oCelluScope (BioSense Solution, Denmark).

[0128] For stability and dispersion into the oil it was measured that dispersion of crystals in the size of 5 pm to 2 mm in diameter created the best distribution in the oil and resulted in improved viability of the microorganisms.

[0129] Viability of the microorganisms in the crystals were determined by image analysis in the oCelluScope. Oil is smeared in a thin layer in 6 well microtiter plates 10-20 pm thick and a thin layer of liquid MRS medium was added on top of the gel, out growth from the crystals was followed by image analysis. References

[0130] Favaro-Trindade et al., (2011) CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 6: 1-8 WO18185432

Claims

Claims1. A closed container for storage of live microorganisms in a formulation for topical application comprising a three-phase system, the three-phase system comprising : a) microcrystals comprising live microorganism; b) an anhydrous carrier; and c) an air phase in contact with a desiccant.

2. A container according to claim 1, wherein the microcrystals comprising microorganisms are in a lyophilized form in a water soluble cryoprotectant.

3. A container according to claim 1, wherein the anhydrous phase comprises an antioxidant.

4. A container according to any of the preceding claims, wherein the microorganism is bacteria or a mixture of bacterial strains.

5. A method of prolonging the stability of cosmetic products for topical application comprising a) adding microcrystals of live microorganisms to an anhydrous carrier to create a two-phase system; b) dispensing the two-phase composition to a container; and c) sealing with an air phase of at least 0.1 cm3air in direct contact with a desiccant.

6. The method of claim 5, wherein the anhydrous carrier is an oil with a water content of 1% (w / w) or less.

7. The methods of claim 5 to 6 wherein the water activity in the air phase is in the range of about 0.1 to about 0.75.

8. The method of anyone of claim 5 to 7, wherein the anhydrous carrier is an oil comprising tocopherol as an antioxidant.

9. The method of anyone of claim 5 to 8, wherein the microcrystals have an average diameter of less than 1 mm.

10. The method of anyone of claim 5 to 9, wherein the anhydrous carrier is a vegetable oil.

11. The method of anyone of claim 5 to 10 wherein the anhydrous carrier is selected from almond oil, sunflower oil, hemp oil, CBD oil, cannabis oil, Evening prim rose, Borage oil, acai oil, Almond sweet oil, baobab oil, Rose Hip oil, jojoba oil, Jojoba Golden oil, Camomile oil, Calendula oil, Sea buck-thorn oil, Jafflower oil, castor oil, olive oil, linseed oil, apricotkernel oil, argan oil, camelina oil, comfrey oil, grape seed oil, kiwi seed oil, mullein oil, peach kernel oil, thistle oil, sesame oil or any combinations thereof.

12. The method of claim 5 to 11 wherein the oil is dried by using a desiccant, by vacuum tray drying, dehydrated air before forming a two-phase composition.

13. A method for storing live microorganisms maintaining the viability of the microorganisms with less than about 2 log reduction per month at about 25 degrees C, wherein the method comprises storing the live microorganisms in a container with a three- phase composition with an internal relative humidity of about 30 percent or less.

14. A method of preparing an anhydrous topical composition comprising a) providing viable microorganisms in water soluble crystals of at least one probiotic bacterial strain in an amount of at least 105CFU / g, b) providing a two-phase composition, c) dispensing the two phase system into a container, and d) closing the containing with an air phase in contact with a desiccant.

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