[001] The present disclosure relates to a microencapsulated probiotic. More particularly, the present disclosure relates to a probiotic that comprises a polymeric slurry of freeze-dried probiotic(s) encased with a nutrient composition in a protective barrier layer. Still more particularly, the disclosure relates to a probiotic particulate that can be incorporated in a large variety or products without being compromised by the chemistry within the carrier compositions.

The present disclosure further relates to products containing the

microencapsulated probiotic and methods of making and using the same.

[002] Probiotics are live bacteria and yeast that are known to provide a variety of health benefits, particularly in the digestive system. The digestive system, being such a harsh environment, has spurred much research on ways to deliver probiotics to a host. Because probiotics are living cells, they must be protected if they are to remain viable until they reach the host that can receive the expected health benefit.

[003] Probiotics have found many uses outside of the digestive system. Recently, it has been discovered that an imbalance in the human biome can be a substantial cause of conditions, including for example, skin irritations and eczema. Adding beneficial bacteria back to the skin through contact with probiotics has shown to improve skin barrier function, counteract inflammatory diseases such as eczema, and reduce the microbes that cause acne.

[004] Currently many cosmetics and pharmaceuticals are marketed as containing live probiotics; however, what might have been formulated using live bacteria, do not end up retaining live bacteria. Modern cosmetics and

pharmaceuticals must contain preservatives and other shelf stabilizing

compositions that can kill the probiotic. Worse yet, even if the probiotic is protected and does remain viable until application, the contact between the probiotic and the surrounding carrier composition results in the same

preservatives killing the probiotic before it can have the expected health benefits.

[005] In addition to interfering with probiotic viability, the high level of preservatives that are required for adequate shelf-life of a cosmetic can also damage the human biome by killing naturally occurring microbes. The residual activity of preservatives found in products like skin care lotions can kill large numbers of beneficial bacteria that are naturally found on the skin. As a result, areas with a dearth of healthy bacteria, provide an opportunity for pathogens like Clostridium difficille, Methicillin Resistant Staphylococcus aureus (MRSA), or Vancomycin-Resistant Enterococci (VRE) to grow. Skin is believed to follow the lush lawn theory: if a lawn is lush and full, it is difficult for weeds to grow. If skin is full of healthy vibrant beneficial natural (flora) microbes, it is more difficult for pathogens to become established.

[006] In some cases, the probiotic that is added to the cosmetic is in a lysed form. Lysed probiotics are those that have been chemically cleaved into many pieces and accordingly, they are not alive, so the issue of viability would seem less urgent. Without comment on whether or not there are benefits derived from bacteria parts, the cellular parts delivered to the host will likely also be compromised by the preservatives or other ingredients in the carrier cosmetic formulation.

[007] Probiotic encapsulation has been contemplated for a number of years, to improve the survival of living probiotics in a range of formulations.

Probiotic survival can be affected by a number of factors including, by way of example, pH and temperature. Encapsulation of the probiotic creates a physical barrier between the living probiotic and its surroundings, be they stomach acid, or pharmaceutical excipients. Current probiotic encapsulation technology (PET) includes encapsulation, entrapment and immobilization within a variety of biocompatible materials. While substantial research has been conducted on ways to maintain the viability of a living probiotic until it can reach the point of release and benefit, the currently available solutions remain wholly inadequate.

[008] The present disclosure provides a method for producing

microencapsulated, living probiotics, that remain alive until they are delivered to the area of the host in need of the health benefit. In addition, the

microencapsulated delivery vehicle allows living microbes to be delivered to the skin in a cosmetic or pharmaceutical composition, without the microbe death experienced by prior art products.

SUMMARY OF THE DISCLOSURE

[009] The present disclosure relates to a composite material comprising a freeze-dried probiotic and a low-water-activity carrier. [010] The present disclosure further relates to a microencapsulated probiotic including a core containing a freeze-dried probiotic and a polymer; a nutrient layer surrounding the freeze-dried core; and a protective layer surrounding the nutrient layer.

[011] The present disclosure further relates to a microencapsulated probiotic including a core comprising a freeze-dried probiotic, a polymer and a nutritive composition; and a protective layer surrounding the core.

[012] In one embodiment, the present disclosure relates to a composition for delivering a probiotic to a host including a freeze-dried probiotic suspended in a polymeric core and further including a probiotic nutrient; and an excipient having a low-water-activity (or water activity below 0.3).

[013] According to another embodiment, the disclosure relates to a method for making an encapsulated probiotic comprising, freeze drying a probiotic, mixing the freeze-dried probiotic and a prebiotic sugar with a polymer or oil to create a composite, and encapsulating the composite in a protective coating.

[014] According to yet another embodiment, the instant disclosure relates to skin and skin care products that comprise microencapsulated probiotics

[015] Additional advantages of the described methods and products will be set forth in part in the description which follows, and in part will be obvious from the description or may be learned by practice of the disclosure. The advantages of the disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. [016] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description, serve to explain the principles of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[017] FIGURE 1 illustrates one embodiment of a microencapsulated probiotic according to the disclosure.

[018] FIGURE 2 illustrates another embodiment of a microencapsulated probiotic according to the disclosure.

DESCRIPTION

[019] Reference will now be made in detail to certain exemplary embodiments, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like items.

[020] The present disclosure relates to a probiotic microcapsule, a process for microencapsulating the probiotic, compositions containing the probiotic microcapsule, and uses and treatments using the probiotic

microcapsule. [021] The probiotic microcapsule as described herein includes a probiotic material that is encased in a manner that prevents death of the live probiotic material before it can be delivered to a host in need of the probiotic. The microcapsule is made up of a number of layers, each of which serves a different purpose in protecting the probiotic material. The microcapsule may include all or a subset of a polymeric core that contains the probiotic material, a nutritive composition dispersed in the core or as a layer around the core, an

encapsulating layer that protects the core of probiotic and nutritive material, a moisture protective layer and a fugitive layer. The moisture protective layer and fugitive layers are optional and may or may not be used in combination with all of the embodiments.

[022] As used herein,“encapsulation layer,”“encapsulation coating,” “encapsulation layers,”“shell,” and“encapsulate shell” are interchangeable and refer to the protective layer that surrounds the probiotic and nutritive composition.

[023] In the description that follows, a layered microcapsule will be described. Each of the layers may be a single distinct coating or layer or may be made up of a number of layers. Unless indicated to the contrary, whether the term“layer” or“layers” is used, it should be understood that all embodiment can include either the plural and singular.

[024] As seen in FIG. 1 , the probiotic microcapsule of the instant disclosure 10 comprises a probiotic that has been freeze-dried. Freeze drying is a multi-step dehydration process in which a material is frozen and water is minimized by sublimation, leaving a dehydrated and preserved product. In the instant product, this preservation technique puts the probiotic into a state of stasis.

[025] Probiotics for use in the instant disclosure can be chosen from any art recognized probiotic that one would want to protect until administration of the probiotic to the appropriate host. Such probiotics include one or more of

Bifidobacterium, Pediococcus, Leuconostoc, Micrococcus, Escherichia,

Staphylococcus, Streptocococcus, Cadida, Bacillus, and combinations thereof. Exemplary probiotics may include Staphylococcus, including S. epidermidis and S hominis; Propionibacterium including P. acnes, P. australiense, P. avidum, P. cyclohexanicum, P. granulosum, P. jensenii, PI. microaerophilum, P.

propionicum, P. thoenii, P. freudenreichif, Micrococci including M. antarcticus, M.luteus, M. lylae, M. roseus, M. agilis, M. kristinae, M. sedentarius, M. halobius; Cornebacterium including C. diphtheriae, C. efficiens, C. glutamicum; Malassezia (Yeast) including M. furfur, and combinations thereof. Preferred probiotics include those that can grow on, adhere to, or release beneficial proteins or DNA to the skin. Appropriate probiotics and prebiotics for use in the instant disclosure can be found, for example, in the Handbook or Probiotics and Prebiotics by Yuan Kun Lee and Seppo Alminen, second edition.

[026] Some probiotics can be analogized to small biochemical manufacturing facilities where each microbe is a biochemical plant that keeps producing beneficial biochemicals. Microbes that naturally grow on the skin are those most often thought of in this way. According to one embodiment, the biochemicals of the probiotic can safely and naturally disinfect the skin. According to another embodiment, the biochemicals can reduce the inflammation response and reduce or eliminate eczema or skin irritation. According to yet another embodiment, the biochemicals of the probiotics can strengthen the skin and induce natural ceramide production - which is to say that skin is rejuvenated, made younger, and made more resistant to the effects of pollution and aging.

[027] Other probiotics can be understood as delivery vehicles that drop off the beneficial ingredients and then move on to deliver the ingredients somewhere else. These are generally the microbes that cannot grow on the skin. Instead they release beneficial peptides and DNA delivering unique benefits to the skin. The DNA and peptides carried by these probiotics are proteins, and as such, they interact with the chemistry of the surrounding delivery vehicle or composition base. Specifically, proteins cannot function properly when they chemically attach to cationic ingredients or become misshapen due to acids or bases. The only way to successfully deliver these proteins to a host is to deliver the proteins in a viable bacteria that naturally breaks when exposed to air. These probiotic bacteria (e.g. anaerobic bacteria) can be likened to a

microcapsule that is, according to the described invention, then

microencapsulated within another microcapsule. According to one embodiment, anaerobic bacteria delivered to the skin may enter the pores and survive naturally, delivering the beneficial proteins to the host.

[028] The freeze-dried probiotic is suspended in a polymer to form the composite 40 making up the core of the microcapsule. Any art recognized polymer(s) or combination of polymers useful for pharmaceutical applications can be used for suspending the freeze-dried probiotic including, but not limited to, polyethylene glycols (PEGs), polyvinyl pyrrolidone ((PVPs)- preferably with molecular weights between about 40,000 to about 360,0000), polyvinyl alcohol (PVAs), polyacrylamides, N-(2-hydroxypropyl) methacrylamide (HPMA), xanthan gum, guar gum, pectins, dextran, carrageenan, sodium carboxyethyl cellulose, polyacrylic acid polymers, hyaluronic acid, carboxyvinyl polymers, hydroxyethyl cellulose, cellulose, hydroxypropylmethyl cellulose, carboxyvinyl polymer.

[029] According to one embodiment, polyacrylic acid polymers, for example, Carbopol® Ultrez 20, Carbopol® Ultrez 21 , both from Lubrizol, as well as HivisWako® a carboxyl vinyl polymer, from Wako Chemicals Ltd, can be used to suspend the freeze-dried probiotic.

[030] According to one embodiment, the microcapsule core 40 has a low- water-activity. Water activity is a measure of how much water is available to hydrate materials. Maintaining a low-water-activity minimizes the need for preservatives, keeps the freeze-dried probiotic from becoming hydrated, and improves the long term survival of the probiotic.

[031] According to one embodiment, the polymer is treated with a hygroscopic agent to bind and remove free water. The lower the water content, the more stable the freeze-dried probiotic. The hygroscopic agent can be any material that will bind free water and not interfere with the stasis or release of the probiotic. The hygroscopic material may be chosen from prebiotic sugars (e.g. dried (glucose corn syrup, fructose, manose, mannitol, maltodextrin, lactulose, treehalos, and sorbitol) oligosaccharides (e.g. Fructo-oliosaccharides - Raftilose P95 Orafti, Belgium), galactooligosaccharises, resistant starch-rich whole grains (e.g. oat b-glucan, flaxseed gum, fenugreek gum, and matured gum Arabic), and mannan oligosaccharide-rich yeast cell wall material is demonstrated to be a valuable prebiotic, and certain proteins (e.g. lactoferrin), certain plant extracts (e.g. luteins and black current extract powered). Prebiotic sugars or sugar alcohols have the advantage that they can not only reduce the water activity, but also act as a nutritive composition 20 for the probiotics since they include compounds such as glucose, fructose, oligosaccharides, mannose,

glucomannans hydrolyzate, xylitol, erythitol, or sorbitol, all of which encourage the growth of the probiotic microbes.

[032] According to another embodiment, the water activity may be modified by adding a humectant (miscible solvent) such as glycerin, propylene glycol, butylene glycol, and the like.

[033] According to another embodiment as seen in FIG. 2, the nutritive composition 20 may be coated around the core 40. In this embodiment, the coating 20 is made from any material that can provide nutritive compounds, e.g., glucose, fructose, etc. to the probiotic microbes. Appropriate coatings 20 can be made, for example, from combinations of fat and sugar. Such coatings are well known and are most often found in the candy industry, for example, the coating on an M&M. This coating can be from about 0.1 pm to about 10 pm, for example, from about 0.5 pm to about 5 pm, for example, from about 1 pm to about 5 pm, for example from about 1.5 pm to 5 pm. [034] According to yet another embodiment, prebiotic sugars may be added to the core to scavenge water and provide some nutritive benefit, while a nutritive coating layer is also applied around the microcapsule core (not shown).

[035] According to one embodiment, the core may be a non-active around which a probiotic containing polymer layer, a nutritive layer can be created. According to this embodiment, (not shown) the non-active core may be chosen from any material that is compatible and capable of being coated with the probiotic material. Appropriate cores may be made of materials such as cellulose or other filling agents.

[036] The polymer core 40 with the incorporated nutritive composition 30 of FIG. 1 or the polymer core 40 with the nutritive coating 30 of FIG. 2 further includes a protective encapsulation coating 20. The encapsulation coating 20 may be from 1 to 30 layers thick, for example, from about 1 to about 20 layers thick, for example, from about 1 to about 10 layers thick. The coating weight of the encapsulation coating 20 may be from about 1 % to about 50% of the microcapsule 10 weight, for example, from about 1 % to about 40% of the microcapsule 10 weight, for example, from about 1 % to about 30% of the microcapsule 10 weight, for example, from about 1 % to about 20% of the microcapsule 10 weight, for example, less than 15% of the microcapsule 10 weight.

[037] According to one embodiment, the probiotic core may be further coated with an oil layer before being subjected to encapsulation. The oil layer may provide insulation between the probiotic core and the encapsulation layer allowing higher temperature materials to be used during the microencapsulation process.

[038] According to one embodiment, freeze-dried probiotic may be dispersed in oil. Optionally, the oil may be thickened with a polymer or copolymer (e.g. Adjinimoto AJK-OD2046 (Octyl Dodecanol and Dibutyl Lauroyl Glutamide and Dibutyl ethylhexanoly Glutamide). The slurry can be dispersed in water and encapsulated with gelatin.

[039] Suitable materials for the encapsulation coating 20 include those appropriate for application at about 120°C or less, for example, less than about 100°C, for example, less than about 90°C, to facilitate the coating of the freeze- dried probiotic slurry. Waxes may be chosen from organic esters and waxy compounds derived from animal, vegetable, and mineral sources including modifications of such compounds in addition to synthetically produced materials having similar properties. Specific examples that may be used alone or in combination include glyceryl tristearate, glyceryl distearate, vegetable oils such as canola wax, hydrogenated cottonseed oil, hydrogenated soybean oil, castor wax, rapeseed wax, beeswax, camauba wax, candelilla wax, microwax, polyethylene, polypropylene, epoxies, long chain alcohols, long chain esters, long chain fatty acids such as stearic acid and behenic acid, hydrogenated plant, and animal oils such as fish oil, tallow oil, and soy oil, microcrystalline waxes, metal stearates, white grease, yellow grease, and brown grease, and metal fatty acids. According to one embodiment, hydrophobic wax materials include for use in the instant disclosure include Dynasan™ 110, 114, 116, and 118 (commercially available from DynaScan Technology Inc., Irvine, Calif.),

Sterotex™ (commercially available from ABITEC Corp., Janesville, Wisconsin.

[040] The encapsulation coating layer 20 may be comprised of a polymeric material, a crosslinked polymeric material, a metal, such as Ca ²⁺ , a ceramic or a combination thereof, that results in a shell material that may be formed during manufacturing. Specifically, the encapsulation coating layer may be comprised of crosslinked sodium alginate, anionic dispersed latex emulsions, crosslinked polyacrylic acid, crosslinked polyvinyl alcohol, crosslinked polyvinyl acetate, silicates, carbonates, sulfates, phosphates, borates, polyvinyl pyrollidone, PLA/PGA, thermionic gels, urea formaldehyde, melamine

formaldehyde, polymelamine, crosslinked starch, nylon, ureas, hydrocolloids, and combinations thereof. According to one embodiment, the crosslinked polymeric system is crosslinked sodium alginate.

[041] The encapsulation coating layer 20 generally has a thickness of from about 0.1 micrometers to about 500 micrometers, for example, from about from about 1 micrometer to about 100 micrometers, for example, from about 1 micrometer to about 50 micrometers, for example, from about 1 micrometer to about 20 micrometers, for example, from about 10 micrometers to about 20 micrometers. Suitable methods for measuring the thickness of the encapsulation layer 20 (once fractured), and the other optional layers described herein, include Scanning Electron Microscopy (SEM) and Optical Microscopy.

[042] At these thicknesses, the encapsulation coating layer 20 has a sufficient thickness to achieve its intended function. The barrier coating can serve one or more functions. First, the barrier can act to protect the contents of the microcapsule from low water pressure. Preferably, the water activity inside the microcapsule should be approximately equal to water activity outside the microcapsule. However, practically, the balance is usually not perfect, and the microcapsule coating layer will protect the capsule contents against small osmotic pressure differences. In this way, the encapsulation coating protects the bacteria from being prematurely activated due to water exposure.

[043] Second, the encapsulation coating protects the live bacteria from low levels of preservative. Although low water systems many not need a preservative, it is likely that a minimal level of preservative may be needed to maintain shelf stability. If a small amount of preservative is used, the

encapsulation coating would protect the probiotic bacteria. According to one embodiment, the preservative would be present in low enough level either to disappear during storage or to be insufficient to cause any damage to the bacterial during dispersion.

[044] According to one embodiment, the encapsulation coating layer is a single discrete layer. According to another embodiment, the encapsulation coating 20 comprises multiple layers added in one or more steps.

[045] According to some embodiments, a moisture protective layer (not shown) may also be included. The moisture protective layer generally surrounds the encapsulation coating 20. The moisture protective layer can comprise one or more of the following compositions, alone or in combination. The materials are chosen from polyols in combination with isocynate, styrene-acrylate, vinyl tolueneacrylate, styrene-butadiene, vinyl-acrylate, polyvinylbutyral, polyvinyl acetate, polyethylene terephthalate, polypropylene, polystyrene, polymethyl methacrylate, polylactic acid, polyvinylidene chloride, polyvinyldichloride, polyethylene, alkyd polyester, carnauba wax, hydrogenated plant oils,

hydrogenated animal oils, fumed silica, silicon waxes, titanium dioxide, silicon dioxide, metals, metal carbonates, metal sulfates, ceramics, metal phosphates, and microcrystalline waxes. When the moisture protective layer is used, it may be from about 5% to about 35% of the microcapsule weight, for example, from about 5% to about 30% of the capsule weight, for example, from about 5% to about 20%, for example, from about 5% to about 15%.

[046] According to another embodiment, one or more fugitive layers (not shown) may be added to protect the microcapsule from process damage. The fugitive layer may be comprised of any one of several suitable materials including polylactic acid, polymers of dextrose, hydrocolloids, alginate, zein, and

combinations thereof. According to one embodiment, the fugitive layer is starch. The fugitive layer protects the microcapsule during production. The layer may be applied to any of the layers of the microcapsule. The fugitive layer may be something that is eliminated during processing or something that may remain as part of the end product.

[047] The probiotic microcapsules 10 of the instant disclosure can be produced using any art recognized methods. According to one embodiment, the first step in the production of the encapsulated probiotic 10 is to select and freeze dry the probiotic materials. [048] According to one embodiment, the probiotic is selected from a combination of bacteria and/or yeast. To decrease production losses, the probiotic can be cooled to a temperature of 5°C to 15°C and dried by sublimation of the water from the organism. In some embodiments, the probiotic may be added to an excipient before being cooled. Such an excipient may contain an oil and/or a prebiotic sugar. Probiotics can be freeze-dried using any art recognized process. According to one embodiment, the probiotic powder is produced using standard freeze drying, spray drying, or chemical drying.

[049] The freeze-dried probiotic is next encapsulated in a polymeric material to form the core of the microcapsule 40. According to one embodiment, the polymer is dissolved in water at a high enough concentration to suspend the freeze-dried probiotic, but at a low enough concentration to allow the solution to flow through the microencapsulation process equipment. Polymer

concentrations are generally between 5 % and 60%, for example, from about 40 % to about 60%, when using a sugar/protein or wax matrix, and for example, from about 5% to about 15% when using a polymeric matrix.

[050] While the polymer is diluted with water to form a slurry, the polymer is highly hygroscopic so it quickly reduces the water activity by absorbing the water preventing the probiotic materials from being hydrated. Hydration of the freeze-dried probiotic will interfere with the probiotic and reduce its ability to survive long-term. According to one embodiment, after addition of the probiotic to the slurry, the water activity of the polymer slurry is then lowered as low as possible, preferably below 0.1. Measurement of water activity is taught in U.S. Patent 4,886,664, which is incorporated herein by reference.

[051] As used herein“low-water-activity” means a water activity of 0.6 or less measured using a standard water activity monitor, for example, Goldenwall Model HD-3A, available from Great Wall Instruments.

[052] To reduce the water activity to less than 0.6, for example, less than 0.3, for example, less than 0.1 , simple drying will not be sufficient. Glycerin or other humectant can be added to the water reducing the water activity further. The polymer slurry may be dried using any art recognized technique that won’t interfere with the viability of the probiotics and which will reduce the water content, as measured by water activity to below about 0.6. For example, the material can be heat dried at low temperature, sealed and subjected to heat and/or vacuum with a desiccant, spray dried or dried in a fluidized bed at appropriate temperatures, and/or combined with hygroscopic materials. Other drying techniques will be readily apparent to the skilled artisan.

[053] According to one embodiment, a hygroscopic composition that scavenges water can be added. According to a specific embodiment, the hygroscopic scavenging composition is a prebiotic sugar. When added in sufficient amounts, the water activity of the composition can approach zero. The amount of hygroscopic material needed can be calculated based upon the measured water activity of the system. Once the polymer solution has achieved a low-water-activity, care should be taken to prevent the reintroduction of water to the system before, during or after the encapsulation coating. [054] According to one embodiment, the freeze-dried probiotic slurry can be blended with a wax. In this embodiment, the freeze-dried probiotic slurry is hydrophilic and would form droplets inside the wax at an elevated temperature. The selected temperature should be sufficient to melt the hydrophobic wax material, but maintained for a sufficiently short time to keep the freeze-dried probiotic viable. Temperatures can range from 80°F to about 150°F. To maintain the viability of the freeze-dried probiotics, the temperature should not be maintained at or above 140°F, for longer than required to create the desired thickness of microcapsule. Appropriate times are based upon the specific materials being used and would be well understood by the skilled artisan.

[055] In this embodiment, after the mixing of the freeze-dried probiotic is complete, the mixture is cooled to room temperature to allow the wax to solidify on the freeze-dried probiotic slurry. Once the coated particles have cooled, they can be ground to the desired size prior to incorporation into a carrier composition. After grinding the mixture to the desired size, one can optionally subject the ground material to an after process to ensure that the hydrophobic wax material coating is substantially continuous around the freeze-dried probiotic slurry.

[056] Suitable after processes include, for example, spheroidization (high heat fluidization slightly below the melt temperature of the hydrophobic wax material) and ball milling. These after processes should cause the hydrophobic wax material to coat the freeze-dried probiotic slurry particles in a substantially continuous manner. [057] According to another embodiment, the core 40 may be

encapsulated by including an activator or crosslinker in the core 40. The encapsulating activator may be any activator capable of initiating a crosslinking reaction in the presence of a crosslinkable compound. Suitable encapsulating activators include polyvalent ions of calcium, polyvalent ions of copper, polyvalent ions of barium, silanes, aluminum, titanates, chelators, acids, or combinations of these. According to one embodiment, the activator is calcium chloride or calcium combined with any number of anions.

[058] According to this embodiment, encapsulating the freeze-dried probiotic/polymer slurry in the presence of an activator in the core composition allows for almost instantaneous crosslinking when the core composition is introduced into the solution containing the crosslinkable compound. This immediate crosslinking reduces the potential for unwanted freeze-dried probiotic hydration. According to one embodiment, the freeze-dried probiotic/polymer slurry may be added dropwise into the liquid containing the crosslinkable compound and the beads that form when the drops contact the liquid will form an encapsulating coating. Stirring can provide sufficient disruption to maintain the individual beads separate during the crosslinking reaction. Agglomerated masses can be susceptible to numerous defects and while they may be physically separated, it is preferable that they not be formed. Any art recognized method, physical or chemical, for achieving bead separation may be used. The drops added to the liquid solution may have a diameter of from about 0.05 millimeters to about 1 millimeter, for example from about 0.1 millimeters to about 1 millimeter.

[059] When the core composition including the encapsulating activator is introduced into the liquid containing the crosslinkable compound, the

encapsulating activator migrates to the interface between the core composition and the liquid solution and initiates the crosslinking reaction on the surface of the core composition to allow the encapsulation layer to grow outward toward the liquid solution.

[060] The thickness of the resulting encapsulation layer 20 surrounding the core 40 composition can be controlled by (1 ) controlling the amount of encapsulating activator included in the core composition; (2) controlling the amount of time the core composition including the encapsulating activator is exposed to the liquid solution including the crosslinkable compound; and/or (3) controlling the amount of crosslinkable compound in the liquid solution. By way of example, a solution including alginate in a range of from about 1 to about 500 mg/ml, CaCL in a range of from about 0.1 to about 100 mg/ml level and at a temperature between about 4°C and about 37°C, would produce a thickness of between 1 -20pm of alginate.

[061] According to another embodiment, the core composition may be introduced or poured into a liquid solution including the crosslinkable compound and then subjected to shear sufficient to break the paste into small beads for crosslinking. Any art recognized method of applying the shear may be used. [062] According to one embodiment, the liquid solution includes a crosslinkable compound that can be crosslinked in the presence of the

encapsulating activator and a surfactant to form the outer encapsulate shell. The surfactant can be chosen from one or more sugar or sugar-based surfactants, e.g., Tween 20, or amino acid or protein-based materials

[063] According to another embodiment, the encapsulation coating 20 may can be formed using a process known as coacervation, which may not require a chemical encapsulating activator to be present in the core composition. Coacervation processes can utilize a change in pH, a change in temperature, and/or a change in ionic strength of the liquid solution to initiate the formation of the encapsulating layer around the core composition.

[064] Although it is generally desirable to locate the encapsulating activator in the core 40, according to one embodiment, the encapsulating activator may be in the liquid solution. In this embodiment, the encapsulating activator chemically reacts with the crosslinkable compound also contained in the liquid solution. The resulting microencapsulated freeze-dried probiotic slurry may be free from any encapsulating activator or it may contain a small amount of encapsulating activator not consumed in the crosslinking reaction.

[065] According to some embodiments, microencapsulated freeze-dried probiotics are subjected to a process to impart a moisture protective layer that surrounds the encapsulated layer that comprises the crosslinked compound.

This moisture protective layer provides the microencapsulated freeze-dried probiotic with increased protection from water; that is, it makes the microencapsulated freeze-dried probiotic substantially fluid impervious and allows the microencapsulated freeze-dried probiotic to survive long term in an aqueous environment and not degrade until the moisture protective layer is ruptured by mechanical action. The moisture protective layer may be a single layer applied onto the microencapsulated freeze-dried probiotic, or may comprise several layers one on top of the other.

[066] The moisture protective layer may be applied to the

microencapsulated freeze-dried probiotic utilizing any number of suitable processes including, atomizing, or dripping a moisture protective material onto the microencapsulated freeze-dried probiotic. Additionally, a Wurster coating process may be utilized. When a solution is used to provide the moisture protective coating, the solids content of the solution is generally from about 5% to about 40%, for example, from about 5% to about 30%, for example from about 5% to about 20%, for example from about 10% to about 20%. Generally, the viscosity of the solution is from about 20 cp to about 500 cp, for example from about 20 cp to about 80 cp, for example, from about 30 cp to about 70 cp.

[067] According to one embodiment, a fluidized bed process can be utilized to impart the moisture protective layer on the microencapsulated freeze- dried probiotic. The fluidized bed is a bed or layer of microencapsulated freeze- dried probiotic through which a stream of heated or unheated carrier gas is passed at a rate sufficient to set the microencapsulated freeze-dried probiotic in motion and cause them to act like a fluid as the microcapsules are fluidized, a spray of a solution comprising a carrier solvent and the moisture protective material is injected into the bed and contacts the vehicles imparting the moisture protective material to the outside of the microcapsule. The treated

microcapsules 10 are collected when the desired moisture protective layer thickness is achieved. The microencapsulated freeze-dried probiotic 10 can be subjected to one or more fluidized bed processes to impart the desired level of moisture protective layer.

[068] According to some embodiments, the microencapsulated freeze- dried probiotic 10, can be subjected to a process for imparting a fugitive layer surrounding the outermost layer. The fugitive layer could be applied on the freeze-dried probiotic 10 such that it substantially completely covered the moisture protective layer. The fugitive layer may be applied to the

microencapsulated freeze-dried probiotic 10 utilizing any number of suitable processes including, atomizing, or dripping a fugitive material onto the

microencapsulated freeze-dried probiotic. When a solution is used to provide the fugitive coating, the solids content of the solution is from about 10% to about 60%, for example, from about 10% to about 50%, for example, from about 20% to about 50%. The pH of the solution is from about 2.5 to about 11. The viscosity of the solution may be from about 20 cp to about 100 cp, for example from about 20 cp to about 80 cp, for example, from about 30 cp to about 70 cp. The preferred method of applying the fugitive layer utilizes a fluidized bed reactor. Alternatively, any art recognized coating process may be used, including a Wurster coating process. [069] According to another embodiment, an alternative method of producing a preserved encapsulated probiotic may comprise microencapsulating the live microbes that have not been freeze-dried into a slurry with prebiotic materials and, optionally, a polymer, and then coating the microcapsules with alginate or another crosslinked system. After those microcapsules are formed, they would be subjected to freeze drying to preserve the probiotics. Further, the freeze-dried microcapsule may then be coated with a water impermeable coating.

[070] These probiotic microcapsules can be used in a vast number of ways and in a variety of compositions. For example, they can be used to 1 ) deliver living probiotic microbes to the skin to improve skin barrier function, 2) deliver living probiotic microbes to the skin to reduce inflammation associated with acne, eczema, rosacea, or contact dermatitis, 3) strengthen the human biome so pathogenic bacteria do not have a chance to colonize, 4) deposit probiotics on skin that produce anti-pathogenic peptides which kill pathogens; 5) improve the skins ability to ward off pathogen colonization near medical devices such as insulin pumps or catheters; 6) reduce or eliminate the need for preservatives in cosmetic products; 7) reduce skin ulcers on bedridden, diabetic, or otherwise compromised individuals, 8) protects skin from and fights cancer.

[071] Further, having microencapsulated probiotics that remain viable will allow further development of 1 ) cosmetic formulations that are hospitable to probiotic microcapsules; 2) a prebiotic media that encourages the growth of probiotics after deposition on the skin: 3) a prebiotic media that extends the shelf- life of encapsulated probiotic bacteria; 4) microcapsules that increase the shelf- life of consumer products.

[072] The system as described herein further comprises a method for determining suitable microbes for use in the disclosed microencapsulation process. The process comprises testing microbes for their ability to withstand freezing, testing microbes to ascertain their ability to withstand temperatures up to 140°F; and testing microbes for their ability to survive at various water activities. Appropriate microbes will be those that can handle the extremes of freezing and the encapsulation temperature. Further preferred microbes will remain viable even if the water activity of the composition is increased.

[073] Preferred microbes for use in health and beauty products may be tested for their ability to adhere to human skin, their ability to produce peptides that are anti-pathogenic, their ability to grow on human skin, their ability to respond to probiotic media, and, for commercial purposes, their ability to be grown on a macro scale.

[074] Micro-encapsulated probiotics can be used in compositions including, for example, moisturizing lotion, sunscreen, lip balm, oral care product, shampoo, soap, hair conditioner, baby wipes, perineal wipes, facial cleaning wipes, feminine hygiene pad or tampon, diaper or adult incontinence product, deodorant (roll-on liquid, spray, or stick), pet food, food additives, food

condiments, and the like.

[075] In use, the microcapsules are broken by physical forces that are applied to the capsules. Accordingly, depending upon the end use, the size and thickness of the microcapsules can play a big role in the release of the probiotics. Thinner capsule walls would generally result in more delicate capsules.

[076] According to one embodiment of the disclosure, leave-on

applications such as lotion, sunscreen, deodorants, lip balm or hair conditioners may be made in systems with low-water-activity. According to this embodiment, the water activity is reduced not only in the microencapsulated probiotic, but also in the surrounding carrier composition or cosmetic product. When the carrier composition has low-water-activity, the osmotic pressure on the microcapsules is minimized which would allow microcapsules to be smaller, thinner, and less noticeable. In some applications, the microcapsules could be undetectable by the by the consumer.

[077] Additionally, since water activity is necessary for microbial growth, low-water-activity stunts microbial growth and can thereby reduce the need for preservatives in the carrier composition. Minimizing preservatives in the carrier composition further reduces the chance that a preservative could kill the probiotics immediately upon release to the skin.

[078] Low preservative levels are likely to increase the shelf life of the cosmetic or health product. According to one embodiment, decreasing the water activity of the cosmetic decreases through the incorporation of prebiotic sugars in the carrier composition. Adding sufficient prebiotic sugar may reduce the water activity to nearly 0, or undetectable levels.

[079] The reduction of elimination of preservatives can have a significant impact on the functions of cosmetic and/or health products. Preservatives can do significantly more damage to the skin biome than antimicrobial soaps. The same preservatives that give hand lotion a long shelf life, will kill both the microbes in the skin’s biome, as well as any probiotics that may be delivered to the skin biome in a viable state.

[080] According to one embodiment, the encapsulated microbes may be chosen from those that can produce peptides that kill pathogenic bacteria. When such a product is applied to the skin, it provides anti-bacterial properties that may extend beyond the point of first application.

[081] Below Tables 1 provides a description of probiotic materials that can be used in the processes described herein. Many are preferred for use on the skin.

Table 1 : Beneficial Skin Microorganisms

[082] Below, Table 2 lists probiotics that are not harmful to skin, but that create peptides that target non-native bacteria (pathogens). Besides targeting C. difficile, MRSA, and VRE, probiotics can be used to target other pathogens of concern are listed below: Table 2: Pathogenic Skin Microorganisms.

[083] After understanding the information disclosed herein, the

production of other carrier compounds suitable for use with the described microencapsulated product would be readily understood by the skilled artisan.

[084] In addition to low-water activity carrier compositions, oxygen transmissive packaging may allow the microencapsulated probiotics to remain viable without refrigeration. Containers for the products described herein may be any standard art recognized packaging. Packaging compositions with the described microencapsulated probiotic in packages with low oxygen transmission may reduce shelf-like, but would not otherwise interfere with the product.

[085] According to one embodiment, compositions comprising the microencapsulated probiotic(s) are packaged in high oxygen transmission packaging which can improve both shelf-like and/or bacterial viability without refrigeration. Oxygen transmission rate (OTR) is well understood and high OTR packaging is used for products that require substantial oxygen concentrations, such as contact lenses. See, for example, U.S. Patent No. US 9,062,180.

However, current technology surrounding cosmetic products is to provide a good oxygen barrier. See, for example, U.S. Patent No. 8,124,204. Probiotics benefit from high oxygen environments and for that reason their packaging should be produced from high OTR materials. Like the prebiotic sugar, high oxygen may extend the life of microbes without refrigeration thereby creating a shelf-stable product at room temperature. Further, carrier compositions that have very low- water-activity will have much less degradation based upon exposure to oxygen. [086] According to one embodiment, the probiotic container could be a single layer of any number of materials including:

Table 3

[087] Oxygen requirements for various microbes can be found in the literature. According to one embodiment, for oxygen loving microbes, LDPE packaging could be used. For microbes that respond better with lower oxygen conditions, the packaging could be selected with lower OTR per the list above. For all packaging, the oxygen transmission should not be so high that the probiotic leaves stasis. [088] According to yet another embodiment of the instant description, a non-encapsulated freeze-dried probiotic can be incorporated into a carrier composition that has low-water-activity and is free from microbial contamination and is free from preservatives. According to one embodiment, the carrier composition, for example, a lotion, sunscreen, deodorant, could be irradiated to assure it was free from microbial contamination. The low-water-activity of the composition would prevent the growth or reintroduction of microbes. The irradiated product could be blended with freeze-dried probiotic. The product could them be packaged in an appropriate OTR material and sold. The freeze- dried probiotic should stay in stasis until water is introduced at the time of use of the product.

[089] The methods and products described herein should not be limited to the examples provided. Rather, the examples are only representative in nature.

EXAMPLES

Example 1

Formulations and uses for the probiotic microcapsules will be discussed below. The described microencapsulated probiotics are particularly useful in low- water-activity compositions. Examples of low-water-activity skin care lotions are set forth below in Tables 4 to 7. Examples of hand cleaner, hand soap and shampoo can be found in Tables 8, 9, and 10, respectively.

Table 4

Additional Ingredients

Table 5: Example Lotion Formulation (Oil-in-Water #2)

Additional Ingredients

Table 6: Example Lotion Formulation (Water-in-Oil #1)

Table 7: Example Lotion Formulation (Water-in-Oil #2)

Table 8: Waterless Hand Cleaner Example

Table 9: Hand Soap Example

Table 10: Shampoo Example:

Example 2 - Prophetic Example

[090] The encapsulated probiotic product as described can be delivered to the gut of a user by incorporation of the encapsulated probiotic into or onto a food source. According to this Example, microencapsulated probiotic is sprinkled onto a food, such as bread or pastry and is consumed by the user. The microcapsules should protect the probiotic until it can be released and provide benefit to the user.

[091] Additionally, other embodiments will be apparent from

consideration of the specification and practice of the present disclosure. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.