Aerosol spray containing live bacterial species

Abstract

The present invention relates to an aerosol spray containing one or more bacterial species and at least one siloxane suitable for applying the one or more bacterial species to a surface or environment.

Description

Technical Field

[0001] This invention relates to aerosol sprays containing one or more live bacterial species and one or more volatile siloxanes suitable for applying the one or more live bacterial species to a surface or environment. Background Technology

[0002] Aerosol sprays are very useful for applying different reagents to surfaces or environments, and they offer an interesting way to formulate live bacterial species. However, aerosol sprays typically contain aqueous components that are incompatible with live bacterial species formulations. In particular, the presence of water usually necessitates the presence of preservatives to prevent the growth of harmful pathogens and control microbial growth. Preservatives are detrimental to the survival of co-formulated microorganisms. Furthermore, even a limited amount of co-formulated water is sufficient to activate dormant bacteria, thereby significantly reducing the shelf life of such compositions. Therefore, typical aerosol sprays are not suitable for formulations of live bacterial species, and thus one object of the present invention is to provide an aerosol composition that solves these problems in formulating and obtaining live bacterial species for long-term storage.

[0003] Furthermore, it was surprisingly found that aerosol sprays containing at least one volatile siloxane were very useful for the stated purpose.

[0004] In particular, silicone fluids and siloxanes are commonly used in sprays containing crop protection products, insect repellents, cosmetics, and other personal care products. The most commonly used silicone fluids are dimethicone, cyclomethicone, and phenyltrimethicone.

[0005] These silicone oils are excellent carrier oils when incorporated into products, offering numerous benefits such as improved slip properties, reduced tack, and lubrication. Despite their low solubility and viscosity, cyclomethylsiloxanes are considered particularly useful when formulated into aerosol sprays because they tend to provide the aforementioned benefits without contributing to oiliness or greasiness. Furthermore, cyclomethylsiloxanes are known to have relatively high volatility and do not excessively clog the antiperspirant active ingredients in deodorants. Finally, cyclomethylsiloxanes are excellent dispersing agents and spreaders, typically water-white, low-odor, volatile, and resistant to chemical and oxidative degradation.

[0006] Regardless of their widespread use in aerosol sprays, these silicone resin solutions are generally considered to have antimicrobial activity against a broad range of (aerobic) bacteria, and therefore their use in formulating bacteria has not been further investigated. Nevertheless, we have now surprisingly discovered that silicone resin solutions, particularly volatile siloxanes such as cyclomethylsiloxanes, can be used for the long-term storage of (dormant) lactic acid bacteria in aerosol sprays. These dormant bacteria were found to be unaffected by the volatile siloxanes and thus remained stable during storage. After spraying, the dormant bacteria became active due to contact with water or vapor, and by then, the co-formulated volatile siloxanes had evaporated and no longer affected the survival of the activated bacteria. Invention Overview

[0008] In a first aspect, the present invention provides an aerosol spray comprising 1-20 wt% of one or more live probiotic strains, one or more volatile siloxanes, and a propellant under pressure.

[0009] In a specific implementation, the one or more live bacterial species, more specifically, live probiotic strains, are dormant bacterial species, more particularly dormant lactic acid bacteria.

[0010] In specific embodiments of the present invention, the one or more siloxanes are selected from the following list: linear siloxanes, such as dimethylsiloxane, dimethiconol, phenyltrimethicone and their derivatives; or cyclic siloxanes, such as cyclomethylsiloxane and their derivatives; more particularly, cyclic siloxanes, such as cyclomethylsiloxane and their derivatives.

[0011] In another specific embodiment, the present invention provides an aerosol spray comprising a mixture of one or more live (preferably dormant) bacterial species and one or more volatile siloxanes, wherein the mixture is in the form of a powder, suspension or emulsion; wherein the aerosol spray of the present invention may further comprise a propellant.

[0012] The propellant (under pressure) is particularly selected from the list including: liquefied gas, more particularly one or more hydrocarbons, such as methane, ethane, propane, isopropane, butane, n-butane, pentane and n-pentane; or, the propellant may be a permanently compressed and / or soluble gas, such as nitrogen, carbon dioxide, compressed air or nitrous oxide gas.

[0013] In yet another embodiment, the aerosol spray of the present invention may further comprise an anti-settling agent and / or an anti-caking agent. The anti-settling agent and / or anti-caking agent is particularly selected from inorganic polymers, such as organoclay gels containing bentonite or lithium montmorillonite; and / or fumed silica, such as aerosil.

[0014] In another specific embodiment, the aerosol spray of the present invention may further comprise one or more lubricants, suspending agents, wetting agents, antioxidants, humectants, emulsifiers, viscosity modifiers, fragrances and / or other excipients.

[0015] In a specific embodiment, the present invention provides an aerosol spray; wherein its contents comprise or consist of: at least 50% propellant, 1-20% siloxane, 1-20% lactic acid bacteria and 0.1-10% antisettling agent and / or anticaking agent.

[0016] In yet another embodiment, the present invention provides an aerosol spray containing, or comprising, at least 75% propellant, 5-10% cyclomethylsiloxane, 1-10% lactic acid bacteria, and 0.1-5% bentonite gel. Alternatively, the present invention provides an aerosol spray containing, or comprising, at least 75% propellant, 5-10% cyclomethylsiloxane, 1-10% lactic acid bacteria, and 0.1-5% aerosil.

[0017] In specific embodiments, the aerosol spray of the present invention is substantially free of water and preservatives.

[0018] In another aspect, the present invention provides the use of the aerosol spray of the present invention for applying the one or more live bacterial species, specifically, live probiotic strains, to a surface or into the environment.

[0019] In specific embodiments, the present invention provides an aerosol spray as defined herein for restoring and / or maintaining a healthy skin microbiome. Therefore, the present invention also provides the use of the aerosol spray of the present invention for restoring and / or maintaining a healthy skin microbiome.

[0020] Brief description of the attached figures

[0021] Referring now specifically to the accompanying drawings, it is important to emphasize that the details shown are merely exemplary and are intended only to illustrate different embodiments of the invention. They are presented to provide what is considered most useful and readily apparent in describing the principles and concepts of the invention. In this regard, no attempt is made to show the structural details of the invention in more detail than is necessary for a basic understanding of it. The description, through the accompanying drawings, enables those skilled in the art to understand how several forms of the invention will be embodied in practice.

[0022] Figure 1The stability of self-cultured (undried) Lac4 and Lac7 strains suspended in silicone oil CF 1406-OH (1 / 10 m / m%) for one year compared to LGG commercial freeze-dried strains was compared. The figure clearly demonstrates that freeze-drying probiotics and suspending them in an anhydrous silica mixture is an ideal way to prevent a rapid decline in the viability of live bacteria. Suspending freeze-dried bacteria in a silica mixture containing dimethylsiloxane and polydimethylsiloxane alcohol provides this *Lactobacillus* genus (… Lactobacillus ( ) types of long-term storage.

[0023] Figure 2 Stability of self-cultured (undried) Lac4 and Lac7 strains suspended in silicone oil CF 6570-DM (1 / 10 m / m%) for one year compared to commercially freeze-dried strains from LGG. The figure clearly demonstrates that freeze-drying probiotics and suspending them in an anhydrous silica mixture is an ideal way to prevent a rapid decline in the viability of live bacteria. Suspending freeze-dried bacteria in a silica mixture containing dimethylsiloxane, disiloxane, and cyclopentasiloxane provides long-term storage for this *Lactobacillus* species.

[0024] Figure 3 : Freeze-dried *Lactobacillus plantarum* suspended in different formulations (F1-F8) and stored in pressurized tanks at room temperature (20°C) along with hydrocarbon propellants. L. Plantarum The survival curves of the probiotics were observed. After a small initial decline in viability during the first month, viability remained fairly stable in the following months, demonstrating that storage in pressurized tanks is a suitable method for the long-term survival of probiotics.

[0025] Figure 4 The 6-month stability of freeze-dried *Lactobacillus plantarum* in different formulations compared to the same formulation stored in a pressurized tank was investigated. After 6 months of storage, there was a statistically significant difference in survival between the formulation stored in the pressurized tank with the propellant and the formulation stored without propellant. Better survival was observed in all 8 formulations tested, demonstrating that storing freeze-dried bacteria suspended in silicone oil with the propellant is a better method for long-term stability than simply suspending the freeze-dried bacteria in silicone oil.

[0026] Figure 5 Stability of freeze-dried LAC_15 after 3 months in different silicones and silicone mixtures. Although small differences were noted, most were considered insignificant. There appears to be a trend that mixtures of different silicone oils provide better stability than one or two oil components. This may indicate that lower concentrations of different silicone oils offset the adverse effects of higher concentrations of a single silicone component. Invention Details

[0028] In a first aspect, the present invention provides an aerosol spray comprising 1-20 wt% of one or more live probiotic strains, one or more volatile siloxanes, and a propellant under pressure.

[0029] Typically, the present invention provides an aerosol spray comprising a mixture of one or more live (preferably dormant) bacterial species and one or more volatile siloxanes, wherein the mixture is in the form of a powder, suspension, or emulsion. In a specific embodiment, the one or more live bacterial species are dormant bacterial species; more particularly, dormant lactic acid bacteria.

[0030] In the context of this invention, the term "aerosol spray" refers to a distribution system that produces an aerosol mist. This is used with a can or bottle containing the mixture of the invention and, preferably, a propellant under pressure. When the valve of the container is opened, the mixture is forced out of the can through the valve and appears as an aerosol or mist. While a small amount of propellant expands to expel the mixture, a large amount of propellant remains inside the can to maintain a constant pressure. Outside the can, the propellant evaporates rapidly.

[0031] In the context of this invention, the term "mixture" refers to a system consisting of two or more different substances mixed but not chemically bonded, such as a combination of volatile siloxanes and dormant bacterial species. In such mixtures, the individual properties of each component are preserved, and they are mixed in the form of solutions, suspensions, emulsions, colloids, or powders.

[0032] In the context of this invention, the term "powder" refers to a dry, bulk solid consisting of a large number of very fine particles that can flow freely when shaken or tilted. Although the terms powder and granules are sometimes used to distinguish different categories of materials, powder is a special subclass of granular materials.

[0033] In the context of this invention, the term "dormant" refers to a state in which living / active bacteria temporarily cease growth, development, reproduction, etc. They are in a state of metabolic dormancy, where metabolic processes are slowed to the point where large amounts of metabolites are no longer produced. Many bacterial species can survive adverse conditions such as low temperatures by forming (endospores), sporangia, conidia, or by a state of reduced metabolic activity due to a lack of specialized cellular structures. Dormant bacteria can be reactivated upon exposure to "normal" conditions, for example, after contact with water or exposure to a certain temperature or vibration. In the context of this invention, techniques known in the art, such as freeze-drying, spray drying, vacuum drying, fluidized bed drying, nitrogen freeze-drying, etc., can be used to induce a dormant state in the bacteria. Preferably, the bacteria of this invention are freeze-dried.

[0034] In the context of this invention, the term "volatility" is used for the disclosed siloxanes to indicate their tendency to evaporate. Volatility is directly related to the vapor pressure of a substance. At a given temperature, a substance with a higher vapor pressure evaporates more readily than a substance with a lower vapor pressure. Conversely, a substance with a lower heat of vaporization (kJ / kg) will evaporate faster at a given temperature than a substance with a higher heat of vaporization. The term is primarily applicable to liquids; however, it can be used to describe sublimation processes associated with solid substances, such as dry ice (solid carbon dioxide), which can directly change from a solid to a vapor without becoming a liquid. The vapor pressure of a substance is the pressure at which its gaseous phase is in equilibrium with its condensed phase (liquid or solid). It is a measure of the tendency of molecules and atoms to escape from a liquid or solid. At a given temperature, the higher the vapor pressure of a liquid, the higher its volatility, and the lower its normal boiling point. Preferably, the vapor pressure of the volatile siloxane of the present invention is less than 2500 kJ / kg at room temperature (25°C), more preferably less than 1000, and even more preferably less than 900, 800, 700, 600, 500, 400, 300, or 200 kJ / kg.

[0035] Cyclomethylsiloxanes are a group of methylsiloxanes, a class of liquid organosilicones (cyclic polydimethylsiloxane polymers) characterized by low viscosity and high volatility, and are useful as skin lubricants and cleaning solvents in certain situations. Unlike dimethylsiloxanes, which are linear siloxanes that do not evaporate rapidly, cyclomethylsiloxanes are cyclic: both groups consist of a polymer characterized by a monomer backbone of a silicon atom bonded to two oxygen atoms, but not a very long "linear" backbone surrounded by a series of methyl groups (producing transparent, non-reactive, non-volatile liquids ranging from water-like thinness to toffee-like density). Cyclomethylsiloxanes have short backbones that can form closed or nearly closed rings (rings or "cycles") with their methyl groups, thus giving them many of the same properties as dimethylsiloxanes, but making them more volatile. Therefore, depending on the desired degree of evaporation, a more volatile siloxane, such as cyclomethylsiloxane, can be chosen, or alternatively, a less volatile siloxane, such as dimethylsiloxane. However, more volatile siloxanes are preferred, such as cyclomethylsiloxanes and their derivatives.

[0036] Silicones are commonly used in the personal care industry (and many other industries) and are perceived as smooth, soft, non-greasy, and non-sticky upon skin contact (Handbook of Cosmetic Science and Technology Fourth Edition 2014; New York CRC Press; 204 AD. pp. 321-329). They have low surface energy, are resistant to moisture and temperature, are biocompatible, volatile, and permeable, and are non-toxic and non-irritating. Silicones are polymers derived from silicon (Si), carbon, oxygen, and hydrogen, and can be linear, cyclic, or organically functionalized. The most common silicone (dimethylsiloxane) has an inorganic Si-oxide backbone with organic (2xmethyl) side groups on the Si. Silicon polymers have 2 to thousands of monomers, and their viscosity ranges from 0.65 cSt (hexamethyldisiloxane) to +1 million cSt cps. High-viscosity fluids can form protective films, while low-viscosity fluids can be emulsified and have better solubility with other components. Organic groups can be modified to provide surface-active properties. The Si-O bonds are highly mobile (Si-O rotational energy 0.2 kcal / mol; cc bond rotational energy 3.3 kcal / mol), possess strong bonding energies (Si-O bond strength 117 kcal / mol; cc bond strength 85 kcal / mol), and are elongated (Si-O angle 130°, length 0.163 nm; cc bond angle 112°, length 0.154 nm). All of this contributes to a very flexible and robust backbone. It reacts with ethanol (50 mM / m) and water (72 mM / m). 2 In comparison, polydimethylsiloxane (PDMS) also has a very low surface tension of 20 mM / m. This provides complete surface coverage and excellent spreading properties, both of which are useful in aerosol formulation. Lower molecular weight silicones are highly volatile, leaving no residue and providing a light skin feel. Due to their low thermal evaporation (per gram), they do not require a significant amount of heat from the skin for evaporation compared to water- or ethanol-based products, thus not feeling cold (see Table 1).

[0037] Table 1. Heat of vaporization of some volatile fluids used in cosmetics.

[0038]

[0039] PDMS polymers exhibit high gas permeability, which is related to solubility and diffusion coefficient (see Table 2).

[0040] Table 2. Permeability data of some volatile fluids used in cosmetics

[0041]

[0042] This high gas diffusion rate allows the skin to "breathe" and is described as non-clogging. Cyclic silicones (cyclomethylsiloxanes, D4-D5-D6) are excellent solvents, non-staining, colorless, odorless, and highly volatile. They are most commonly used in antiperspirant products as delivery media for the active ingredients. They do not feel cold on the skin due to low-heat evaporation and have a pleasant skin feel, making them a preferred suspension medium for pressurized aerosol cans. Due to the lubricating properties of silicones, they also prevent the formation of aggregates at the valve. Other suspension media commonly used in antiperspirants are ethanol, water, and mineral oil.

[0043] In the context of this invention, the term "siloxane" refers to a molecule containing a functional group having Si-O-Si bonds. Parent siloxanes include those having the formula H(OSiH2). n OH and (OSiH2) n Oligomeric and polymeric hybrids. Siloxanes also include branched compounds, characterized by each pair of silicon centers being separated by an oxygen atom. Siloxane functional groups form the backbone of the organosilicon. In the context of this invention, suitable volatile siloxanes may be selected from the following list: linear siloxanes, such as dimethylsiloxane, polydimethylsiloxane alcohol, phenyltrimethylsiloxane, and their derivatives; or cyclic siloxanes, such as cyclomethylsiloxane and its derivatives; particularly cyclic siloxanes, such as cyclomethylsiloxane and its derivatives.

[0044] The inventors have discovered that by pressurizing the aerosol can, a more improved stability is achieved compared to the unpressurized composition. Therefore, in another specific embodiment, the aerosol spray of the present invention may also contain a propellant.

[0045] In the context of this invention, the term "propellant" refers to a pressurized gas used to generate fluid or powder motion. Specifically, they are simply readily evaporable liquids (or gases). Cold gas propellants can be used to fill pressurized distribution systems, such as aerosol sprays, to force material through nozzles. In aerosol spray canisters, the propellant is typically a pressurized gas in equilibrium with its liquid or powder (at its saturated vapor pressure). As some gas escapes to expel the payload, more liquid evaporates, thus maintaining a uniform pressure. Suitable propellants useful within the scope of this invention can be selected from a list including: liquefied gases, more particularly one or more hydrocarbons such as methane, ethane, propane, isopropane, butane, n-butane, pentane, and n-pentane; or permanently compressed and / or soluble gases such as nitrogen, carbon dioxide, compressed air, or nitrous oxide gas. Other liquefied gases that can be used in the context of this invention are DME (dimethyl ether) and HFC (hydrofluorocarbons), such as 1,1,1,2-tetrafluoroethane and 1,1-difluoroethane.

[0046] In yet another embodiment, the aerosol spray of the present invention may further comprise an anti-settling agent and / or an anti-caking agent.

[0047] In the context of this invention, the term "anti-settling agent" refers to a substance that prevents suspended particles from settling out of the fluid that carries them and abutting against a barrier such as the bottom of an aerosol can. A good anti-settling agent has the property of forming a porous, bulky, and easily resuspended suspension, which is desirable for uniform dosage. In the context of this invention, the term "anti-caking agent" refers to a substance that prevents components from clumping or agglomerating. Suitable anti-settling and / or anti-caking agents within the scope of this invention may be selected from a list including: inorganic polymers, such as organoclay gels containing bentonite or lithium montmorillonite; and / or ignited silica, such as aerosil®. Aerosils® can be chemically modified to obtain a better 3D structural network in a selected siloxane support. Aerosils are typically hydrophilic ignited silica (SiO2). More suitable for the present invention is hydrophobic silica, typically silica post-treated with a hydrophobic substance, such as, but not limited to: dimethyldichlorosilane, polydimethylsiloxane, organosilane, hexadecylsilane, octamethylcyclotetrasiloxane, silane methacrylate, silicone oil, aminosilane and its derivatives.

[0048] In another specific embodiment, the aerosol spray of the present invention may contain any other substances that can be used in the context of the present invention. These other substances include, but are not limited to, one or more lubricants, humectants, antioxidants, moisturizers, suspending agents, emulsifiers, viscosity modifiers, polarity modifiers, fragrances, electrolytes, color modifiers, and other excipients. Suitable antioxidants may be, but are not limited to, BHT, BHA, propyl gallate, ascorbic acid, and sodium metabisulfite. Suitable viscosity modifiers may be, but are not limited to, various colloids, silicone resins, propylene glycol, organic gels, aerosols, etc.

[0049] Other suitable components in the aerosol spray of the present invention are, for example, Tegosoft®, namely polypropylene glycol butyl ether or ppg-14-butyl ether. Tegosoft® is a light cosmetic oil with excellent spreadability and is commonly used as a lubricant in skin care products such as creams, lotions, and bath oils. It also has excellent suspension properties for the active ingredients in aerosol formulations. The PPG-butyl ether component in cosmetic formulations enhances the appearance of dry or damaged skin by reducing exfoliation and restoring softness.

[0050] Whenever percentages are mentioned in the context of this invention, they refer to wt / wt percentages relative to the total composition. For example, 80% propellant means that 80 g of propellant is present in a composition with a total weight of 100 g.

[0051] In a specific embodiment, the present invention provides an aerosol spray whose contents comprise or consist of the following: at least 10% propellant, 10-40% siloxane, 10-40% lactic acid bacteria, 0-5% antisettling agent and / or anticaking agent, and 0-5% other additives.

[0052] In a specific embodiment, the present invention provides an aerosol spray whose contents comprise or consist of the following: at least 50% propellant, 1-20% siloxane, 1-20% lactic acid bacteria, 0.1-5% antisettling agent and / or anticaking agent, and 0-5% other additives.

[0053] Preferably, the aerosol spray of the present invention contains 1-20% lactic acid bacteria; more particularly 1-10%; and even more particularly about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% lactic acid bacteria.

[0054] Furthermore, the aerosol spray of the present invention preferably contains a small amount of anti-settling agent, such as less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or, if feasible, even no anti-settling agent and / or anti-caking agent.

[0055] In a specific embodiment, the present invention provides an aerosol spray containing or composed of the following: at least 75% propellant, 5-10% siloxane, 1-10% lactic acid bacteria, and 0-5% anti-settling agent and / or anti-caking agent.

[0056] In another specific embodiment, the present invention provides an aerosol spray whose contents comprise or consist of: at least 80% propellant, 1-8% siloxane, 1-8% lactic acid bacteria and 0-4% antisettling agent and / or anticaking agent.

[0057] In another specific embodiment, the present invention provides an aerosol spray whose contents comprise or consist of: at least 90% propellant, 1-4% siloxane, 1-4% lactic acid bacteria and 0-2% antisettling agent and / or anticaking agent.

[0058] In specific embodiments of the compositions defined above, the antisettling agent and / or anticaking agent are selected from bentonite gel and aerosil®.

[0059] In yet another embodiment, the present invention provides an aerosol spray containing or composed of: at least 75% propellant, 5-10% cyclomethylsiloxane, 1-10% lactic acid bacteria, and 0-5% bentonite gel. Alternatively, the present invention provides an aerosol spray containing or composed of: at least 75% propellant, 5-10% cyclomethylsiloxane, 1-10% lactic acid bacteria, and 0-5% aerosil.

[0060] Obviously, to ensure long-term storage of the composition, harmful products such as preservatives and activating substances such as water are preferably avoided in formulations containing dormant bacteria. Therefore, in specific embodiments, the aerosol spray of the present invention is substantially free of water and preservatives. The term "substantially free" means less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, preferably less than 1%. Where multi-compartment aerosol sprays are used, the compartments containing bacteria are preferably substantially free of water and preservatives.

[0061] On the other hand, the present invention provides the use of the aerosol spray of the present invention in applying the one or more species of live bacteria to a surface or environment. In the context of the present invention, the term "surface" should be interpreted in its broadest sense, i.e., it can include the surface of an object; however, it can also include the skin of a person or animal. Therefore, the aerosol spray of the present invention can also be a topical aerosol spray, wherein the term "topical" refers to local delivery at a specific location on the body, particularly application to a specific location on or inside the body. In particular, it includes application to mucous membranes via aerosol.

[0062] In specific embodiments, the present invention provides an aerosol spray as defined herein for restoring and / or maintaining a healthy skin microbiome. Therefore, the present invention also provides the use of the aerosol spray of the present invention for restoring and / or maintaining a healthy skin microbiome.

[0063] Example

[0064] Example Description

[0065] Currently, antifungal agents such as miconazole are used to treat athlete's foot and other fungal skin infections. Miconazole interferes with ergosterol synthesis. A well-known commercial product containing miconazole is daktarin® spray, preferred for its ease of use. There is a known trend of increasing resistance to both antibiotics and antifungal agents. Therefore, alternative treatments, such as the use of probiotics to treat localized lesions, would be beneficial to patients. Most probiotics exert beneficial effects on the host (human) by preventing pathogens from invading and colonizing. Some probiotics, such as species of Lactobacillus, are able to combat these pathogens by releasing organic acids, hydrogen peroxide, and antimicrobial peptides into the environment. Common fungal skin infections (such as athlete's foot) are caused by different species, such as Trichophyton (…). Trichophyton Epidermophyton ( ) Epidermophyton Microsporum spp. Microsporum ) and Candida spp. Candida This study investigated whether aerosols (pressurized containers) are suitable formulations for the preservation and long-term stability of probiotics.

[0066] As has been demonstrated, probiotics exhibit better long-term stability when properly dried, unlike undried bacteria (Morgan et al., 2006 - Preservation of micro-organisms by drying; areview – Journal of microbiological Methods 66 (2006) 183-193). The drying process renders the probiotics metabolically inactive (i.e., dormant). The residual water activity (Aw) of the powder, along with storage conditions (temperature, oxygen, light), will primarily determine the powder's stability.

[0067] Example 1: Stability of probiotics in organosilicon

[0068] In this embodiment, we investigated the long-term stability of live probiotics in different organosilicon / siloxane mixtures.

[0069] Materials and methods

[0070] Stability Study

[0071] This stability study used three strains:

[0072] Self-cultured Lactobacillus pentosus ( L. pentosus ) (i.e., lac4 or Lp)

[0073] Self-cultivated LGG (i.e., lac7 or LGG)

[0074] Purchased freeze-dried LGG (Dupont® LR-32)

[0075] Two organosilicon-based suspension media for preserving bacteria were tested at room temperature (+ / - 20 °C):

[0076] Organosilicon:

[0077] ○ Cosmetic Liquid 1406-OH (Polydimethylsiloxane & Dimethylsiloxane) – Chemsil®

[0078] ○ Cosmetic Liquid 6570-DM (Disiloxane, Cyclopentasiloxane and Dimethylsiloxane) – Chemsil®

[0079] Obtain samples at the predetermined time point:

[0080] T0 = ​​before being suspended in the suspension medium

[0081] T1 = 1 day after suspension

[0082] T2 = 1 week after suspension

[0083] T3 = 2 weeks after suspension

[0084] T4 = 1 month after suspension

[0085] T5 = 2 months after suspension

[0086] T6 = 6 months after suspension

[0087] T7 = 12 months after suspension

[0088] All materials used are sterilized by autoclaving before use. If possible, all used components and ingredients are autoclaved; otherwise, they are filtered in a sterile environment and then suspended to allow bacteria to suspend.

[0089] T0 CFU Measurement

[0090] Lac 4 and Lac 7 were cultured in liquid MRS (de MAN, Rogosa & Sharpe-Carl Roth) medium (37°C) until full growth was achieved. The medium was centrifuged at 2780G for 10 minutes. The supernatant was discarded, and the bacterial pellet was used for further processing.

[0091] Weigh 100 mg and dilute with physiological water (0.85% NaCl) to obtain a total volume of 10 ml. Prepare a series of dilutions (10-fold serial dilutions) using three strains (lac4, lac7, and LGG) and perform plate counting according to the spread plating method (derived from Pharmacopeia methods 2.6.12 and 2.6.13) (European Pharmacopeia 8.0). Perform measurements in triplicate; results are expressed as CFU / g powder.

[0092] Bacteria are suspended in a suspension medium.

[0093] Uniform 1 / 10 dilutions (m / m%) of bacterial blocks were prepared using different anhydrous organosilicon-based suspension media. One gram of the suspension was weighed in a Falcon tube and sealed in an aluminum bag (RH = 20%). Each sample thus contained 100 mg of bacteria for further testing purposes. The aluminum bag was opened at regular intervals for viability testing.

[0094] Test after storage time

[0095] The sealed bag was opened at a specific time point for stability testing. Falcon tubes containing 1 gram of anhydrous material were further treated by adding 1 gram of an emulsifier mixture (composed of polysorbate 80 and dehydrated sorbitan sesquioleate) and 8 grams of physiological water. This formed an emulsion through which bacteria could come into contact with water and be reactivated. Further sample preparation and plate counting were performed as described above, and derived from Pharmacopoeia (8.0) Method 2.6.12: Microbiological Examination of Non-Sterile Products: Microbial Count Test, and 2.6.13: Microbiological Examination of Non-Sterile Products: Test for Specified Microorganisms.

[0096] Results and discussion

[0097] As expected, the freeze-dried powder showed significantly better stability compared to the unfreeze-dried strain. This was observed after only one month, and regardless of the type of silica mixture used. Figure 1 :CF1406; Figure 2The strain CF6570 showed an average reduction of 3 log values, while no significant reduction was detected in the freeze-dried strain. Figure 1 and 2 Therefore, it is important to begin formulation with a suitable freeze-dried (or other drying method) powder to ensure that the live microbial preparation is stable over time. Stable dried probiotic powders are easier to handle for further formulation purposes.

[0098] Stability results of silicon mixtures ( Figure 1 and 2 This indicates that they are suitable as suspension media. Although the stability of anhydrous organosilicon media decreased by an average of 1 log after 1 year, this left enough live microorganisms to be applied topically and produce beneficial effects on the skin.

[0099] Therefore, it has been demonstrated that a suspension with stable probiotics can be obtained by using appropriately freeze-dried powder suspended in an anhydrous organosilicon medium.

[0100] Example 2: Stability of probiotics in pressurized aerosol formulations

[0101] In this embodiment, we examined the long-term stability of live probiotics formulated in a pressurized aerosol composition.

[0102] Materials and methods

[0103] Freeze-dried Lactobacillus plantarum ( L. Plantarum The bacterial strain (using maltodextrin, THT® - freeze-dried) was sieved to obtain a powder fraction with a particle size <50 micrometers to facilitate passage through valves and nozzles and prevent clogging. The bacteria were suspended in Mirasil CM5® (decamethylcyclopentasiloxane, a volatile cyclomethylsiloxane) and / or Tegosoft PBE® (propoxylated alcohol-ppg 14-butyl ether), both serving as suspension media. An organic clay component, namely bentonite gel vs5PC V® (cyclopentasiloxane, lithium distearate, lithium montmorillonite, and propylene carbonate), was used as an anti-settling and anti-caking agent.

[0104] Before filling the aerosol cans with propellant, a total of eight different concentrations of formulations (concentrates) were prepared (see Table 3).

[0105] Table 3. Composition of different formulations: Fill the tank with concentrate (20%), then add 80% propellant to dilute the concentrate 5 times.

[0106]

[0107] For sample 6, unlike all other formulation samples with a powder fraction of <50 micrometers, a sieve fraction of <30 micrometers was used.

[0108] Assess the bacterial viability of the canister, as well as the spray properties, residue, and settling properties of the suspended powder. Excessive settling can lead to clumping, resulting in product release failure or potentially frequent nozzle clogging. Fill the canister with 20% formulation and 80% propellant. The propellant used is a mixture of LPG (propane / butane / isobutane-29 / 69 / 2) and butane (55 / 45). It exists as a liquefied propellant at pressure (±2-4 bar).

[0109] Reference fractions (1 gram) of different formulations were kept in sealed aluminum bags at room temperature (T = 20°C) for CFU determination. CFUs obtained from aerosol containers also stored at room temperature (20°C) were compared with the reference fractions to evaluate the effectiveness of filling and storage. Bacterial load assays of the powders were performed directly before formulation preparation and container filling, yielding 5 and 9.0E+11 CFU / g bacterial powder as initial concentrations. Further sampling was conducted after 1, 2, 3, 4, 6, and 12 months of storage. Results were calculated in CFU / g bacterial powder.

[0110] The CFU of the formulation was determined by adding 1 gram of emulsifier mixture (70% Tween 80 / 30% Span 83) and 8 grams of physiological water, shaking until dissolved, and then performing a 10-fold serial dilution. Plates were prepared according to the "Copacabana method" (glass bead spreading), and CFU counts were performed after incubation at 37°C for 3 days. All materials and liquids used were sterilized by autoclaving.

[0111] The CFU of the aerosol can was determined by spraying 5 grams from the can and waiting (±1 hour) until all the propellant had evaporated. Therefore, 1 gram of the formulation remained in the container. Further sample preparation was performed as described above.

[0112] All samples were tested with 3 biological replicates (3 samples) and 3 technical replicates (plate counts), and the mean and bias were calculated using the following formula:

[0113]

[0114] An additional set of cans for each formulation was kept unopened at 20°C, emptied after 6 months (after vibration), and their spray properties and residues in the cans were evaluated after emptying.

[0115] Results and discussion

[0116] Survival curves of *Lactobacillus plantarum* in filled containers showed that storage stability decreased by an average of 1 log after 1 month. Figure 3 This indicates a slight decrease in viability immediately after or during can filling. There are several possible explanations for this phenomenon. Before filling, the propellant gas is maintained at its partial pressure (±4 bar). Under slight overpressure, the liquid is filled into the can, where it mixes with the formulation of this invention, thus subjecting the bacteria of this invention to pressure shock. Another explanation may be that during can use, by opening the valve nozzle, aerosol formation accompanied by high shear forces ejects bacterial powder from the can. A final explanation may be that the endothermic reaction accompanying propellant evaporation causes a sudden drop in temperature of the remaining formulation. All three stresses (pressure shock, shear force, and temperature shock) are fully described in the literature as detrimental to bacteria and affect viability.

[0117] On the other hand, viability in the filling tank remained interestingly constant over a period of 1 to 4 months post-filling, with a maximum decrease of 0.5–1 log. For stability over a period of 4 to 6 months, viability decreased by several logs for some formulations. The decrease in viability from start to finish (6 months) was: 1 log for F3, 2 logs for F4 and F5, 3 logs for F8, F1, and F7, and 5 logs for F6 and F2. These data suggest that stability can be guaranteed for 4 months for all pressurized formulations, and that appropriate formulations can be selected to ensure stability for 6 months or longer, depending on the required stabilization time. Based on these results ( Figure 4 It is clear that containing the formulation in a pressurized hydrocarbon propellant tank has a positive effect on the viability of *Lactobacillus plantarum* in the formulation. This indicates stabilization of the formulation after the inevitable initial decrease in viability during the first month of storage. The hypothesis is that the hydrocarbon propellant mixture creates an anhydrous and anaerobic environment, thereby supporting the survival of freeze-dried *Lactobacillus* species. Some studies in the literature describe increased survival of *Lactobacillus* bacteria when stored under inert gases such as nitrogen and carbon dioxide or under vacuum. However, there are no reports of survival of *Lactobacillus* bacteria in hydrocarbon gas / liquid media.

[0118] After storage, the viability of *Lactobacillus plantarum* was highest in formulation 3 (25% probiotics), and formulation 4 (20% probiotics) also showed good viability. Clearly, formulations with high bacterial concentrations (F3, F4) exhibited good stability, even after correction for the initial loading percentage. This may be attributed to the mutual shielding effect of the harsh conditions of the external medium. When evaluating the effect of bentonite gel in the pressurized tank (F5 vs. F1 and F2 – at gradually increasing concentrations of 0 – 5 – 25%), it showed a slight difference in logarithmic reduction between F5 (2.2 [1.9–2.9] log) and F1 (2.8 [2.6–3.1] log), and a larger difference compared to F2 (5.5 [5.3–5.9] log). Therefore, it can be concluded that formulations preferably contain no or only a small amount of antisettling agents, such as bentonite gel.

[0119] The viability of F1 (50 µm) was significantly higher (2 logarithms) than that of F6 (30 µm), indicating that smaller particle size is less conducive to survival. Smaller particle size results in a larger surface area, thus making the bacteria more susceptible to adverse effects from the environment (formulation). Therefore, the particle size of the formulation of the present invention is preferably greater than 30 micrometers.

[0120] The addition of propoxylated ppg-14-butyl ether (oils, volatile skin care products, and lubricants), such as Tegosoft® (F7 and F8), has no adverse effect on stability in filled cans. Figure 3 Therefore, it can be concluded that these components can be added without adverse effects, but can be used, for example, to improve aerosol formation and help to completely clear aerosols.

[0121] Figure 4 The data show a comparison of different formulations under pressure, either as is or in a filled container. These data clearly demonstrate that pressurizing the formulations significantly improves bacterial viability for all formulations over a 6-month period.

[0122] This experiment demonstrates that a freeze-dried bacterial formulation (suspended in volatile organosilicon and maintained under pressure using a hydrocarbon propellant) is a suitable delivery system for the long-term preservation of probiotics at room temperature. After the spray is expelled from the canister, the volatile components (propellant and organosilicon) evaporate, leaving the freeze-dried bacteria on the sprayed skin or surface. At this administration site, the formulation comes into contact with both the skin and the environment. The hygroscopic probiotic powder absorbs moisture from the surrounding environment, and the bacteria retain their metabolic activity, enabling them to perform their beneficial functions.

[0123] Example 3: Stability of probiotics in different organosilicones and organosilicon mixtures

[0124] The purpose of this embodiment is to obtain a screening platform in which the effects of different organosilicones and organosilicon mixtures on the viability of freeze-dried *Lactobacillus plantarum* are evaluated. Cyclic organosilicon molecules and linear organosilicon molecules are compared. Furthermore, chain lengths (#siloxanes) are compared. Viscosity), because smaller molecules may permeate more through cells, thus disrupting metabolic processes upon reactivation. Finally, the hypothesis that organosilicon mixtures may offset the adverse effects of different individual organosilicones was evaluated, unless the adverse effects are cumulative. These extensive and comparable stability studies enabled us to formulate more stable aerosol formulations for topical application containing lyophilized Lactobacillus species suspended in silicone oil.

[0125] For this experiment, freeze-dried Lactobacillus plantarum powder was suspended at 1 / 25 (m / m %) in different organosilicones or mixtures of organosilicones commonly used in antiperspirants (see Table 4).

[0126] Table 4. Different organosilicon compositions in which freeze-dried Lactobacillus plantarum is suspended at 1 / 25 (m / m %)

[0127]

[0128] A higher viscosity silicone, CF 6570, was used as a reference silicone because this mixture was previously used in the stability study of Example 1. One gram of each fraction was weighed in a falcon tube and stored in a laminated aluminum bag (Daklapack® – PET / ALU / NY / LDPE) protected from light, oxygen, and relative humidity at room temperature (20°C). The T0 of the powder was determined prior to suspension preparation, yielding (5.85 ± 0.71) E+11 CFU / g. Further sampling was performed at 1 week, 2 weeks, 1 month, 3 months, and 6 months. During sampling, three falcon tubes of the same sample were taken, and the plates were triplicated for counting as described in 2.1. The Blanc sample (reference) was stored as 100 mg of powder (unsuspended).

[0129] Results and discussion

[0130] from Figure 5 It is clear that for all formulations, high stability of the composition was achieved during a 3-month storage period (10). 9 (log CFU / g), some of the compositions scored slightly better than others, such as the reference powder, F11, and the lesser F10. The differences were too small to draw reliable conclusions about the effects of cyclic versus linear organosilicones and siloxane chain lengths; this may indicate that these aspects are not highly relevant to the usefulness of siloxanes in this invention.

[0131] Single organosilicon component shown Figure 5 On the left, more complex mixtures (3-5 components) are located on the right. Typically, Figure 5 The organosilicon mixture on the right showed better survival compared to the single organosilicon on the left, suggesting that the mixture of different components (partially) offset some of the adverse effects observed in the single-component organosilicon.

[0132] Tegosoft® (ppg-14 butyl ether) is a biodegradable and moderately volatile propylene glycol butyl ether commonly found in antiperspirants. Compared to cyclopentasiloxanes, it has no adverse effect on the stability of freeze-dried bacteria in pressurized aerosol cans. Figure 3-4 It appears that once mixed with other organosilicones, it can even protect the survival of freeze-dried bacteria. Figure 5 -F11 and F13). In addition to their good suspension and spray properties and low residue in the can, this suggests that the addition of polypropylene glycol ethers may be another valuable ingredient in the formulation of probiotic aerosols.

Claims

1. An aerosol spray comprising 1-20 wt% of one or more live dormant lactic acid bacteria, 1-20 wt% of one or more volatile siloxanes, 0.1-5 wt% of an antisettling agent and / or an anti-caking agent, and at least 50 wt% of a liquefied gas propellant under pressure, wherein the lactic acid bacteria and the volatile siloxanes form a mixture in the form of a powder or suspension, and wherein the combination of the one or more volatile siloxanes with the liquefied gas propellant under pressure maintains the survival of the one or more live dormant lactic acid bacteria.

2. The aerosol spray according to claim 1, wherein the one or more volatile siloxanes are linear siloxanes or cyclic siloxanes.

3. The aerosol spray according to claim 2, wherein the linear siloxane is selected from the group consisting of: dimethylsiloxane, polydimethylsiloxane alcohol, phenyltrimethylsiloxane and its derivatives, and the cyclic siloxane is selected from cyclomethylsiloxane and its derivatives.

4. The aerosol spray of claim 1, wherein the liquefied gas propellant comprises one or more hydrocarbons selected from the group consisting of methane, ethane, propane, butane, and pentane.

5. The aerosol spray according to claim 4, wherein the butane is n-butane and the pentane is n-pentane.

6. The aerosol spray according to claim 1, wherein the anti-settling agent and / or anti-caking agent is selected from the group consisting of: inorganic polymers, organic clay gels containing bentonite or lithium montmorillonite, and / or igneous silica.

7. The aerosol spray according to claim 6, wherein the ignited silica is aerosil.

8. The aerosol spray of claim 1, wherein the spray further comprises one or more excipients selected from the group consisting of: lubricants, suspending agents, wetting agents, antioxidants, humectants, emulsifiers, viscosity modifiers, or fragrances.

9. The aerosol spray of claim 1, wherein its contents consist of at least 50 wt% propellant, 1-20 wt% volatile siloxane, 1-20 wt% lactic acid bacteria, 0.1-5 wt% antisettling agent and / or anticaking agent and 0-5 wt% excipient of claim 8.

10. The aerosol spray according to claim 1, wherein its contents consist of at least 75 wt% propellant, 5-10 wt% cyclomethylsiloxane, 1-10 wt% lactic acid bacteria and 0.1-5 wt% bentonite gel.

11. The aerosol spray of claim 1, wherein its contents consist of at least 75 wt% propellant, 5-10 wt% cyclomethylsiloxane, 1-10 wt% lactic acid bacteria and 0.1-5 wt% aerosil.

12. The aerosol spray according to claim 1, wherein the spray is free of water and preservatives.

13. The aerosol spray according to any one of claims 1-12, for restoring and / or maintaining a healthy skin microbiome.

14. Use of the aerosol spray of any one of claims 1-12 in the preparation of a medicament for restoring and / or maintaining a healthy skin microbiome.

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