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 to the environment. [Selection diagram] None

Description

[Technical field]

[0001] The present invention relates to an aerosol spray containing one or more live bacterial species and one or more volatile siloxanes suitable for applying said one or more live bacterial species to a surface or to the environment. [Background technology]

[0002] Aerosol sprays are very useful for applying various agents to surfaces or to the environment and can be an interesting way to incorporate live bacterial species. However, aerosol sprays generally contain aqueous components, which are incompatible with the incorporation of live bacterial species. In particular, the presence of water usually prevents the growth of harmful pathogens and also requires the presence of preservatives to control the growth of microorganisms. Preservatives are detrimental to the survival of the co-incorporated microorganisms. Moreover, even a limited amount of co-incorporated water is sufficient to activate dormant bacteria, thereby significantly reducing the storage period of such compositions. Thus, typical aerosol sprays are not suitable for the incorporation of live bacterial species, and it was an object of the present invention to provide an aerosol composition that solves these problems of incorporation of live bacterial species and achieving their long-term storage.

[0003] In addition, it has been surprisingly discovered that aerosol sprays containing at least one volatile siloxane are highly useful for the above purposes.

[0004] Silicone fluids and siloxanes are particularly often used in sprays, including crop protection products, repellents, toiletries and other personal care products. The most frequently used silicone fluids are dimethicone, cyclomethicone and phenyltrimethicone.

[0005] These silicone fluids incorporated into products are very suitable carrier oils with a wide range of benefits, such as improving slip, reducing tack and providing skin softness.Despite the low solubility and viscosity of cyclomethicone, these silicone fluids have been considered to be particularly useful when formulated into aerosol sprays, since they tend to provide the above benefits without contributing to oiliness or greasiness.In addition, cyclomethicone is known to have a relatively high volatility and not excessively block antiperspirant active ingredients in deodorants.Finally, cyclomethicone is an excellent dispersant and diffusant, is generally colorless and transparent, has low odor, is volatile, and is resistant to chemical and oxidative degradation.

[0006] Despite the widespread use of these silicone fluids in aerosol sprays, no further research has been carried out to incorporate bacteria into them, since they are generally considered to have antibacterial effects against a wide range of (aerobic) bacteria. However, the present inventors have now surprisingly discovered that silicone fluids, especially volatile siloxanes such as cyclomethicone, can be used for long-term storage of (dormant lactic acid) bacteria in aerosol sprays. Such dormant bacteria are found to be unaffected by volatile siloxanes and therefore remain stable during storage. After being sprayed, the dormant bacteria become active due to contact with water or steam, by which time the volatile siloxanes incorporated at the same time have evaporated and no longer affect the survival of the activated bacteria. Summary of the Invention

[0007] In a first aspect, the present invention provides an aerosol spray comprising 1% to 20% by weight of one or more live probiotic bacterial strains, one or more volatile siloxanes, and a propellant under pressure.

[0008] In a particular embodiment, said one or more live bacterial species, more particularly live probiotic strains, are dormant bacterial species, more particularly dormant lactic acid bacteria.

[0009] In a particular embodiment of the invention, the one or more siloxanes are selected from the list comprising linear siloxanes such as dimethicone, dimethiconol, phenyltrimethicone and their derivatives, or cyclic siloxanes such as cyclomethicone and its derivatives, more particularly cyclic siloxanes such as cyclomethicone and its derivatives.

[0010] 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 said mixture is in the form of a powder, suspension or emulsion, and the aerosol spray of the present invention may further comprise a propellant.

[0011] Said propellant (under pressure) is in particular a liquefied gas, more particularly selected from a list comprising 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 gas and / or a soluble gas such as nitrogen, carbon dioxide, compressed air or nitrous oxide gas.

[0012] In yet a further embodiment, the aerosol spray of the invention may further comprise an anti-settling and / or anti-agglomerating agent, in particular selected from the list of inorganic polymers such as organoclay gels containing bentonite or hectorite, and / or fumed silicas such as aerosil.

[0013] In another specific embodiment, the aerosol sprays of the present invention may further comprise one or more emollients, suspending agents, humectants, antioxidants, humectants, emulsifiers, viscosity modifiers, fragrances, and / or other excipients.

[0014] In certain embodiments, the present invention provides an aerosol spray, the contents of which comprise or consist of at least 5% propellant, 1%-20% siloxane, 1%-20% lactic acid bacteria, and 0.1%-10% anti-settling and / or anti-agglomerating agents.

[0015] In yet a further embodiment, the present invention provides an aerosol spray, the contents of which comprise or consist of at least 75% propellant, 5%-10% cyclomethicone, 1%-10% lactic acid bacteria and 0.1%-5% bentone gel. Alternatively, the present invention provides an aerosol spray, the contents of which comprise or consist of at least 75% propellant, 5%-10% cyclomethicone, 1%-10% lactic acid bacteria and 0.1%-5% aerosil.

[0016] In certain embodiments, the aerosol spray according to the present invention is substantially free of water and preservatives.

[0017] In a further aspect, the present invention provides the use of an aerosol spray according to the invention for applying said one or more live bacterial species, in particular said live probiotic bacterial strains, to a surface or to the environment.

[0018] In a particular embodiment, the present invention provides an aerosol spray as defined herein for use in restoring and / or maintaining a healthy skin microflora. Thus, the present invention also provides the use of an aerosol spray according to the present invention for restoring and / or maintaining a healthy skin microflora.

[0019] With specific reference now to the drawings, it is emphasized that the items shown are exemplary and are intended solely for the purpose of an explanatory discussion of various embodiments of the invention. These drawings are presented to provide what is believed to be the most useful and simplest explanation of the principles and conceptual aspects of the invention. In this regard, no attempt has been made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. This description, taken together with the drawings, will make apparent to one skilled in the art how some forms of the invention may be practically embodied. [Brief description of the drawings]

[0020] [Figure 1] Figure 1 shows the stability of autologous (non-dried) Lac 4 and Lac 7 versus commercial freeze-dried strains of LGG suspended in silicone oil CF1406-OH (1 / 10 m / m%) over a one-year period. This figure clearly shows that freeze-drying the probiotic and suspending it in anhydrous silicone mixtures is an ideal method to prevent the rapid loss of viability of viable bacteria. Suspension of the freeze-dried bacteria in silicone mixtures containing dimethicone and dimethiconol provides long-term storage of this Lactobacillus species. [Diagram 2] Figure 1 shows the stability of autologous (non-dried) Lac 4 and Lac 7 versus commercial freeze-dried strains of LGG suspended in silicone oil CF6570-DM (1 / 10 m / m%) over a one-year period. This figure clearly shows that freeze-drying the probiotic and suspending it in anhydrous silicone mixtures is an ideal method to prevent the rapid loss of viability of live bacteria. Suspension of the freeze-dried bacteria in silicone mixtures containing dimethicone, disiloxane and cyclopentasiloxane provides long-term storage of this Lactobacillus species. [Diagram 3]Figure 1. Survival curves of freeze-dried L. plantarum suspended in different formulations (F1-F8) stored in pressurized canisters with hydrocarbon propellants at room temperature (20°C). After a small initial decrease in viability during the first month, viability remained rather constant over the following months, proving that storage in pressurized canisters is a suitable method for long-term survival of probiotics. [Figure 4] Figure 6-month stability of freeze-dried L. plantarum in various formulations versus the same formulations stored in pressurized canisters. After 6 months of storage, a statistically significant difference in survival is seen for the formulations stored in pressurized canisters with propellant compared to the formulations stored without propellant. Survival was better for all eight tested formulations, proving that suspending freeze-dried bacteria in silicone oil and storing with propellant is a better method for long-term stability than simply suspending freeze-dried bacteria in silicone oil. [Diagram 5] Figure 3 shows the stability of freeze-dried LAC_15 in various silicones and silicone mixtures after 3 months.Slight differences were observed, but most were judged to be insignificant.It seems that the mixture of various silicone oils tends to provide better stability than one or two oil components.This may show that the lower concentration of various silicone oils offsets the negative effect of higher single concentration of silicone component. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] In a first aspect, the present invention provides an aerosol spray comprising 1% to 20% by weight of one or more live probiotic bacterial strains, one or more volatile siloxanes, and a propellant under pressure.

[0022] More generally, 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, said mixture being in the form of a powder, suspension or emulsion. In a particular embodiment, said one or more live bacterial species are dormant bacterial species, more particularly dormant lactic acid bacteria.

[0023] In the present invention, the term "aerosol spray" is meant to be a type of dispensing system that creates an aerosol mist. It is used with a can or bottle that contains the mixture of the present invention and a propellant, preferably under pressure. When the valve of the container is opened, the mixture is forced out of the canister through the valve and exits as an aerosol or mist. A small amount of propellant expands to expel the mixture, while a large amount of propellant remains inside the can, maintaining a constant pressure. Outside the can, the propellant evaporates quickly.

[0024] In the present invention, the term "mixture" refers to a system composed of two or more different substances that are mixed but not chemically associated, such as a combination of a volatile siloxane and a dormant bacterial species. In such a mixture, the individual identities of the components are preserved and they are mixed in the form of a solution, suspension, emulsion, colloid or powder.

[0025] For the purposes of this invention, the term "powder" is a dry bulk solid composed of many fine particles that can flow freely when shaken or tilted. Powders are a special subclass of granular materials, although the terms powder and granules are sometimes used to distinguish between separate classes of materials.

[0026] In the present invention, the term "dormant" refers to a state of live / viable bacteria where growth, development, reproduction, etc. are temporarily stopped. Live / viable bacteria are metabolically dormant in which metabolic processes are decelerated to such an extent that they no longer produce equivalent amounts of metabolic products. Many bacterial species can survive adverse conditions, such as low temperatures, by forming a state of reduced metabolic activity lacking (endophytic) spores, cysts, conidia, or specialized cell structures. Dormant bacteria can be reactivated when subjected to "normal" conditions, for example after they come into contact with water or after they are subjected to certain temperatures or vibrations. In the present invention, bacteria can be made dormant using techniques known in the art, such as freeze-drying, spray-drying, vacuum drying, fluidized bed drying, nitrogen cryo-drying, etc. The bacteria of the present invention are preferably freeze-dried.

[0027] In the present invention, the term "volatile" is used in relation to the disclosed siloxanes to indicate that they have a tendency to vaporize. Volatility is directly related to the vapor pressure of a substance. At a given temperature, a substance with a higher vapor pressure will vaporize more quickly than a substance with a lower vapor pressure. Otherwise, a substance with a lower heat of vaporization (kJ / kg) will evaporate more rapidly at a given temperature than one with a higher heat of vaporization. Although the term is written primarily as applied to liquids, the term may also be used to describe the process of sublimation associated with solid substances, such as dry ice (solid carbon dioxide), which can change directly from a solid state to a vapor without becoming a liquid. The vapor pressure of a substance is the pressure at which its gas phase is in equilibrium with its condensed phase (liquid or solid). The vapor pressure of a substance is a measure of the tendency of molecules and atoms to leave a liquid or solid. The higher the vapor pressure of a liquid at a given temperature, the more volatile it is and the lower the normal boiling point of the liquid. Preferably, the vapor pressure (heat of vaporization) of the volatile siloxanes of the present invention is less than 2500 kJ / kg at room temperature (25° C.), more preferably less than 1000 kJ / kg, and even more preferably less than 900 kJ / kg, 800 kJ / kg, 700 kJ / kg, 600 kJ / kg, 500 kJ / kg, 400 kJ / kg, 300 kJ / kg, or 200 kJ / kg.

[0028] Cyclomethicone is a group of methylsiloxanes, a type of liquid silicone (cyclic polydimethylsiloxane polymer) with the characteristics of low viscosity and high volatility, which makes it an emollient and a useful cleaning solvent in certain situations.Unlike dimethicone, a linear siloxane that does not evaporate quickly, cyclomethicone is cyclic, and both groups are made of polymers with a monomer skeleton with one silicon and two oxygen atoms bonded together, but instead of having a very long "linear" skeleton surrounded by a series of methyl groups (which creates a clear non-reactive non-volatile liquid that ranges from watery to viscous), cyclomethicone has a short skeleton that creates a closed or nearly closed ring or "cycle" in addition to those methyl groups, giving them many of the same properties of dimethicone, but making them more volatile.As a result, depending on the desired degree of evaporation, a more volatile siloxane such as cyclomethicone, or a less volatile siloxane such as dimethicone, can be selected. However, more volatile siloxanes such as cyclomethicone and its derivatives are preferred.

[0029] Silicones are commonly used in the personal care industry (and many other industries) to provide a smooth, velvety, nongreasy, and nontacky feel when in contact with the skin (Handbook of Cosmetic Science and Technology Fourth Edition 2014; New York CRC Press; 204 AD. Pg. 321-329). Silicones have low surface energy, are moisture and temperature resistant, biocompatible, volatile and permeable, nontoxic and nonirritating. Silicones are polymers derived from silicon (Si), carbon, oxygen, and hydrogen, and can be linear, cyclic, or organofunctional. The most common silicones (dimethicones) have an inorganic Si-oxide backbone with organic (2x methyl) pendant groups on the Si. Silicone polymers have monomers ranging from 2 to several thousand, with viscosities that can range from 0.65 cSt (hexamethyldisiloxane) to +1 million cSt cps. High viscosity fluids can form protective films, whereas low viscosity fluids can be emulsified and have good solubility with other ingredients. The organic groups can be modified to provide surface active properties. The Si-o bonds are very free to move (Si-o has a rotational energy of 0.2 kcal / mol, cc has a rotational energy of 3.3 kcal / mol), have strong bond energies (Si-o has a bond strength of 117 kcal / mol, cc has a bond strength of 85 kcal / mol), are flat, and are long (Si-o angle is 130° and length is 0.163 nm, cc bond has an angle of 112° and length is 0.154 nm). All of this creates a very flexible and strong backbone. Polydimethylsiloxane (PDMS) also has a very low surface tension of 20 mM / m compared to ethanol (50 mM / m) and water (72 mM / m), resulting in complete surface coverage and excellent agent spreading, both of which are useful properties in aerosol formulations.Low molecular weight silicones are highly volatile, leave no residue, and provide a light skin feel. Due to their low heat of evaporation (per gram), low molecular weight silicones do not require much heat to evaporate from the skin, and as a result, do not feel as cold as water or ethanol based products (see Table 1).

[0030] Table 1. Heat of vaporization for some volatile fluids used in cosmetics. [Table 1]

[0031] PDMS polymer is highly permeable to gases, with permeability being related to the solubility and diffusion coefficient (see Table 2).

[0032] Table 2. Permeation data for some volatile fluids used in cosmetics. [Table 2]

[0033] This high gas diffusion rate allows the skin to "breathe" and is described as non-occlusive. Cyclic silicones (cyclomethicone, D4-D5-D6) are very good solvents, non-staining, colorless, odorless, and highly volatile. Cyclic silicones are most commonly used in antiperspirant products as delivery vehicles for active ingredients. Cyclic silicones do not feel cold on the skin due to their low heat of vaporization, have a pleasant skin feel, and are the optimal suspending medium for pressurized aerosol canisters. Cyclic silicones can also prevent the formation of agglomerates at the valve due to the lubricating properties of silicone. Other suspending media commonly used in antiperspirants are ethanol, water, and mineral oil.

[0034] In the present invention, the term "siloxane" means a molecule containing a functional group having a Si-O-Si bond. The parent siloxane has the formula H(OSiH2) n OH and (OSiH2) n Siloxanes include oligomers and polymer hybrids having the formula: ##STR1## Siloxanes also include branched compounds, the defining feature of which is that each pair of silicon centers is separated by one oxygen atom. Siloxane functional groups form the backbone of silicone. Suitable volatile siloxanes within the present invention can be selected from a list including linear siloxanes such as dimethicone, dimethiconol, phenyltrimethicone and their derivatives, or cyclic siloxanes such as cyclomethicone and its derivatives, more particularly cyclic siloxanes such as cyclomethicone and its derivatives.

[0035] The inventors have discovered that by pressurizing the aerosol can, improved stability is achieved over non-pressurized compositions. Thus, in another particular embodiment, the aerosol spray of the present invention may further comprise a propellant.

[0036] In the present invention, the term "propellant" means a pressurized gas used to create the movement of a fluid or powder. In particular, a propellant is simply a liquid (or gas) that can be easily vaporized. Cold gas propellants can be used to fill pressurized dispensing systems such as aerosol sprays and push the material through a nozzle. In an aerosol spray can, the propellant is usually a pressurized gas that is in equilibrium (at its saturated vapor pressure) with the liquid or powder. As some gas is released and the load is discharged, more liquid evaporates to maintain further pressure. Suitable propellants useful in the present invention may be selected from a list including liquefied gases, more specifically 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 gas and / or a soluble gas such as nitrogen, carbon dioxide, compressed air or nitrous oxide gas. Other liquefied gases that may be used in the present invention are DME (dimethyl ether) and HFCs (hydrofluorocarbons) such as 1,1,1,2-tetrafluoroethane and 1,1-difluoroethane.

[0037] In yet a further embodiment, the aerosol spray of the present invention further comprises an anti-settling agent and / or an anti-agglomerating agent.

[0038] In the present invention, the term "anti-settling agent" means a substance that prevents particles in suspension from settling out of the fluid and becoming trapped and remaining on a barrier such as the bottom of an aerosol can. A good anti-settling agent has the properties of forming a porous, bulky, easily resuspendable suspension required for homogeneous dosing. In the present invention, the term "anti-agglomerating agent" means a substance that prevents caking or clumping of the components. Suitable anti-settling agents and / or anti-agglomerating agents within the present invention are inorganic polymers such as organoclay gels containing bentonite or hectorite, and / or aerosils. (商標)and fumed silica such as Aerosil. (商標) can be chemically modified to obtain a better 3D structure network in the selected siloxane carrier. Aerosil is usually a hydrophilic fumed silica (SiO2). Hydrophobic silicas, usually silicas post-treated with hydrophobic materials such as, but not limited to, dimethyldichlorosilane, polydimethylsiloxane, organosilane, hexadecylsilane, octamethylcyclotetrasiloxane, methacrylsilane, silicone oil, aminosilane and their derivatives, are particularly suitable for the present invention.

[0039] In another specific embodiment, the aerosol spray of the present invention may contain any additional material that may be useful in the present invention. Such additional material may include, but is not limited to, one or more emollients, moisturizers, antioxidants, humectants, suspending agents, emulsifiers, viscosity modifiers, polarity modifiers, fragrances, electrolytes, color modifiers, and other excipients. Suitable antioxidants may include, but are not limited to, BHT, BHA, propyl gallate, ascorbic acid, and sodium metabisulfite. Suitable viscosity modifiers may include, but are not limited to, various colloids, silicone resins, propylene glycol, organogel, aerosil, and the like.

[0040] Suitable further components in the aerosol spray of the invention are, for example, Tegosoft (商標) , i.e. polypropylene glycol butyl ether or ppg-14-butyl ether. (商標) Tegosoft is a lightweight cosmetic oil with very good spreading properties that is often used as an emollient in skin care products such as creams, lotions, and bath oils. (商標)It also has excellent suspending properties for active ingredients in aerosol formulations. PPG-butyl ether components in cosmetic formulations improve the appearance of dry or damaged skin by reducing flaking and restoring softness.

[0041] In the present invention, when percentages are mentioned, they are meant to be wt / wt percentages of the total composition, for example, 80% propellant means that 80 g of propellant is present in the total weight of 100 g of the composition.

[0042] In a particular embodiment, the present invention provides an aerosol spray whose content comprises or consists of at least 10% propellant, 10%-40% siloxane, 10%-40% lactic acid bacteria, 0%-5% anti-settling and / or anti-agglomerating agents, and 0%-5% other additives.

[0043] In a particular embodiment, the present invention provides an aerosol spray whose content comprises or consists of at least 50% propellant, 1%-20% siloxane, 1%-20% lactic acid bacteria, 0.1%-5% anti-settling and / or anti-agglomerating agents, and 0%-5% other additives.

[0044] Preferably, the aerosol spray of the present invention contains 1% to 20% lactic acid bacteria, more particularly 1% to 10% lactic acid bacteria, and even more particularly about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% lactic acid bacteria.

[0045] Furthermore, the aerosol sprays of the present invention preferably contain small amounts of anti-settling agents, such as less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or even possibly no anti-settling and / or anti-agglomerating agents.

[0046] In a particular embodiment, the present invention provides an aerosol spray whose content comprises or consists of at least 75% propellant, 5%-10% siloxane, 1%-10% lactic acid bacteria, and 0%-5% anti-settling and / or anti-agglomerating agent.

[0047] In another specific embodiment, the present invention provides an aerosol spray whose content comprises or consists of at least 80% propellant, 1%-8% siloxane, 1%-8% lactic acid bacteria, and 0%-4% anti-settling and / or anti-agglomerating agent.

[0048] In another specific embodiment, the present invention provides an aerosol spray whose content comprises or consists of at least 90% propellant, 1%-4% siloxane, 1%-4% lactic acid bacteria, and 0%-2% anti-settling and / or anti-agglomerating agent.

[0049] In certain embodiments of the composition as defined herein above, the anti-settling and / or anti-flocculating agent is bentonite gel and aerosil (商標) is selected from.

[0050] In a further embodiment, the present invention provides an aerosol spray whose contents comprise or consist of at least 75% propellant, 5%-10% cyclomethicone, 1%-10% lactic acid bacteria, and 0%-5% bentone gel. Alternatively, the present invention provides an aerosol spray whose contents comprise or consist of at least 75% propellant, 5%-10% cyclomethicone, 1%-10% lactic acid bacteria, and 0%-5% aerosil.

[0051] Obviously, in the formulation of dormant bacteria, harmful products such as preservatives and activating substances such as water are preferably avoided to ensure long-term storage of the composition. Thus, in certain embodiments, the aerosol spray according to 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%. When using a multi-compartment aerosol spray, it is preferred that the compartment containing the bacteria is substantially free of water and preservatives.

[0052] In a further aspect, the present invention provides the use of the aerosol spray according to the present invention for applying one or more live bacterial species to a surface or to the environment. In the present invention, the term "surface" should be interpreted in its broadest sense, i.e., a surface may include the surface of an object, but also the skin of a human or animal. Thus, the aerosol spray of the present invention may also be a topical aerosol spray, where the term "topical" means local delivery at a designated location of the body, in particular application to a specific place on or in the body. In particular, topical delivery includes application to mucous membranes by aerosol.

[0053] In a particular embodiment of the invention, the invention provides an aerosol spray as defined herein for use in restoring and / or maintaining a healthy skin microflora.The invention therefore also provides the use of an aerosol spray according to the invention for restoring and / or maintaining a healthy skin microflora. EXAMPLES

[0054] Overview of the Examples Athlete's foot and other fungal skin infections are currently treated with antifungal drugs such as miconazole, which interfere with ergosterol synthesis. A well-known commercial product containing miconazole is Daktarin (商標)It is a spray and is preferred due to its ease of use. Both antibiotics and antifungals are experiencing an increasing trend of resistance. As a result, alternative treatments such as the use of probiotics for topical diseases are beneficial for patients. Most probiotics exert beneficial effects on the host (human) by preventing the invasion and colonization of pathogens. Some probiotics, such as Lactobacillus species, are able to combat these pathogens by excreting organic acids, hydrogen peroxide and antimicrobial peptides into the environment. Common skin fungal infections such as tinea pedis are caused by various species of Trichophyton, Epidermophyton, Microsporum and Candida. This study investigates whether aerosols (pressurized canisters) are a suitable formulation for the storage and long-term stability of probiotics.

[0055] As has been demonstrated, probiotics, when properly dried, have a fairly good long-term stability in contrast to non-dried bacteria (Morgan et al., 2006 - Preservation of micro-organisms by drying; a review -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 mainly determines its stability, as well as the storage conditions (temperature, oxygen, light).

[0056] Example 1: Stability of probiotic bacteria in silicone In this example, we investigated the long-term stability of live probiotic bacteria in various silicone / siloxane mixtures.

[0057] Materials and Methods Stability studies Three strains were used in this stability study: Autologous L. pentosus (i.e., lac4 or Lp) Autologous cultured LGG (i.e., lac7 or LGG) Freeze-dried LGG (Dupon (商標) LR-32)

[0058] Two different silicone-based suspending media for preserving bacteria were tested at room temperature (+ / - 20°C): silicone: Cosmetic Fluid 1406-OH (Dimethiconol and Dimethicone) - Chemsil (商標) Cosmetic Fluid 6570-DM (Disiloxane, Cyclopentasiloxane and Dimethicone) - Chemsil (商標)

[0059] Samples were obtained at the indicated time points: T0 = ​​before suspension in suspending medium T1 = 1 day after suspension T2 = 1 week after suspension T3 = 2 weeks after suspension T4 = 1 month after suspension T5 = 2 months after suspension T6 = 6 months after suspension T7 = 12 months after suspension

[0060] All materials used were sterilized prior to use by autoclaving. All components, ingredients, etc. used were autoclaved if possible, or filtered under sterile conditions before suspending the bacteria.

[0061] T0 CFU determination Lac 4 and Lac 7 were cultured in liquid MRS (de MAN, Rogosa & Sharpe-Carl Roth) medium (37°C) until complete growth. The medium was centrifuged at 2780 G for 10 min. The supernatant was discarded and the bacterial pellet was used further.

[0062] 100 mg was weighed and diluted with saline (0.85% NaCl) to obtain a total volume of 10 ml. From the three strains (lac4, lac7 and LGG), serial dilutions were made (10-fold serial dilutions) and plate counts were performed according to the smear plate method (based on pharmacopoeial methods 2.6.12 and 2.6.13 (European Pharmacopoeia 8.0)). Measurements were repeated in triplicate. Results are expressed as cfu / gram of powder.

[0063] Suspension of bacteria in a suspending medium The resulting bacterial mass was used to make homogenous 1 / 10 dilutions (m / m%) with various anhydrous silicone-based suspension media. One gram of the above suspension was weighed into a Falcon tube and sealed in an aluminum bag (RH=20%). Thus, each sample contained 100 mg of bacteria for further experimental purposes. The aluminum bag was opened at certain time intervals for viability experiments.

[0064] Experiments after storage period The sealed pouches were opened at certain time points for stability studies. Falcon tubes containing 1 gram of anhydrous material were further treated by adding 1 gram of emulsifier mixture (consisting of polysorbate 80 and sorbitan sesquioleate) and 8 grams of saline. This formed an emulsion and allowed the bacteria to come into contact with water and become reactivated. Further sampling and plate counts were performed as described above and based on Pharmacopoeia (8.0) methods 2.6.12: Microbiological examination of non-sterile products: microbial enumeration tests and 2.6.13: microbiological examination of non-sterile products: test for specified micro-organisms.

[0065] Results and Discussion The stability of the freeze-dried powders was, as expected, much better compared to the stability of the non-freeze-dried strains. After only one month, an average of 3 log reduction was observed, regardless of the type of silicone mixture used (Figure 1: CF1406, Figure 2: CF6570), whereas no significant reduction was detectable for the freeze-dried strains (Figures 1 and 2). Thereby, it is proven that it is important to start the formulation with a properly freeze-dried (or other drying method) powder to guarantee a stable formulation of live microorganisms over time. Stable dry probiotic powders are easy to handle for further formulation purposes.

[0066] Stability results for the silicone mixtures (Figures 1 and 2) show that they are suitable as suspending media. After one year of stability for the anhydrous silicone vehicles, an average of one log reduction is seen, leaving enough viable microorganisms to exert a beneficial effect on the skin when applied topically.

[0067] This proves that in conjunction with properly freeze-dried powders suspended in anhydrous silicone medium it is possible to obtain a stable probiotic-bearing suspension.

[0068] Example 2: Stability of probiotic bacteria in pressurized aerosol formulations In this example, we investigated the long-term stability of live probiotic bacteria formulated in a pressurized aerosol composition.

[0069] Materials and Methods Lyophilized strain of L. plantarum (THT dried using maltodextrin) (商標) The powder fraction was sieved to obtain a powder fraction with a particle size of less than 50 microns to facilitate passage through the valves and nozzles and prevent clogging. The bacteria were then sieved with Mirasil CM5, a volatile cyclomethicone, which both acted as a suspending medium. (商標) (Decamethylcyclopentasiloxane) and / or Tegosoft PBE (商標)(propoxylated alcohol-ppg 14 butyl ether). Organoclay components Bentone gel vs5 PC V (商標) (Cyclopentasiloxane, disteardimonium hectorite and propylene carbonate) were used as anti-settling and anti-flocculating agents.

[0070] A total of eight formulations (concentrates) were made at various concentrations (see Table 3) before loading the propellants into an aerosol canister.

[0071] Table 3. Composition of various formulations: The concentrate is filled into a canister (20%), then 80% of the propellant is added to make a 5-fold dilution of the concentrate. [Table 3] * A sieve fraction of less than 30 microns was used for Sample 6, in contrast to all other formulation samples which had a powder fraction of less than 50 microns.

[0072] The canisters were evaluated for bacterial viability and for the spray properties of the suspended powder as well as for the residue and sedimentation properties. Too much sedimentation can result in caking and therefore failure to release the product or frequent clogging of the nozzle. The canisters are filled with 20% formulation and 80% propellant. The propellant used is a mixture of LPG (propane / butane / isobutane-29 / 69 / 2) and butane (55 / 45). Under pressure (± 2 bar to 4 bar), the propellant used is present as a liquefied propellant.

[0073] Reference fractions (1 gram) of the different formulations were kept in sealed aluminum bags at room temperature (T=20° C.) for CFU determination. The CFU obtained from aerosol canisters, also stored at room temperature (20° C.), was compared with the reference to evaluate the impact of filling and storage. Just before formulation preparation and filling of the canisters, a bacterial load determination of the powder was performed, which gave a starting concentration of 5.90 CFU / g of bacterial powder. Further sampling was performed after 1, 2, 3, 4, 6 and 12 months of storage. The results were calculated as CFU / g of bacterial powder.

[0074] CFU determination of the formulations was performed by adding 1 gram of emulsifier mixture (Tween 80 70% / Span 83 30%) and 8 grams of saline, shaking until dissolved, and performing 10-fold serial dilutions. Plating was performed according to the "Copacabana method" (smear plating with glass beads) followed by CFU counting after 3 days of incubation at 37°C. All materials and liquids used were sterilized by autoclaving.

[0075] CFU determinations of the aerosol canisters were performed by aerosolizing 5 grams from the canister and waiting until all propellant had evaporated (± 1 hour). As a result, 1 gram of the formulation remained in the recipient. Further sample preparation was performed as described above.

[0076] All samples were tested with three biological replicates (three samples) and three technical replicates (plate count) values, and then the mean and deviation were calculated using the following formula: CFU / g powder = (CFU x dilution count / g on plate (ml)) x ((g powder in sample + g dilution medium) / g powder)

[0077] A reserve set of canisters of each formulation were kept unopened at 20° C. and emptied (after shaking) after 6 months and evaluated for their spray properties and residue in the canisters after emptying.

[0078] Results and Discussion The survival curves of L. plantarum in filled canisters show an average 1-log decrease after one month of storage stability (Figure 3). This indicates a slight decrease in viability immediately after or during filling of the canisters. There are several possible explanations for this phenomenon. The propellant gas is kept under its partial pressure (± 4 bar) before filling. The liquid is filled into the canister where it is mixed with the formulation of the invention under a slight overpressure, and thus the bacteria of the invention are subjected to a pressure shock. Another explanation could be that during use of the canister, by opening the valve nozzle, the aerosol formation is combined with high shear forces, causing the bacterial powder to be released from the canister. A final explanation could be that the endothermic reaction combined with the evaporation of the propellant causes a rapid temperature drop of the remaining formulation. All three stresses (pressure shock, shear forces and temperature shock) are well documented in the literature to be harmful to bacteria and affect their viability.

[0079] On the other hand, in the 1-4 month time frame after filling, viability in the filled canisters remains somewhat interestingly constant, with a maximum decrease in viability of 0.5 Log to 1 Log. In the 4-6 month time frame of stability, viability decreased by several more logs for some formulations. The viability decrease from start to finish (6 months) was a 1-log reduction for F3, a 2-log reduction for F4 and F5, a 3-log reduction for F8, F1 and F7, and a 5-log reduction for F6 and F2 canisters. These data indicate that 4 months of stability can be guaranteed for all pressurized formulations, and that appropriate formulations can be selected to guarantee stability over 6 months, depending on the desired time of stability. It is clear from these results (Figure 4) that placing the formulations in a hydrocarbon propellant pressurized canister has a positive impact on the viability of L. plantarum in the formulations. This indicates stabilization of the formulation after the inevitable initial loss of viability during the first month of storage. The hypothesis is that the hydrocarbon propellant mixture creates a water- and oxygen-free environment to support the survival of freeze-dried Lactobacillus species. Several studies in the literature have described improved viability for Lactobacillus bacteria when stored under inert gases such as nitrogen and carbon dioxide, or when stored under vacuum. However, no reports of survival under hydrocarbon gases / liquids in siloxane-based media have been published to date.

[0080] The viability of L. plantarum after storage was highest in formulation 3 (25% probiotics), and furthermore formulation 4 (20% probiotics) also showed good viability. Apparently, formulations with high concentrations of bacteria (F3, F4) have good stability, even when corrected for the percentage of initial load. This may be due to a mutual shielding effect against the harsh conditions of the external medium. Evaluating the impact of Benton gel (F5 vs. F1 and F2, with increasing concentrations of 0%-5%-25%) in pressurized canisters showed a small difference in log reduction between F5 (2.2 [1.9-2.9] LOG) and F1 (2.8 [2.6-3.1] log), and a larger difference for F2 (5.5 [5.3-5.9] log). It can therefore be concluded that formulations that do not contain or only small amounts of anti-settling agents such as Benton gel are preferred.

[0081] The viability in F1 (50 μm) is significantly (2-log) higher than F6 (30 μm), indicating that smaller particle size is worse for survival. Smaller particle size results in a larger surface area and therefore more negative influences from the environment (formulation) can harm the bacteria. Therefore, it is preferred that the formulation of the present invention has a particle size larger than 30 microns.

[0082] Tegosoft (商標) The addition of propoxylated ppg-14-butyl ether (an oil-like, volatile skin care agent and emollient) such as (F7 and F8) did not have a negative impact on stability in filled canisters (Figure 3). It can therefore be concluded that such components can be added, for example, to improve aerosol formulations and promote complete evacuation of the aerosol without adverse effects.

[0083] Figure 4 shows a comparison of the various formulations under pressure, either on their own or in filled canisters. These data clearly show that pressurizing the formulations significantly increases bacterial viability for all formulations over a six month period.

[0084] In this experiment, a formulation of freeze-dried bacteria suspended in a volatile silicone held under pressure using a hydrocarbon propellant was proven to be a suitable delivery system for long-term storage of probiotics at room temperature. After ejection of the canister's squirt, the volatile components (propellant and silicone) evaporate, leaving the freeze-dried bacteria on the skin or surface where it was sprayed. At this site of administration, the formulation comes into contact with the skin and the environment. The hygroscopic probiotic powder absorbs water from the surroundings, allowing the bacteria to become metabolically active and perform their beneficial functions.

[0085] Example 3: Stability of probiotic bacteria in various silicones and silicone blends The goal of this example was to obtain a screening platform in which the effects of different silicones and silicone mixtures were evaluated on the viability of lyophilized L. plantarum. Comparisons were made on cyclic silicone molecules versus linear silicone molecules. Chain length (# siloxane molecules ~ viscosity) was also compared, since smaller molecules may penetrate the cell more and interfere with metabolic processes once active again. Finally, the hypothesis was evaluated that a mixture of silicones may offset the negative effects of the various individual silicones, as long as the negative effects are not cumulative. These extensive comparative stability studies will enable us to formulate more stable aerosol formulations containing lyophilized Lactobacillus species suspended in silicone oil for topical use.

[0086] For this experiment, freeze-dried L. plantarum powder was suspended at 1 / 25 (m / m%) in various silicones or silicone mixtures commonly used in antiperspirants (see Table 4).

[0087] Table 4. Various silicone compositions in which freeze-dried L. plantarum is suspended at 1 / 25 (m / m%) [Table 4] Dimethicone 1 cSt Dimethicone 5 cSt Mirasil(TM) cm-5 (Decamethylcyclopentasiloxane) Dow Corning™ 345 (Cyclopentasiloxane, Cyclohexasiloxane 70 / 30-6 cSt) Hexamethyldisiloxane (0.65 cSt) CF 6570-DM (Disiloxane, Cyclopentasiloxane and Dimethicone - 300 cSt) Dimethicone 5 cSt-Mirasil(TM) Cm-5 (50 / 50) Dimethicone 5 cP-DC 345 (50 / 50) Dimethicone 5 cP-Hexamethyldisiloxane (50 / 50) DC(TM) 345-Hexamethyldisiloxane (50 / 50) Dimethicone 5 cSt-DC345-PPG-14-Butyl Ether (40 / 40 / 20) Dimethicone 5 cSt-DC345-Hexamethyldisiloxane (40 / 40 / 20) Dimethicone 1 cSt - Dimethicone 5 cSt - Mirasil™ cm5 - DC™ 345 - Hexamethyldisiloxane, 14 ppg Butyl Ether (all at 16.7%)

[0088] The higher viscosity silicone CF 6570 was used as the reference silicone since this mixture was previously used in the stability study of Example 1. 1 gram of the various fractions was weighed into Falcon tubes and stored in laminated aluminum pouches (Dakplapack™-PET / ALU / NY / LDPE) and stored at room temperature (20° C.) protected from light, oxygen and relative humidity. After carrying out the TO determination of the powder, a suspension was made and (5.85±0.71)E+11 CFU / gram was obtained. Further sampling was carried out after 1 week, 2 weeks, 1 month, 3 months and 6 months. During sampling, 3 Falcon tubes of the same sample were taken and the plates were counted in triplicate as described in 2.1. A blank sample (reference) was stored as 100 mg of powder (not suspended).

[0089] Results and Discussion For all formulations, the composition showed high stability (10 9 It is clear from Figure 5 that the log CFU / g) achieved over the 3 month storage period, with some scoring slightly better than others such as reference powders F11 and, to a lesser extent, F10. The differences are too small to make any firm conclusions regarding the effect of cyclic versus linear silicones and the chain length of the siloxane, which may indicate that these properties are not very relevant for a siloxane to be useful in the present invention.

[0090] Single silicone components are shown on the left side of Figure 5, and more complex mixtures (3-5 components) are shown on the right side. In general, the silicone mixtures on the right side of Figure 5 show better survival compared to the single silicones on the left side, indicating that mixtures of various components (partially) offset some of the negative effects observed with single component silicones.

[0091] Tegosoft (商標) (ppg-14 butyl ether) is a biodegradable, moderately volatile propylene glycol n-butyl ether commonly found in antiperspirants.(商標) Tegosoft has no negative effect on the stability of freeze-dried bacteria in pressurized aerosol canisters compared to cyclopentasiloxane (Figures 3 and 4). (商標) It further appears that when mixed with other silicones, it may have a protective effect on the survival of freeze-dried bacteria (Fig. 5-F11 and F13). This indicates that in addition to its good suspension and spray properties and low residue in the canister, the addition of polypropylene glycol ether may be a beneficial further ingredient when formulating probiotic aerosols. [Explanation of symbols]

[0092] Drawing translation Figure 1 Stability of suspended bacteria at 20 ℃ in silicon oil CF 1406-OH CFU / g powder CFU / 1g powder Time(days) Figure 2 Stability of suspended bacteria at 20 ℃ in silicon oil CF 6570-DM CFU / g powder CFU / 1g powder Time(days) Figure 3 CFU / g powder in pressurized canister CFU / g powder in pressurized canister Time(months) Figure 4 Stability of the different formulations versus the pressurized canister 6 month at 20 ℃ CFU / g powder CFU / 1g powder Formulation Canister Figure 5 Viability of Lac 15 after 3 months suspension Log CFU / gram Lac15 powder LogCFU / Lac15 powder 1g Reference powder Different silicones and silicone mixtures

Claims

1. An aerosol spray comprising 1% to 20% by weight of one or more freeze-dried live dormant strains of lactic acid bacteria, one or more volatile siloxanes, and a liquefied gas propellant under pressure, wherein said strains of lactic acid bacteria and said volatile siloxanes form a mixture in the form of a suspension.

2. 2. The aerosol spray of claim 1, wherein the one or more live dormant strains of lactic acid bacteria are L. plantarum.

3. 3. The aerosol spray according to claim 1 or 2, wherein the one or more volatile siloxanes are selected from the list comprising linear siloxanes, represented by dimethicone, dimethiconol, phenyltrimethicone and their derivatives, or cyclic siloxanes, represented by cyclomethicone and its derivatives, or cyclic siloxanes, represented by cyclomethicone and its derivatives.

4. The aerosol spray according to any one of claims 1 to 3, wherein the liquefied gas propellant under pressure is one or more hydrocarbons represented by methane, ethane, propane, isopropane, butane, n-butane, pentane and n-pentane.

5. 5. The aerosol spray of claim 1, further comprising an anti-settling agent and / or an anti-agglomerating agent.

6. 6. The aerosol spray according to claim 5, wherein the anti-settling and / or anti-agglomerating agent is selected from the list of inorganic polymers such as organoclay gels containing bentonite or hectorite, and / or fumed silicas represented by Aerosil.

7. 7. The aerosol spray of any one of claims 1 to 6, further comprising one or more emollients, suspending agents, humectants, antioxidants, humectants, emulsifiers, viscosity modifiers, fragrances, or other excipients.

8. The aerosol spray according to any one of claims 1 to 7, wherein the content of the aerosol spray consists of at least 50% by weight of propellant, 1% to 20% by weight of siloxane, 1% to 20% by weight of lactic acid bacteria, 0.1% to 5% by weight of an anti-settling agent and / or an anti-agglomerating agent, and 0% to 5% by weight of other excipients as listed in claim 7.

9. The aerosol spray according to any one of claims 1 to 8, wherein the content of the aerosol spray consists of at least 75% by weight of a propellant, 5% to 10% by weight of cyclomethicone, 1% to 10% by weight of lactic acid bacteria, and 0.1% to 5% by weight of Bentone gel.

10. The aerosol spray according to any one of claims 1 to 9, wherein the content comprises at least 75% by weight of a propellant, 5% to 10% by weight of cyclomethicone, 1% to 10% by weight of lactic acid bacteria, and 0.1% to 5% by weight of aerosil.

11. 11. The aerosol spray of any one of claims 1 to 10, which is substantially free of water and preservatives.

12. Use of an aerosol spray according to any one of claims 1 to 11 for the manufacture of a therapeutic medicament for use in applying said one or more live dormant strains of lactic acid bacteria to a surface or to the environment.

13. 12. An aerosol spray according to any one of claims 1 to 11 for use in restoring and / or maintaining a healthy skin microflora.

14. Use of an aerosol spray according to any one of claims 1 to 11 for the manufacture of a therapeutic medicament for use in restoring and / or maintaining a healthy skin microflora.

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