EA045049B1 - AQUEOUS COMPOSITION FOR TOPICAL APPLICATION CONTAINING VIABLE PROBIOTIC BACTERIA - Google Patents

Description

Field of invention

The present invention relates to aqueous topical compositions containing at least 1 10 ⁶ colony forming units (CFU)/g viable bacteria of at least one strain of probiotic bacteria that remain viable in the composition for a period of time, for example up to 14 days or even longer at 25°C/60% relative humidity (RH), or 30°C/75% RH, or 5°C. The present invention also relates to methods for preparing such aqueous compositions for topical use.

In particular, the present invention relates to a device suitable for long-term storage of viable bacteria of at least one strain of probiotic bacteria and a water-miscible solvent in separate compartments. At any time during storage, for example up to 3 years, the contents of individual compartments containing said viable bacteria of at least one strain of probiotic bacteria and said water-miscible solvent, respectively, can be combined to obtain ready-to-use said aqueous topical compositions according to the invention.

The present invention also relates to methods for preparing said device and to using said delivery device to provide probiotic bacteria that remain viable in said aqueous topical composition.

State of the art

It is well known in the art that probiotic bacteria in topical compositions do not survive in compositions that are moist (0.400.90 water activity), contain emulsifiers, preservatives, and/or when the compositions are stored at high temperatures.

Some manufacturers have attempted to formulate topical compositions containing viable bacteria of at least one strain of probiotic bacteria, but no commercially available product provides viable bacteria of at least one strain of probiotic bacteria in an amount of at least 1 10 ⁶ CFU/g. which would allow consumers to use it to obtain the desired level of viable bacteria from at least one strain of probiotic bacteria. There are also no topical aqueous products available that can be stored for years at room temperature or even higher temperatures and still maintain a very high level of viable bacteria of at least one strain of probiotic bacteria at the time of use, i.e. at least 1 10 ⁶ CFU/g. Topical compositions that are aqueous provide a pleasant tactile sensation upon application and are therefore often sought after by consumers.

There is therefore a need in the art to provide aqueous topical compositions containing viable bacteria of at least one strain of probiotic bacteria that are stable upon receipt, viable after storage for a period of up to three years at room temperature, or after storage at cool temperatures. place, for example in a refrigerator, that is, at about 25°C/60% RH, for example about 30°C/60% RH and, for example, about 5°C, and which are viable until use within the stated shelf life.

Therefore, an improved topical composition containing viable bacteria of at least one strain of probiotic bacteria is useful, and in particular, a more stable topical composition containing viable bacteria of at least one strain of probiotic bacteria for long-term storage of viable bacteria for at least one strain of probiotic bacteria is beneficial.

Summary of the invention

An object of the present invention relates to a stable aqueous topical composition containing viable bacteria of at least one strain of probiotic bacteria that maintains a desired high level of viable probiotic bacteria in the composition for a significant period of time.

In particular, it is an object of the present invention to provide a topical composition that solves the above-mentioned problems in the prior art with poor viability of probiotic bacteria, poor stability of probiotic bacteria in aqueous compositions and the inability of long-term storage of probiotic bacteria.

One aspect of the present invention relates to a method for preparing an aqueous composition for topical use, comprising:

a) providing viable bacteria of at least one strain of probiotic bacteria and optionally at least one buffer,

b) providing at least one water-miscible solvent containing at least one thickening agent to obtain the mixture; and

c) adding said viable bacteria of at least one probiotic strain

- 1 045049 human bacteria from stage a) to the specified mixture from stage b) to obtain the specified aqueous composition for topical use, having a pH of 3.0-8.0 and a viscosity of more than 2500 mPa-s and the number of said viable bacteria of at least one strain of probiotic bacteria of at least

1-10 ⁶ CFU/g.

Another aspect of the present invention relates to an aqueous composition for topical use containing viable bacteria of at least one strain of probiotic bacteria and at least one water-miscible solvent, wherein viable bacteria of at least one strain of probiotic bacteria in an amount ranging from 1- 106-10 ¹¹ CFU/g, are viable in an aqueous topical composition for a certain period of time, for example up to 14 days at 25°C/60% RH or 30°C/75% RH.

In yet another aspect of the present invention, there is provided a device for delivering probiotic bacteria, comprising:

a first compartment containing viable bacteria of at least one strain of probiotic bacteria and optionally a desiccant, and a second compartment containing at least one water-miscible solvent, wherein the number of viable bacteria of at least one strain of probiotic bacteria in said first compartment is at least 5-10 ⁶ CFU/g for up to 3 years at 25°C/60% RH or 30°C/75% RH.

In another aspect of the present invention, there is provided a method for preparing a delivery device of the present invention, comprising the steps of:

a) providing viable bacteria in the first compartment of at least one strain of probiotic bacteria; and

b) providing in the second compartment at least one water-miscible solvent, where a) and b) together provide said delivery device.

Another aspect of the present invention relates to the use of a delivery device of the present invention to provide viable bacteria of at least one strain of probiotic bacteria in an aqueous topical composition of the present invention for up to 14 days at 25°C/60% RH or 30 °C/75% RH.

Brief description of graphic materials

In fig. 1a is a graph showing the stability of Lactobacillus rhamnosus in the aqueous topical gel composition of the present invention.

In fig. 1b is a graph showing the stability of Lactobacillus paracasei in the aqueous topical gel composition of the present invention.

In fig. 2a is a graph showing the stability of Bifidobacterium animalis subsp lactis in the aqueous topical gel composition of the present invention.

In fig. 2b is a graph showing the stability of Lactobacillus rhamnosus in the aqueous topical lotion composition of the present invention.

In fig. 3a is a graph showing the stability of the Lactobacillus rhamnosus mixture in the first compartment.

In fig. 3b is a graph showing the stability of the Bifidobacterium animalis subsp lactis mixture in the first compartment.

Detailed Description of the Invention

In one aspect of the present invention there is provided a method for preparing an aqueous composition for topical use, comprising:

a) providing viable bacteria of at least one strain of probiotic bacteria in an amount of at least 5-10 ⁶ CFU/g and at least one buffer,

b) providing a mixture containing at least one thickener selected from one or more of carbomer, poloxamer, polyethylene glycol (PEG)/macrogol and/or carboxylic acid, and at least 60% (w/w) water , And

c) adding said viable bacteria of at least one strain of probiotic bacteria from step a) to said mixture from step b) to obtain said aqueous composition for topical use having a pH of 4.0-6.7 and a viscosity of more than 2500 mPa-s , and the number of said viable bacteria of at least one strain of probiotic bacteria is 1-106-10 ¹¹ CFU/g, and where said viable bacteria of at least one strain of probiotic bacteria are viable in an aqueous composition for topical use for up to 7 days at 25 °C/60% RH.

In one embodiment, a method for preparing an aqueous composition for topical use is disclosed, comprising:

a) providing viable bacteria of at least one strain of probiotic bacteria in an amount of at least 5-10 ⁶ CFU/g and at least one buffer,

b) providing a mixture containing at least one thickener selected from one or

- 2 045049 more than one of carbomer, poloxamer, PEG/macrogol and/or carboxylic acid, and at least 60% (w/w) water, and

c) adding said viable bacteria of at least one strain of probiotic bacteria from step a) to said mixture from step b) to obtain said aqueous composition for topical use having a pH of 4.0-6.7 and a viscosity of more than 2500 mPa-s , and the number of said viable bacteria of at least one strain of probiotic bacteria is 1-106-1011 CFU/g, and where said viable bacteria of at least one strain of probiotic bacteria are viable in an aqueous composition for topical use for up to 7 days at 25° C/60% RH.

In one embodiment, a method for preparing an aqueous composition for topical use is disclosed, comprising:

a) providing a dry composition of viable bacteria of at least one strain of probiotic bacteria in an amount of at least 5-106 CFU/g and at least one buffer, wherein said dry composition of viable bacteria of at least one strain of probiotic bacteria is ground into a powder having water activity not more than 0.30 and particle size distribution in the area less than 500 microns,

b) providing a mixture containing at least one thickener selected from one or more poloxamers, and at least 60% (w/w) water to provide said mixture having a viscosity of about 100 mPa-s at a temperature not above 10°C and a viscosity of approximately 1000 mPa-s at temperatures above 25°C, and

c) adding said dry composition from step a) to said mixture from step b) to obtain said aqueous composition for topical use having a viscosity of less than 500 mPa-s at a temperature not exceeding 10°C and a viscosity of more than 2500 mPa-s at a temperature above 30°C, and where the specified aqueous composition for topical application contains the specified viable bacteria of at least one strain of probiotic bacteria in an amount in the range of 1-106-10 ¹¹ CFU/g, and where the specified viable bacteria of at least one strain of probiotic bacteria bacteria are viable in an aqueous composition for topical use for up to 7 days at 25°C/60% RH.

In one aspect of the present invention, an aqueous composition for topical use is disclosed comprising at least 60% (w/w) water and viable bacteria of at least one strain of probiotic bacteria, wherein said viable bacteria of at least one strain of probiotic bacteria in an amount 1-106-10 ¹¹ CFU/g are viable in an aqueous topical composition for up to 7 days at 25°C/60% RH, and wherein said composition contains at least one thickener selected from one or more of a carbomer, poloxamer, PEG/macrogol and/or carboxylic acid, and at least one buffer.

In yet another aspect, a probiotic bacteria delivery device is disclosed, comprising:

a first compartment containing viable bacteria of at least one strain of probiotic bacteria, and at least one buffer, and optionally a drying agent, and a second compartment containing at least one thickener selected from one or more of a carbomer, a poloxamer, PEG/macrogol and/or carboxylic acid, and at least 60% (w/w) water, and at least one buffer, wherein the number of viable bacteria of said at least one strain of probiotic bacteria in said first compartment is at least at least 5-106 CFU/g for up to 3 years at 25°C/60% RH.

In another aspect, a method for producing a delivery device is disclosed, comprising the steps of:

a) providing the first compartment with viable bacteria of at least one strain of probiotic bacteria in an amount of at least 5-106 CFU/g for up to 3 years at 25°C/60% RH and

b) providing in the second compartment at least one thickener selected from one or more of carbomer, poloxamer, PEG/macrogol and/or carboxylic acid, and at least 60% (w/w) water, and at least one buffer, where a) and b) together provide the specified delivery device.

In another aspect, the use of a delivery device for providing viable bacteria of at least one strain of probiotic bacteria in an aqueous topical composition of the present invention for at least 7 days at 25°C/60% RH or 30°C/75% RH is disclosed. OV.

One aspect of the present invention relates to a method for preparing an aqueous composition for topical use, comprising:

a) providing viable bacteria of at least one strain of probiotic bacteria and optionally at least one buffer,

b) providing at least one water-miscible solvent containing at least one thickener to provide the mixture, and

- 3 045049

c) adding said viable bacteria of at least one strain of probiotic bacteria from step a) to said mixture from step b) to obtain said aqueous composition for topical use having a pH of 3.0-8.0 and a viscosity of more than 2500 mPa-s, for example more than 2700 mPa-s, for example more than 3000 mPa-s, for example more than 3300 mPa-s, for example more than 3600 mPa-s, for example more than 3900 mPa-s, for example more than 5000 mPa-s, for example more than 6000 mPa-s, for example more than 7500 mPa-s, for example more than 10000 mPa-s, and the number of said viable bacteria of at least one strain of probiotic bacteria in the range of 1-106-1011 CFU/g, for example at least 106 CFU/g, for example at least 10 ⁷ CFU/g, for example at least 108 CFU/g, for example at least 109, for example at least ¹⁰¹⁰ , for example at least ¹⁰¹¹ CFU/g. In one embodiment of the invention, the pH may be 3-7.5, for example 3-7.0, for example 3.5-7.0, for example 4.0-6.7, for example 4.07.0, for example 4.0- 8.0, for example 4.5-7.0, for example 4.5-6.5, for example 3-4, for example 3.5-4.5, for example 4.55.5, for example more than 3, for example more than 3 ,5, for example more than 4.0, for example more than 5.0, for example more than 5.5. In a preferred embodiment, the pH is 4.0-7.0.

In one embodiment of the present invention, a method for preparing an aqueous topical composition according to the present invention is disclosed, wherein said mixture from step b) has a viscosity of less than 2500 mPa-s, for example less than 2200 mPa-s, for example less than 2000 mPa-s, for example less than 1800 mPa -s, for example less than 1500 mPa-s, for example less than 1000 mPa-s, for example less than 750 mPa-s, for example less than 500 mPa-s.

Typically, the consumer will store the aqueous topical composition of the present invention at room temperature or in a refrigerator in his home in his country of residence. Typical room temperature varies quite a bit depending on the country, time of day, and so on. In a preferred embodiment, the temperature of the aqueous topical composition of the invention or the delivery device containing the aqueous topical composition of the invention may be in the range of about 18°C-32°C, for example 20°C-32°C, for example 22°C С-32°С, for example 24°С-31°С, for example 25°С-30°С, for example 20°С-30°С, for example 22°С-30°С, for example 23°С-28°С , for example 24°C-26°C and preferably 25°C or 30°C. Also, temperatures in refrigerators vary quite greatly and can be approximately 1°C-15°C, for example 1°C-10°C, for example 2°C-9°C, for example 3°C-8°C, for example 4°C -6°C, and preferably 5°C according to one embodiment of the invention.

In one embodiment, the invention relates to a method for preparing an aqueous topical composition of the present invention, wherein said viable bacteria of at least one strain of probiotic bacteria are viable in said aqueous topical composition for at least 5 minutes at 25°C/60 % RH or 30°C/75% RH or 5°C, for example for at least 20 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example for at least 1 hour at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 4 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example at at least 12 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 24 hours at 25°C/60% RH or 30°C/75% RH or 5°C C for example at least 2 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 3 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 4 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 5 days at 25°C/60% RH or 30°C /75% RH or 5°C, for example at least 6 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 7 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 8 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 9 days at 25°C /60% RH or 30°C/75% RH or 5°C, for example at least 10 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 14 days at 25°C/60% RH or 30°C/75% RH or 5°C.

In another embodiment, the invention relates to a method for preparing an aqueous topical composition of the present invention, wherein said viable bacteria of at least one strain of probiotic bacteria are viable in said aqueous topical composition for up to 5 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 20 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 1 hour at 25°C/60 % RH or 30°C/75% RH or 5°C, for example up to 4 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 12 hours at 25°C/60 % RH or 30°C/75% RH or 5°C, for example up to 24 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 2 days at 25°C/60 % RH or 30°C/75% RH or 5°C, for example up to 3 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 4 days at 25°C/60 % RH or 30°C/75% RH or 5°C, for example up to 5 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 6 days at 25°C/60 % RH or 30°C/75% RH or 5°C, for example up to 7 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 8 days at 25°C/60 % RH or 30°C/75% RH or 5°C, for example up to 9 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 10 days at 25°C/60 % RH or 30°C/75% RH or 5°C, for example up to 14 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 18 days at

- 4 045049

25°C/60% RH or 30°C/75% RH or 5°C, for example up to 22 days at 25°C/60% RH or 30°C/75% RH or

5°C, for example up to 26 days at 25°C/60% RH % RH or 30°C/75% RH or 5°C, for example up to 30 days at

25°C/60% RH or 30°C/75% RH or 5°C, for example up to 2 months at 25°C/60% RH or 30°C/75% RH or

5°C.

Another embodiment of the present invention relates to a method of preparing an aqueous topical composition of the present invention, wherein said viable bacteria of at least one strain of probiotic bacteria are viable in said aqueous topical composition for from 5 minutes to 2 months, for example from 5 minutes to 1 month, for example from 5 minutes to 14 days, for example from 5 minutes to 7 days, for example from 5 minutes to 4 days.

Surprisingly, it has been shown that said probiotic bacteria remain viable in said aqueous topical composition from the time the composition is ready for use by the user/consumer for at least 5 minutes at 25°C/60% RH or 30°C/75 % RH or 5°C or for up to 5 minutes at 25°C/60% RH or 30°C/75% RH or 5°C.

Additionally, and even more surprisingly, said probiotic bacteria remain viable in said aqueous topical composition for at least 7 days or even up to 14 days at 25° C./60% RH or 30° C./75% RH or 5° WITH. This provides the consumer with the advantage of having a stable topical composition containing viable bacteria of at least one strain of probiotic bacteria ready for use over an extended period of time.

In one embodiment, the invention relates to a method for preparing an aqueous topical composition of the present invention, wherein said viable bacteria of at least one strain of probiotic bacteria are provided in a dry composition. Using a dry composition has the advantage of having a stable product at room temperature for at least 1 month at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 3 months at 25°C/ 60% RH or 30°C/75% RH or 5°C, for example at least 6 months at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 9 months at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 12 months at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least at least 18 months at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 24 months at 25°C/60% RH or 30°C/75% RH or 5°C , for example at least 30 months at 25°C/60% RH or 30°C/75% RH or 5°C, or even at least 36 months at 25°C/60% RH or 30°C/ 75% RH or 5°C.

In one embodiment, the invention relates to a method for preparing an aqueous topical composition of the present invention, comprising:

a) providing viable bacteria of at least one strain of probiotic bacteria and at least one or more than one buffer,

b) providing at least one water-miscible solvent containing at least one thickener, and one or more acids or one or more bases to provide the mixture, and optionally an emulsifier, and one or more hydrophobic connection, and

c) adding said viable bacteria of at least one strain of probiotic bacteria from step a) to said mixture from step b) and optionally further adding a buffer to obtain said aqueous topical composition having a pH of 3.0-8.0 and viscosity more than 2500 mPa-s, for example more than 2700 mPa-s, for example more than 3000 mPa-s, for example more than 3300 mPa-s, for example more than 3600 mPa-s, for example more than 3900 mPa-s, for example more than 5000 mPa-s, for example more than 7500 mPa-s, for example more than 10000 mPa-s, and the number of said viable bacteria of at least one strain of probiotic bacteria in the range of 1-106-10 ¹¹ CFU/g, for example at least 106 CFU/, for example at least 10 ⁷ CFU/g, for example at least 10 ⁸ CFU/g, for example at least 109 CFU/g, for example at least 10 ¹⁰ CFU/g, for example at least 10 ¹¹ CFU/g.

According to the present invention, an acid or base is used to adjust the pH, while a buffer is used to maintain the pH at the desired level. Surprisingly, it has been shown that by controlling the pH at a desired level using an acid and/or base, the viscosity of the mixture in b) can be controlled using the carbomer thickeners of the invention, thereby providing an aqueous topical composition of the invention having a pH of 3.0- 8.0, for example preferably 4.0-7.0, and a viscosity of more than 2500 mPa-s, when viable bacteria of at least one strain of probiotic bacteria and at least one or more buffers are mixed with the mixture from step b) to invention.

In one embodiment of the invention relating to a method for preparing an aqueous topical composition of the present invention, said viable bacteria of at least one strain of probiotic bacteria are provided in a dry composition. In one embodiment, said dry composition has a particle size of no more than 1000 microns, for example no more than 900 microns, for example no more than 800 microns, for example no more than 700 microns, for example no more than 600 microns, for example no more than 500 microns, for example not more than 400 microns, for example not more than 300 microns, for example not more than 200 microns, for example not more than 150 microns, for example not more than 100 microns in diameter. In a preferred embodiment, said dry composition has a particle size of no more than 200 microns in diameter. In another embodiment, said dry composition may further comprise a thickener.

In one embodiment, the invention relates to a method for preparing an aqueous topical composition of the present invention, wherein the viable bacterial composition of at least one strain of probiotic bacteria contains at least one acid or base. The addition of an acid or base helps maintain the pH of the composition at the desired level so that thickening can be controlled. Preferably, when said viable bacteria of at least one strain of probiotic bacteria contain at least one acid or base, said viable bacteria of at least one strain of probiotic bacteria is a dry composition.

In one embodiment, the invention relates to a method for preparing an aqueous topical composition of the present invention, comprising:

a) providing a dry composition of viable bacteria of at least one strain of probiotic bacteria in an amount of at least 5-106 CFU/g and at least one buffer,

b) providing at least one water-miscible solvent containing at least one thickener to provide a mixture having a pH of 2-3 and a viscosity of less than 2500 mPa-s, and

c) adding said dry composition from step a) to said mixture from step b) to obtain said aqueous composition for topical use having a pH of 3.0-8.0 and a viscosity of more than 2500 mPa-s and a number of said viable bacteria of at least one strain of probiotic bacteria in the range of 1-106-10 ¹¹ CFU/g.

In one embodiment, the invention relates to a method for preparing an aqueous topical composition of the present invention, comprising: a) providing a dry composition of viable bacteria of at least one strain of probiotic bacteria in an amount of at least 5-106 CFU/g, and at least one buffer, and at least one or more than one acid or base,

b) providing at least one water-miscible solvent containing at least one thickener to provide a mixture having a pH of 2-3 and a viscosity of less than 2500 mPa-s, and

c) adding said dry composition from step a) to said mixture from step b) to obtain said aqueous composition for topical use having a pH of 3.0-8.0 and a viscosity of more than 2500 mPa-s and a number of said viable bacteria of at least one strain of probiotic bacteria at least 1-106-109 CFU/g.

In one embodiment, the invention relates to a method for preparing an aqueous topical composition of the present invention, comprising:

a) providing a dry composition of viable bacteria of at least one strain of probiotic bacteria in an amount of at least 5-106 CFU/g and at least one buffer,

b) providing at least one water-miscible solvent containing at least one thickener selected from one or more carbomers to provide a mixture having a pH of 2-3 and a viscosity of less than 2500 mPa-s, and

c) adding said dry composition from step a) to said mixture from step b) to obtain said aqueous composition for topical use having a pH of 3.0-8.0 and a viscosity of more than 2500 mPa-s and a number of said viable bacteria of at least one strain of probiotic bacteria in the range of 1-106-10 ¹¹ CFU/g.

In one embodiment, the invention relates to a method for preparing an aqueous topical composition of the present invention, wherein said at least one thickening agent is one or more carbomers, and wherein the dry composition contains at least one buffer selected from one or more one buffer based on monovalent cations. Monovalent cationic buffers are preferred when using carbomers because monovalent cationic buffers control carbomer swelling well.

In one embodiment, the invention relates to a method for preparing an aqueous topical composition according to the invention, comprising:

a) providing a dry composition of viable bacteria of at least one strain of probiotic bacteria in an amount of at least 5-106 CFU/g and optionally at least one buffer,

b) providing at least one water-miscible solvent containing at least one thickener selected from one or more poloxamers to provide a mixture having a viscosity of about 100 mPa-s at a temperature not exceeding 10°C, for example not exceeding 10-15°C, and a viscosity of about 1000 mPa-s at a temperature above 25°C, for example above 20-25°C, and

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c) adding said dry composition from step a) to said mixture from step b) to obtain said aqueous composition for topical use having a viscosity of not more than 1000 mPa-s or not more than 500 mPa-s at a temperature not exceeding 10°C, for example not higher than 20°C, and a viscosity of more than 2000 mPa-s at a temperature higher than 30°C, for example above 20°C, and wherein said aqueous composition for topical use contains a number of said viable bacteria of at least one strain of probiotic bacteria in the range of 1- 10 ⁶ -10 ¹¹ CFU/g.

An alternative aspect of the present invention relates to a method for preparing an aqueous composition for topical use, comprising:

a) providing a dry composition of at least one moisture-sensitive agent that is not viable bacteria of at least one strain of probiotic bacteria,

b) providing at least one water-miscible solvent, and

c) adding said dry composition from step a) to said water-miscible solvent from step b) to obtain said aqueous topical composition containing said at least one moisture-sensitive agent. In one embodiment, said at least one moisture-sensing agent that is not viable bacteria of at least one strain of probiotic bacteria is active in an aqueous topical composition for up to 14 days at 25°C/60% RH or 30°C/75 % RH or 5°C.

In an alternative embodiment, said viable bacteria of at least one strain of probiotic bacteria are active or alive in the aqueous topical composition for up to 5 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example for up to 20 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 1 hour at 25°C/60% RH or 30°C/75% RH or 5°C , for example up to 4 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 12 hours at 25°C/60% RH or 30°C/75% RH or 5°C , for example up to 24 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 2 days at 25°C/60% RH or 30°C/75% RH or 5°C , for example up to 3 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 4 days at 25°C/60% RH or 30°C/75% RH or 5°C , for example up to 5 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 6 days at 25°C/60% RH or 30°C/75% RH or 5°C , for example up to 7 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 8 days at 25°C/60% RH or 30°C/75% RH or 5°C , for example up to 9 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 10 days at 25°C/60% RH or 30°C/75% RH or 5°C , for example up to 14 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 18 days at 25°C/60% RH or 30°C/75% RH or 5°C , for example up to 22 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 26 days at 25°C/60% RH or 30°C/75% RH or 5°C , for example up to 30 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 2 months at 25°C/60% RH or 30°C/75% RH or 5°C .

Another aspect of the present invention relates to an aqueous composition for topical use containing viable bacteria of at least one strain of probiotic bacteria and at least one water-miscible solvent, wherein viable bacteria of at least one strain of probiotic bacteria in an amount ranging from 1- 10-10 ¹¹ CFU/g, are viable in an aqueous topical composition for a period of time, such as at least 5 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, such such as at least 20 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, such as at least 1 hour at 25°C/60% RH or 30°C/75% RH or 5°C, such as at least 4 hours at 25°C/60% RH or 30°C/75% RH or 5°C, such as at least 12 hours at 25°C/60% RH or 30 °C/75% RH or 5°C, such as at least 24 hours at 25°C/60% RH or 30°C/75% RH or 5°C, such as at least 2 days at 25°C /60% RH or 30°C/75% RH or 5°C, such as at least 3 days at 25°C/60% RH or 30°C/75% RH or 5°C, such as at least 4 days at 25°C/60% RH or 30°C/75% RH or 5°C, such as at least 5 days at 25°C/60% RH or 30°C/75% RH or 5°C , such as at least 6 days at 25°C/60% RH or 30°C/75% RH or 5°C, such as at least 7 days at 25°C/60% RH or 30°C/75 % RH or 5°C, such as at least 8 days at 25°C/60% RH or 30°C/75% RH or 5°C, such as at least 9 days at 25°C/60% RH or 30°C/75% RH or 5°C, such as at least 10 days at 25°C/60% RH or 30°C/75% RH or 5°C, such as at least 14 days at 25 °C/60% RH or 30°C/75% RH or 5°C, for example up to 5 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 20 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 1 hour at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 4 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 12 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 24 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 2 days at 25°C/60% RH or 30°C/75% RH or 5°C , for example up to 3 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 4 days at 25°C/60% RH or 30°C/75% RH or 5 °C, for example up to 5 days at 25°C/60% RH or 30°C/75% RH or 5°C,

- 7 045049 for example up to 6 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 7 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 8 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 9 days at 25°C/60% RH or 30°C/75 % RH or 5°C, for example up to 10 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 14 days at 25°C/60% RH or 30°C /75% RH or 5°C, for example up to 18 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 22 days at 25°C/60% RH or 30 °C/75% RH or 5°C, for example up to 26 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 30 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 2 months at 25°C/60% RH or 30°C/75% RH or 5°C.

Another aspect of the present invention relates to an aqueous composition for topical use containing viable bacteria of at least one strain of probiotic bacteria and at least one water-miscible solvent, wherein viable bacteria of at least one strain of probiotic bacteria in an amount ranging from 1- 106-10 ¹¹ CFU/g, are viable in an aqueous topical composition for a period of time, such as 5 minutes to 2 months, such as 5 minutes to 1 month, such as 5 minutes to 14 days, such as 5 minutes to 7 days, for example from 5 minutes to 4 days.

In one embodiment of the invention, at least 1-106 CFU/g, for example at least 5-106 CFU/g, for example at least 10 ⁷ CFU/g, for example at least 10 ⁸ CFU/g, for example at least 109 CFU/g, for example at least 10 ¹⁰ , for example at least 10 ¹¹ CFU/g of said viable bacteria of at least one strain of probiotic bacteria are viable in an aqueous topical composition for up to 14 days at 25°C/60 % RH or 30°C/75% RH or 5°C.

Surprisingly, it has been shown that said probiotic bacteria remain viable in said aqueous topical composition for at least 5 minutes at 25°C/60% RH or 30°C/75% RH or 5°C. Additionally, and even more surprisingly, said probiotic bacteria remain viable in said aqueous topical composition for at least 7 or even 14 days at 25°C/60% RH or 30°C/75% RH or 5°C. This provides the consumer with the advantage that the aqueous topical composition is stable and has a large number of viable bacteria of at least one strain of probiotic bacteria.

In one embodiment, the invention relates to an aqueous topical composition of the present invention, wherein said viable bacteria of at least one strain of probiotic bacteria are provided in a dry composition containing viable bacteria of at least one strain of probiotic bacteria in an amount of at least 5-106 CFU /G.

In one embodiment, the invention relates to an aqueous topical composition of the present invention, wherein said composition contains one or more active ingredients.

In one embodiment, the invention relates to an aqueous topical composition of the present invention, wherein said composition contains at least one thickener and/or at least one buffer. By using said thickener and/or at least one buffer in said aqueous topical composition of the present invention, it has surprisingly been shown that said probiotic bacteria remain viable in said aqueous topical composition for at least 5 minutes at 25° C. /60% RH or 30°C/75% RH or 5°C. Additionally, and even more surprisingly, said probiotic bacteria remain viable in said aqueous topical composition for at least 7 or even at least 14 days at 25°C/60% RH or 30°C/75% RH or 5 °C or for up to 7 or even up to 14 days at 25°C/60% RH or 30°C/75% RH or 5°C. This provides the consumer with the advantage that the aqueous topical composition is stable and has a large number of viable bacteria of at least one strain of probiotic bacteria. Also, said aqueous composition for topical use has a very acceptable shelf life/shelf life of at least 7 or even at least 14 days at 25°C/60% RH or 30°C/75% RH or 5°C, or it has a very acceptable shelf life of up to 7 or even up to 14 days at 25°C/60% RH or 30°C/75% RH or 5°C, thereby providing a very useful product for the consumer.

In one embodiment, the invention relates to an aqueous topical composition of the present invention, wherein said composition contains at least one carbomer, and at least one buffer, and optionally at least one acid and/or at least one base.

In one embodiment, the invention relates to an aqueous topical composition of the present invention, wherein said composition contains at least one poloxamer.

In one embodiment, the invention relates to an aqueous topical composition of the present invention, wherein said composition contains at least one emulsifier.

In one embodiment, the invention relates to an aqueous topical composition of the present invention, wherein said composition contains at least one buffer.

In one embodiment, the invention relates to an aqueous topical composition of the present invention, wherein said composition contains one or more hydrophobic compounds.

In one embodiment, the invention relates to an aqueous topical composition of the present invention, wherein said hydrophobic compound is selected from one or more of oils and waxes that are solid and/or fluid at room temperature.

In one embodiment, the invention relates to an aqueous topical composition of the present invention, wherein said composition has a pH of 3.0-8.0 and a viscosity greater than 2000 mPa-s, for example a viscosity greater than 2250 mPa-s, for example a viscosity greater than 2500 mPa-s , for example a viscosity of more than 2700 mPa-s, such as more than 3000 mPa-s, such as more than 3300 mPa-s, such as more than 3600 mPa-s, such as more than 3900 mPa-s, such as more than 4000 mPa-s, such such as more than 5000 mPa-s, such as more than 6000 mPa-s, such as more than 7500 mPa-s, such as more than 8500 mPa-s, such as more than 10000 mPa-s. In one embodiment of the invention, the pH may be 3.0-4.0, for example 3.5-4.5, for example 4.5-5.5, for example more than 3.0, for example more than 3.5, for example more than 4.0 for example more than 5.0, for example more than 5.5.

In one embodiment, the invention relates to an aqueous topical composition of the present invention, wherein the viable bacteria of at least one strain of probiotic bacteria are selected from one or more than one strain or combination of strains of any of the strains of Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, any strain belonging to the genus Lactobacillus, including Lactobacillus acidophilus, Lactobacillus casei subsp. casei, L. casei (Gynophilus), L. colehominis, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus crispatus, Lactobacillus fermentum, L. fornicalis, L. gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus lactis, L. mucosae, L. paracasei, L plantarum, L. salivarius, L. reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, L. vaginalis, but not limited to, any strain belonging to the genus Bifidobacterium, including Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum, but not limited to, or from the genera Akkermansia, Anaerostipes, Butyricicoccus, Christensenella, Clostridia, Coprococcus, Dorea, Eubacterium, Faecalibacterium, Cutibacterium, for example Cutibacterium acnes, or Roseburia, Staphylococcus, for example Staphylococcus epidermis or Staphylococcus hominis, Weissella viridescens, or the family Coriobacteriaceae.

In one embodiment, the invention relates to an aqueous topical composition of the present invention, wherein the viable bacteria of at least one strain of probiotic bacteria are selected from one or more than one strain or combination of strains belonging to the genus Bifidobacterium, including Bifidobacterium animalis subsp. lactis, for example Bifidobacterium animalis subsp. lactis, Bifidobacterium breve and Bifidobacterium infantis, but not limited to, strains belonging to the genus Cutibacterium, for example Cutibacterium acnes, any strain belonging to the genus Lactobacillus, including L. casei (Gynophilus), L. colehominis, L. delbrueckii, L. fornicalis, L. gallinarum, L. iners, L. mucosae, L. paracasei, but not limited to them, for example Lactobacillus paracasei subsp. paracasei, L. plantarum, L. rhamnosus, for example Lactobacillus rhamnosus, L. salivarius, L. reuteri, L. vaginalis, strains of the genus Staphylococcus, for example Staphylococcus epidermis and Staphylococcus hominis, and Weissella viridescens.

In a preferred embodiment, the probiotic strain included in the dry composition or aqueous topical composition of the present invention is selected from one or more strains belonging to the genus Bifidobacterium, including Bifidobacterium animalis subsp. lactis, for example Bifidobacterium animalis subsp. lactis, Bifidobacterium breve and Bifidobacterium infantis, but not limited to, strains belonging to the genus Cutibaterium, for example Cutibacterium acnes, any strain belonging to the genus Lactobacillus, including L. casei (Gynophilus), L. colehominis, L. delbrueckii, L. fornicalis, L. gallinarum, L. iners, L. mucosae, L. paracasei, for example Lactobacillus paracasei subsp. paracasei, L. plantarum, L. rhamnosus, for example Lactobacillus rhamnosus, L. salivarius, L. reuteri, L. vaginalis, but not limited to, strains of the genus Staphylococcus, such as Staphylococcus epidermis and Staphylococcus hominis, and Weissella viridescens.

In currently preferred embodiments of the invention, one, two, three, four, five, six, seven or eight different strains are present in the composition of the present invention.

An alternative aspect of the present invention relates to an aqueous topical composition comprising at least one moisture-sensing agent that is not viable bacteria of at least one strain of probiotic bacteria, and at least one water-miscible solvent, wherein at least 10% at least a humectant agent is active in the aqueous topical composition for at least minutes or up to 14 days at 25°C/60% RH or 30°C/75% RH or 5°C.

In yet another aspect of the present invention, there is provided a device for delivering probiotic bacteria, comprising:

a first compartment containing viable bacteria of at least one strain of probiotic bacteria and optionally a desiccant, and a second compartment containing at least one water-miscible solvent, wherein the number of viable bacteria of at least one strain of probiotic bacteria in said first compartment is at least 5-106 CFU/g for up to 3 years at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 10 ⁷ CFU/g, for example at least 10 ⁸ CFU/g, for example at least 10 ⁹ CFU/g for up to 3 years at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 30 months at 25°C /60% RH or 30°C/75% RH or 5°C, for example up to 24 months at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 18 months at 25 °C/60% RH or 30°C/75% RH or 5°C, for example up to 12 months at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 6 months at 25°C/60% RH or 30°C/75% RH or 5°C or for example up to 3 months at 25°C/60% RH or 30°C/75% RH or 5°C.

In an alternative aspect of the present invention, there is provided a delivery device comprising:

a first compartment containing at least one moisture-sensitive component that is not viable bacteria of at least one strain of probiotic bacteria, and a second compartment containing at least one water-miscible solvent, wherein at least 10% of the moisture-sensitive component remains active for up to up to 3 years at 25°C/60% RH or 30°C/75% RH or 5°C.

In one embodiment, the invention relates to a delivery device according to the present invention, wherein said first and second compartments are separate elements.

In one embodiment, the invention relates to a delivery device of the present invention, wherein each of said first compartment and said second compartment is hermetically sealed with a lid, and wherein each of said compartments and said lids provides storage for said viable bacteria of at least one a strain of probiotic bacteria and said water-miscible solvent without any leakage of said viable bacteria of at least one strain of probiotic bacteria and/or leakage of said water-miscible solvent during storage.

In one embodiment, the invention relates to a delivery device of the present invention, wherein both said first and second compartments have lids that are reattachable.

In one embodiment, the invention relates to a delivery device of the present invention, wherein said second compartment is an aluminum, plastic or glass tube with a reusable screw cap or pump lid.

In one embodiment, a device according to the invention is provided, wherein said device is a glass or plastic bottle with a dropper, pipette or pump cap. Said plastic bottle may contain a foil that protects against high humidity, such as an aluminum inner layer.

In one embodiment, the invention relates to a delivery device according to the present invention, wherein said first and/or second compartment is a sachet and/or ampoule.

In a preferred embodiment, the invention relates to a delivery device of the present invention, wherein said first compartment is an ampoule or sachet, and wherein said second compartment is an aluminum or plastic tube with a reusable screw cap or pump cap. In an even more preferred embodiment of the invention, said first compartment is a reusable plastic lid which is screwed onto a second compartment made of glass, wherein said reusable plastic lid has a membrane between the compartments that can be broken so that the contents of the compartments can be mixed, in while the delivery device is closed/hermetically sealed from the environment.

In one embodiment, the invention relates to a delivery device of the present invention, wherein said viable bacteria of at least one strain of probiotic bacteria are presented in a dry composition.

In one embodiment, the invention relates to a delivery device according to the present invention, wherein said first compartment contains one or more active ingredients and/or drying agents.

In one embodiment, the invention relates to a delivery device of the present invention, wherein said first compartment contains one or more hydrophobic components.

In one embodiment, the invention relates to a delivery device of the present invention, wherein said first compartment contains one or more buffers.

- 10 045049

In one embodiment, the invention relates to a delivery device of the present invention, wherein said second compartment contains one or more active components.

In one embodiment, the invention relates to a delivery device of the present invention, wherein said second compartment contains one or more hydrophobic components.

In one embodiment, the invention relates to a delivery device of the present invention, wherein said second compartment contains one or more thickeners.

In one embodiment, the invention relates to a delivery device of the present invention, wherein said second compartment contains one or more emulsifiers.

In one embodiment, the invention relates to a delivery device of the present invention, wherein the viable bacteria of at least one strain of probiotic bacteria are selected from one or more than one strain or combination of strains from any of the strains belonging to Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, any strain belonging to the genus Lactobacillus, including Lactobacillus acidophilus, Lactobacillus casei subsp. casei, L. casei (Gynophilus), L. colehominis, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus crispatus, Lactobacillus fermentum, L. fornicalis, L. gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus lactis, L. mucosae, L. paracasei, L plantarum, L. salivarius, L. reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, L. vaginalis, but not limited to, any strain belonging to the genus Bifidobacterium, including Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum, but not limited to, or from the genera Akkermansia, Anaerostipes, Butyricicoccus, Christensenella, Clostridia, Coprococcus, Dorea, Eubacterium, Faecalibacterium, Cutibacterium, for example Cutibacterium acnes, or Roseburia, Staphylococcus, for example Staphylococcus epidermis or Staphylococcus hominis, Weissella viridescens, or the family Coriobacteriaceae.

In one embodiment, the invention relates to a delivery device of the present invention, wherein the viable bacteria of at least one strain of probiotic bacteria are selected from one or more than one strain or combination of strains of any of the genus of Bifidobacterium, including Bifidobacterium animalis subsp. lactis, for example Bifidobacterium animalis subsp. lactis, Bifidobacterium breve and Bifidobacterium infiantis, but not limited to, strains belonging to the genus Cutibacterium, for example Cutibacterium acnes, any strain belonging to the genus Lactobacillus, including L. casei (Gynophilus), L. colehominis, L. delbrueckii, L. fornicalis, L. gallinarum, L. iners, L. mucosae, L. paracasei, for example Lactobacillus paracasei subsp. paracasei, L. plantarum, L. rhamnosus, for example Lactobacillus rhamnosus, L. salivarius, L. reuteri, L. vaginalis, but not limited to, strains of the genus Staphylococcus, for example Staphylococcus epidermis and Staphylococcus hominis, and Weissella viridescens.

In currently preferred embodiments of the invention, one, two, three, four, five, six, seven or eight different strains are present in the composition of the present invention.

In one embodiment, the invention relates to a delivery device according to the present invention, wherein said first compartment maintains the water activity of said viable bacteria of at least one strain of probiotic bacteria at a level of no more than 0.3 for up to 3 years at 25°C/60 % RH or 30°C/75% RH or 5°C.

In one embodiment, the invention relates to a delivery device of the present invention, wherein said first and second compartments are connected in a male-female configuration.

In one embodiment, the invention relates to a delivery device of the present invention, wherein said protrusion-recess configuration includes a tight-fitting configuration.

In another aspect of the present invention, there is provided a method for preparing a delivery device of the present invention, comprising the steps of: a) providing viable bacteria of at least one strain of probiotic bacteria in a first compartment, and

b) providing at least one water-miscible solvent in the second compartment to provide said delivery device.

In one embodiment, the invention relates to a method for producing a delivery device of the present invention, wherein said first compartment contains one or more buffers.

In one embodiment, the invention relates to a method for producing a delivery device according to the present invention, wherein said first compartment contains one or more active components and/or drying agents.

In one embodiment, the invention relates to a method for producing a delivery device of the present invention, wherein said first compartment contains one or more hydrophobic components.

In one embodiment, the invention relates to a method for preparing a delivery device of the present invention, wherein said second compartment contains one or more active components.

In one embodiment, the invention relates to a method for producing a delivery device according to the present invention, wherein said second compartment contains one or more hydrophobic components.

In one embodiment, the invention relates to a method for producing a delivery device of the present invention, wherein said second compartment contains one or more than one thickener and/or one or more emulsifier.

In one embodiment, the invention relates to a method for producing a delivery device of the present invention, wherein the viable bacteria count of at least one strain of probiotic bacteria in said delivery device is at least 5-10 ⁶ CFU/g for up to 3 years at 25 °C/60% RH or 30°C/75% RH or 5°C, for example at least 10 ⁷ CFU/g, for example at least 10 ⁸ CFU/g, for example at least 10 ⁹ CFU/g for up to 3 years at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 30 months at 25°C/60% RH or 30°C/75% RH or 5°C , for example up to 24 months at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 18 months at 25°C/60% RH or 30°C/75% RH or 5 °C, for example up to 12 months at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 6 months at 25°C/60% RH or 30°C/75% RH or 5°C, or for example up to 3 months at 25°C/60% RH or 30°C/75% RH or 5°C.

In one embodiment, the invention relates to a method for producing a delivery device of the present invention, wherein viable bacteria of at least one strain of probiotic bacteria are selected from one or more than one strain or combination of strains of any of the strains belonging to Lactococcus lactis subsp. lactis biovar. diacety lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, any strain belonging to the genus Lactobacillus, including Lactobacillus acidophilus, Lactobacillus casei subsp. casei, L. casei (Gynophilus), L. colehominis, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus crispatus, Lactobacillus fermentum, L. fornicalis, L. gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus lactis, L. mucosae, L. paracasei, L. plantarum, L. salivarius, L. reuteri , Lactobacillus rhamnosus, Lactobacillus salivarius, L. vaginalis, but not limited to, any strain belonging to the genus Bifidobacterium, including Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum, but not limited to, or from the genera Akkermansia, Anaerostipes, Butyricicoccus, Christensenella, Clostridia, Coprococcus, Dorea, Eubacterium, Faecalibacterium, Cutibacterium, for example Cutibacterium acnes, or Roseburia, Staphylococcus, for example Staphylococcus epidermis or Staphylococcus hominis, Weissella viridescens, or the family Coriobacteriaceae.

In one embodiment, the invention relates to a method for producing a delivery device of the present invention, wherein the viable bacteria of at least one strain of probiotic bacteria are selected from one or more than one strain or combination of strains of any of the genus of Bifidobacterium, including Bifidobacterium animalis subsp. lactis, for example Bifidobacterium animalis subsp. lactis, Bifidobacterium breve and Bifidobacterium infantis, but not limited to, strains belonging to the genus Cutibacterium, for example Cutibacterium acnes, any strain belonging to the genus Lactobacillus, including L. casei (Gynophilus), L. colehominis, L. delbrueckii, L. fornicalis, L. gallinarum, L. iners, L. mucosae, L. paracasei, for example Lactobacillus paracasei subsp. paracasei, L. plantarum, L. rhamnosus, for example Lactobacillus rhamnosus, L. salivarius, L. reuteri, L. vaginalis, but not limited to, strains of the genus Staphylococcus, for example Staphylococcus epidermis and Staphylococcus hominis, and Weissella viridescens.

In currently preferred embodiments, one, two, three, four, five, six, seven or eight different strains are present in the composition of the present invention.

In one embodiment, the invention relates to a method for producing a delivery device of the present invention, wherein said first compartment maintains the water activity of said viable bacteria of at least one strain of probiotic bacteria at a level of no more than 0.3 for up to 3 years at 25°C /60% RH or 30°C/75% RH or 5°C, for example at least 10 ⁷ CFU/g, for example at least 10 ⁸ CFU/g, for example at least 10 ⁹ CFU/g for up to 3 years at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 30 months at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 24 months at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 18 months at 25°C/60% RH or 30°C/75% RH or 5°C , for example up to 12 months at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 6 months at 25°C/60% RH or 30°C/75% RH or 5 °C, or for example up to 3 months at 25°C/60% RH or 30°C/75% RH or 5°C.

In yet another aspect, the present invention relates to the use of a delivery device of the present invention to provide viable bacteria of at least one strain of probiotic bacteria in an aqueous topical composition of the present invention for at least 14 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example for at least 5 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example for at least 20 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 1 hour at 25°C/60% RH or 30°C/75% RH or 5°C, for example at at least 4 hours at 25°C/60% RH or

- 12 045049

30°C/75% RH or 5°C, for example at least 12 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 24 hours at 25°C/ 60% RH or 30°C/75% RH or 5°C, for example at least 2 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 3 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 4 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least at least 5 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 6 days at 25°C/60% RH or 30°C/75% RH or 5°C , for example at least 7 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 8 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 9 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 10 days at 25°C/60% RH or 30°C /75% RH or 5°C, for example at least 14 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 5 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 20 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 1 hour at 25°C/ 60% RH or 30°C/75% RH or 5°C, for example up to 4 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 12 hours at 25° C/60% RH or 30°C/75% RH or 5°C, for example up to 24 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 2 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 3 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 4 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 5 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to up to 6 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 7 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 8 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 9 days at 25°C/60% RH or 30°C/75% RH or 5° C, for example up to 10 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 14 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 18 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 22 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 26 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 30 days at 25°C/60% RH or 30°C/ 75% RH or 5°C, for example up to 2 months at 25°C/60% RH or 30°C/75% RH or 5°C.

When a consumer desires to obtain a finished aqueous topical composition of the invention, the contents of the compartments are simply combined, for example by mixing, shaking and/or shaking. Thus, when a consumer uses the delivery device of the present invention, the consumer mixes the contents of the delivery device and thereby obtains an aqueous topical composition of the present invention having at least 1-106 viable bacteria of at least one strain of probiotic bacteria for up to 14 days at 25°C/60% RH or 30°C/75% RH or 5°C. In another embodiment, the consumer mixes the contents of the delivery device and thereby obtains an aqueous topical composition of the present invention having at least 1-106 viable bacteria of at least one strain of probiotic bacteria for at least 14 days at 25° C/60% RH or 30°C/75% RH or 5°C.

In one embodiment, the present invention relates to the use of a delivery device of the present invention to provide at least 1-106 viable bacteria of at least one strain of probiotic bacteria in an aqueous topical composition of the present invention for up to 14 days at 25°C /60% RH or 30°C/75% RH or 5°C.

Definitions

Before discussing the present invention in more detail, the following terms and rules should first be defined.

Probiotic bacteria or a strain of probiotic bacteria.

In the context of the present invention, the expression probiotic bacteria or bacteria of a probiotic bacterial strain refers to a culture of microorganisms, for example bacteria, which, when applied to a person or animal, have a beneficial effect on the host (FAO/WHO, 2001, Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live Lactic Acid Bacteria.Report of a Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria). Unless the context requires otherwise, the terms microorganism, bacterium and probiotic are used interchangeably. The term bacterial strain, as used here, refers to a bacterium that remains genetically unchanged as it grows or reproduces.

In a preferred embodiment, the probiotic strain included in the dry composition or aqueous topical composition of the present invention is selected from one or more than one strain or combination of strains of any of the genus of Bifidobacterium, including Bifidobacterium animalis subsp. lactis, for example Bifidobacterium animalis subsp. lactis, Bifidobacterium breve and Bifidobacterium infantis, but not limited to, strains belonging to the genus Cutibacterium, for example Cutibacterium acnes, any strain belonging to the genus Lactobacillus, including L. casei (Gyno- 13 045049 philus), L. colehominis, L. delbrueckii, L. fornicalis, L. gallinarum, L. iners, L. mucosae, L. paracasei, for example Lactobacillus paracasei subsp. paracasei, L. plantarum, L. rhamnosus, for example Lactobacillus rhamnosus,

L. salivarius, L. reuteri, L. vaginalis, but not limited to, strains of the genus Staphylococcus, e.g.

Staphylococcus epidermis and Staphylococcus hominis, and Weissella viridescens.

Composition for topical use and aqueous composition for topical use.

In the context of the present invention, the expression for topical application is intended to mean application to a specific surface externally or internally of the body, for example application to an external or internal surface of the body, for example the skin or mucous membrane, for example the ear, the internal surface of the nasal cavity, the internal surface of the oral cavity, the lip , urogenital area, such as the urethral, vaginal or rectal area, including the urethral opening, the vaginal opening and the anus. In a preferred embodiment, said topical application is for the urogenital area, for example the urethral, vaginal or rectal area, including the urethral opening, the vaginal opening and the anus. In another preferred embodiment, said topical application is for the skin.

In one embodiment of the invention, said skin or mucous membrane may be intact skin or mucous membrane. In another embodiment, said skin or mucous membrane may be disordered or damaged skin, for example, damaged by dermatitis, such as atopic dermatitis, or having a burn, eczema, one or more psoriatic plaques, wounds, acne, sensitive skin, itchy skin, vitiligo , rosacea, lichen sclerosus.

Thus, in the context of the present invention, the expression aqueous topical composition is intended to mean a topical composition containing at least one water-miscible solvent and viable bacteria of at least one strain of probiotic bacteria capable of surviving in said aqueous topical composition before topical application, i.e. for at least 5 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example for at least 20 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 1 hour at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 4 hours at 25°C /60% RH or 30°C/75% RH or 5°C, for example at least 12 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 24 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 2 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at at least 3 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 4 days at 25°C/60% RH or 30°C/75% RH or 5°C C, for example at least 5 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 6 days at 25°C/60% RH or 30°C/75% RH RH or 5°C, for example at least 7 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 8 days at 25°C/60% RH or 30°C C/75% RH or 5°C, for example at least 9 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 10 days at 25°C/60% RH RH or 30°C/75% RH or 5°C, for example at least 14 days at 25°C/60% RH or 30°C/75% RH or 5°C, or an additional period of up to 5 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 20 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 1 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 4 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to up to 12 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 24 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 2 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 3 days at 25°C/60% RH or 30°C/75% RH or 5° C, for example up to 4 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 5 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 6 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 7 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 8 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 9 days at 25°C/60% RH or 30°C/ 75% RH or 5°C, for example up to 10 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 14 days at 25°C/60% RH or 30° C/75% RH or 5°C, for example up to 18 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 22 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 26 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 30 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example up to 2 months at 25°C/60% RH or 30°C/75% RH or 5°C, or alternatively for 5 minutes to 2 months , for example from 5 minutes to 1 month, for example from 5 minutes to 14 days, for example from 5 minutes to 7 days, for example from 5 minutes to 4 days.

Said topical composition is suitable for topical use in a mammal, particularly a human. All components in the aqueous topical composition of the invention must be suitable for use on the surface of the body in question, that is, they must be approved for topical application to the surface of the body at the concentrations used and/or be dermatologically and cosmetologically acceptable.

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The aqueous topical composition of the present invention can be administered to a mammal, preferably a human, through a wide range of topical forms.

In the context of the present invention, topical compositions are epicutaneous, meaning that they are applied directly to the skin or mucous membranes.

The aqueous topical composition of the invention may be formulated as a cream, drops, foam, gel, lotion, mousse, ointment, paste, poultice, powder, balm, serum, spray, thickened composition, toothpaste or ointment. ). In a preferred embodiment, the aqueous topical composition of the invention is selected from a cream, gel or lotion.

Dry composition.

In the context of the present invention, the expression dry composition is intended to mean that the probiotic bacteria of the present invention are initially provided in a dry composition that is ground into a powder having a water activity of no more than 0.30 and a particle size distribution in the region of less than 500 microns. In one embodiment, there is provided an aqueous topical composition of the invention, wherein said dry composition has a particle diameter of no more than 500 microns, for example no more than 400 microns, for example no more than 300 microns, for example no more than 200 microns, for example no more than 150 µm, for example not more than 100 µm. In a preferred embodiment, said dry composition has a particle diameter of no more than 200 microns. Said dry composition is prepared by conventional freeze-drying and/or spray-drying processes followed by conventional milling and sieving to obtain the desired particle size distribution in the sub-500 micron region.

The dry composition of the present invention contains bacteria in a dried form, which can be obtained, for example, by freeze drying, spray drying or lyophilization.

Before the bacteria are dried, such as freeze-dried, they are typically mixed with a cryoprotectant to achieve high viability. The term cryoprotectant is used in the context of the present invention to define a substance capable of improving survival during freezing and/or drying and improving the stability of bacteria during storage. The cryoprotectant used here preferably contains a saccharide.

The saccharide may be a mono-, di-, oligo- or polysaccharide or a mixture of at least two saccharides. Beneficial monosaccharides include glucose (also known as dextrose), fructose, ribose and galactose, and beneficial disaccharides include sucrose, trehalose, maltose and lactose, among others. The composition may contain one or more mono- or disaccharides, for example one, two or three or even more different saccharides.

As an example, the cryoprotectant may contain a mixture of a disaccharide, such as sucrose, and a polysaccharide, such as maltodextrin.

The cryoprotectant may additionally contain a peptide, protein, protein hydrolysate, or a mixture thereof. Examples of peptides and proteins for use may be casein, pea, whey, albumin, soy protein, glutamic acid or gelatin, and any isolate or hydrolyzate thereof. Other additives may also be present, such as antioxidants such as ascorbate, sodium citrate, propyl gallate.

The dry composition of the present invention can be defined as having no available water and substantially free of water, and these expressions are used interchangeably and are intended to mean that the water present in the first compartment containing viable bacteria of at least one strain of probiotic bacteria , is not able to solvate probiotic bacteria because water is present in too small an amount. This also means that the water activity is no more than 0.30, eg no more than 0.20, eg no more than 0.15 in the dry composition. Preferably, the water activity is no more than 0.30 in the dry composition of the present invention.

In the context of the present invention, the term anhydrous, with respect to viable bacteria of at least one strain of probiotic bacteria, means viable probiotic bacteria substantially free of water. In particular, for such viable bacteria of at least one strain of probiotic bacteria, no significant amount of water has been added, and the amount of unbound water in the viable bacteria of at least one strain of probiotic bacteria is very small. Some components in probiotic bacteria may include some bound water, that is, crystallized water in the form of hydrates or sugars that have water attached to them. However, this water is not directly available to participate in hydrolysis reactions with water-sensitive components, such as lyophilized viable bacteria of at least one strain of probiotic bacteria. In preferred embodiments, the anhydrous viable bacteria of at least one strain of probiotic bacteria of the present invention contain less than 20% (w/w) water, such as less than 16% (w/w) water, such as less than 12% (w/w) water. wt.) water, for example less than 8% (wt./wt.) water, for example less than 7% (wt./wt.) water, for example less than 6% (wt./wt.) water, for example less than 5% (wt./wt.) water ./wt.) water, for example less than 4% (wt./wt.) water, 3% (wt./wt.), 2% (wt./wt.), 1.5% (wt./wt.) ), 1% (w/w),

- 15 045049

0.5% (w/w), for example less than 0.2% (w/w) water, based on the total weight of the composition. Preferably, the amount of water added to the anhydrous viable bacteria of at least one strain of probiotic bacteria is less than 3% (w/w), such as less

2% (w/w), 1% (w/w), 0.5% (w/w), for example less than 0.1% (w/w).

Water activity.

In the context of the present invention, the terms water activity or a _w denote the partial vapor pressure of water in a substance divided by the partial vapor pressure of water in the standard state. Pure distilled water has a water activity of exactly one. As temperature increases, a _w usually increases.

Viable/viable bacteria of at least one strain of probiotic bacteria.

In the context of the present invention, the term viable is intended to mean that the probiotic bacteria of the present invention are capable of normal growth and development, i.e., the term viable, in relation to bacteria of at least one strain of probiotic bacteria, means that the probiotic bacteria are viable and therefore capable of normal growth and development. growth and development. In the present invention, viable bacteria of at least one strain of probiotic bacteria are dried so as to keep the probiotic bacteria viable. Several methods for obtaining viable bacteria of at least one strain of probiotic bacteria are well known in the art.

Buffers.

In the context of the present invention, the term buffer refers to an acid or base used to maintain the acidity (pH) of a solution near a selected value after the addition of another acid or base. Therefore, buffers maintain the pH balance of the aqueous topical composition of the present invention to make the composition less irritating. The one or more buffers are selected from buffers with a pKa in the range 2-9, for example 2-7. The currently preferred buffers are monovalent cations. Even in an even more preferred embodiment of the invention, the one or more buffers are selected from the monovalent cations of carbonates, ascorbates, phosphates, malates, citrates, tartrates, for example sodium bicarbonate, sodium malate, sodium tartrate, potassium tartrate, sodium carbonate and anhydrous trisodium citrate. However, other methods of adjusting the pH of an aqueous topical composition may also be acceptable. By way of example, triethanolamine and sodium hydroxide may be useful for certain embodiments.

In one embodiment, one or more buffers are present in the dry composition and/or are added to a water-miscible solvent when it is desired to control the pH at a certain level. This may be important for controlling viscosity when using, for example, certain carbomers according to the present invention. In a preferred embodiment of the present invention, one or more buffers are present in the final product, that is, the aqueous topical composition of the invention.

The buffer is important for the functioning of the carbomer in the present invention because the carbomer has a low viscosity at low pH, since then the carbomers do not swell (absorb and retain water). When the pH increases, the carbomer swells and thus increases the viscosity. However, a buffer in the presence of carbomer provides the ability to control viscosity.

A water-miscible solvent.

In the context of the present invention, the expression water-miscible solvent means a solvent that can be mixed with water without any phase separation. In particular, water-miscible solvents include hydrophilic solvents. Such solvents include, for example, lower alkyl alcohols, hydrophilic limiting solvents and water. A hydrophilic substance is a molecule or other molecular structure that is attracted to water molecules and tends to dissolve in water. In the context of the present invention, the expression alkyl alcohol means, in the broadest sense, an organic alcohol consisting of a branched or linear alkyl chain and one or more than one hydroxyl group attached thereto. Thus, in a preferred embodiment, the water-miscible solvent is selected from the group consisting of C ₂ -C ₅ alkyl alcohol, glycerol, polyethylene glycol, propylene glycol and C ₂ -C ₅ glycol. In a particularly preferred embodiment, the water-miscible solvent is one or more of water, glycerin, polyethylene glycol and/or propylene glycol. Even in an even more preferred embodiment, said water-miscible solvent is water. The amount of said water is at least 60% (w/w), for example at least 65% (w/w), for example at least 70% (w/w), for example at least 75 % (w/w), for example at least 80% (w/w), for example at least 85% (w/w), for example at least 90% (w/w) , for example, at least 95% (wt./wt.) in the specified mixture according to the invention or in the specified aqueous composition for topical use according to the invention. In the examples of the present invention, said water-miscible

- 16 045049 the solvent is water in an amount greater than 90% (w/w) of the aqueous topical composition of the invention. In a preferred embodiment, the acid or base can be added to the water-miscible solvent to adjust the pH.

Thickener.

The term thickener according to the present invention is defined as a compound capable of increasing the viscosity of an aqueous topical composition as defined herein. A commercially available homogeneous aqueous topical composition, as defined herein, for topical application is formulated at a suitable viscosity for application immediately prior to use. Suitable thickeners are selected from one or more of polyacrylic acid (carbomer) or its derivatives or other rapidly thickening polymers, for example hyaluronic acid, gelatin, pectin, carrageenan and methylcellulose. Preferred thickeners are selected from one or more of polymers, carbomer, poloxamer, PEG/macrogol and/or carboxylic acid. In an even more preferred embodiment, the thickeners are selected from one or more types of acrylic acid comonomer and C10-30 alkyl acrylate and a cross-linked polyacrylic acid copolymer.

Carbomer solutions have the important property that they are able to absorb and retain water and swell with an increase in volume to many times their original volume. The degree of swelling depends on the pH of the solution, so that gelation occurs when the pH rises above a certain point, depending on the particular carbomer that is used. Thus, if the aqueous topical composition of the present invention contains a carbomer and has a pH at which it swells, the aqueous topical composition will have a viscosity that makes it suitable for topical application.

Poloxamer solutions have the important property that they exhibit temperature-dependent self-assembly and thermogelation behavior. This means that aqueous solutions of poloxamers are liquid at low temperatures and form a gel at higher temperatures in a reversible process. Thus, if the aqueous topical composition of the present invention contains a poloxamer, the aqueous topical composition will have a low viscosity at low temperature, for example, when the aqueous topical composition is stored in a refrigerator. Likewise, if said aqueous topical composition is stored at room temperature, said aqueous topical composition will have a viscosity that makes it suitable for topical use.

In a preferred embodiment, one or more thickeners are present in a water-miscible solvent, optionally pH adjusted. Even in an even more preferred embodiment, the one or more thickening agent is one or more carbomer present in the water-miscible solvent. In an alternative embodiment, the one or more thickening agent is one or more poloxamers present in the water-miscible solvent.

Mixture.

According to the present invention, the term mixture is defined as a mixture obtained by mixing at least one water-miscible solvent with at least one thickening agent and optionally a buffer or other components as defined herein, to obtain a thickened mixture, that is, having a high viscosity, than the at least one water-miscible solvent itself.

Viscosity.

The water-miscible solvent should have a low to medium dynamic viscosity and should be flowable, i.e. preferably have a viscosity of less than 2500 cP (mPa-s) when measured at 20 rpm at 20°C/60% RH , for example less than 2400 cP (mPa-s), for example less than 2200 cP (mPa-s), for example less than 2000 cP (mPa-s), for example less than 1000 cP (mPa-s). When a water-miscible solvent is mixed with a dry composition, the viscosity of the mixture increases 31,000 times and takes on medium to high values, that is, 2500 cP (mPa-s.) or more when measured at 20 rpm at 20°C. 60% RH, more preferably greater than 5000 cP (mPas) when measured at 20 rpm 20°C/60% RH. Viscosity is measured using a standard Brookfield laboratory viscometer. An even more precise measurement can be made using the TA Instruments HR-1 Discovery hybrid rheometer with a 40 mm parallel plate on a Peltier steel plate at a constant shear rate of 2.0 s ^-1 .

Colony-forming units (CFU).

In the context of the present invention, the term CFU is intended to mean a colony forming unit, and the number of colony forming units per gram of product (CFU/g) can be measured as disclosed in the Examples section. Most gels, lotions and creams have a density between 0.9 g/ml and 1.1 g/ml, therefore CFU/g is for all practical purposes identical to CFU/ml, and the values for one are comparable to those for the other, mainly for formulations based on water based.

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Oils and wax.

In the context of the present invention, oils and waxes, which are a subset of hydrophobic compounds, as those terms are used herein, are vegetable and/or mineral oils and/or waxes. In one embodiment, the oils and waxes for use in the composition of the invention are selected from oils and waxes that are solid or liquid at room temperature.

Oils and waxes that are liquid at room temperature may be selected from one or more of ethyl oleate, enomer oil, grape seed oil, hydrogenated vegetable (palm) oil, isopropyl isostearate, isopropyl myristate, isopropyl palmitate, jojoba seed oil, liquid lanolin, macadamia oil, medium chain triglycerides, olive oil, paraffin oil/mineral oil, pomegranate seed oil, canola seed oil, rice bran oil, rose hip oil, safflower oil, sesame oil, shea nut butter, soybean oil, sunflower oil, butter from sweet almonds, trimyristin, tripalmitin, tristearin and avocado oil. In a preferred embodiment, the oil or wax, which is liquid at room temperature, is selected from one or more of paraffin oil, almond oil and jojoba oil. In an even more preferred embodiment, the oil and wax that is liquid at room temperature is paraffin oil.

Oils and waxes that are solid at room temperature, as used herein, are selected from one or more of anionic emulsifying wax, candelilla wax, carnauba wax, cetyl palmitate, cocoa butter, gum arabic, tallow, microcrystalline wax, non-ionic emulsifying wax, paraffin, shea butter, artificial beeswax, white wax, xanthan gum, sunflower wax.

In a preferred embodiment, the oil or wax that is solid at room temperature is selected from one or more of anionic emulsifying wax, candelilla wax, carnauba wax, microcrystalline wax, non-ionic emulsifying wax, artificial beeswax, white wax and/or sunflower wax . In an even more preferred embodiment, the oil and waxes, which are solid at room temperature, are selected from one or more of cetyl palmitate, cocoa butter, gum arabic, tallow, paraffin, shea butter and/or xanthan gum.

In an even more preferred embodiment, a combination of liquid and solid oils and waxes is used, such as a combination of at least one solid oil and at least one liquid oil, such as a combination of at least two liquid oils and at least one solid oil, or vice versa, such as a combination of at least three liquid oils and at least one solid oil or vice versa.

Delivery device.

In the context of the present invention, the term delivery device is intended to mean a device suitable for delivering viable bacteria of at least one strain of probiotic bacteria to a user of the device. More specifically, the delivery device includes first and second compartments that provide an interface between the delivery device and a user.

According to the present invention, at least one type of probiotic bacteria must be protected from contamination and/or moisture in a hermetically sealed compartment (called the first compartment), thereby providing a stable environment for long-term storage of the probiotics. Likewise, the at least one water-miscible solvent must be protected from external contamination in a separate hermetically sealed compartment (called the second compartment), thereby providing a stable environment for long-term storage of the water-miscible solvent. Therefore, once the user is ready to use the aqueous topical composition of the invention, the sealing layers of both compartments are destroyed and the contents of the compartments are mixed. In one embodiment of the invention, mixing may be performed in the first compartment or in the second compartment. Also, mixing can be done in a third compartment suitable for such mixing, such as a cup, glass and spoon, or in the hand.

If mixing is performed in the first compartment, then the first compartment also serves as a device for delivering the device to the user. If mixing is performed in the second compartment, then the second compartment also serves as a device for delivering the device to the user. If mixing is performed in a third compartment suitable for receiving and mixing the contents of the first and second compartments, then the third compartment serves as a device for delivering the device to a user.

In a preferred embodiment, the first and second compartments are connected to each other during storage, mixing and delivery to the user of the device. In an alternative embodiment, the first, second and third compartments are connected to each other during storage, mixing and delivery to the user of the device.

In another preferred embodiment, the first and second compartments are separate objects and mixing is performed in a third suitable compartment, such as a cup, glass and spoon, or in the hand.

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In an alternative embodiment, a delivery element, such as a pump, may be located in the first, or second, or third compartment and be adapted to produce a mixture of viable bacteria of at least one strain of probiotic bacteria from the first compartment and at least one water miscible solvent from the second compartment.

In some embodiments, the delivery element is designed such that it can be pushed into the first, or second, or third compartment (push fit). In some embodiments, the delivery element is designed such that it can be articulated with the first, or second, or third compartment. In some embodiments, the delivery element is configured such that it can be screwed into the first, or second, or third compartment. In a preferred embodiment, the delivery element is either a first or a second compartment.

The delivery device or compartment of the present invention may further comprise at least one opening for releasing the contents into the environment. Such openings may extend to the top of the compartment according to the invention, and the lid may protect the contents of the compartments from environmental contamination and prevent the leakage of volatile substances into the environment.

Compartment.

In the context of the present invention, the outer shell of the delivery device is herein designated by the term compartment. In one embodiment, there is no more than one compartment that completely covers the second compartment, however, preferably 2 compartments exist separately, for example 3, for example 4. Preferably, the first and second compartments exist as described here, either separately or tightly connected by a delivery element , or connected to each other in the manner described here, for example with a reusable lid. The compartment is a component that the user holds to use the delivery device. The compartment itself can also serve as a delivery device. The compartment protects the contents inside the compartment from coming into contact with the environment, that is, the compartment can protect the contents of the compartment from contamination from the environment and prevent the leakage of substances into the environment. In one embodiment of the invention, the compartment contains a desiccant.

In embodiments of the invention, the compartment is made of suitable materials such as rubber, plastic (PE, PET), glass or metal such as aluminum or steel. The materials may also be coated, for example, with a heat-resistant metal, such as a rubber or plastic coating. The body material may also form a sachet, ampoule or the like.

The compartment may have a height of less than 20 cm, for example less than 15 cm, preferably less than 12 cm, for example less than 10 cm, for example less than 8 cm, for example less than 6 cm, for example less than 4 cm, and a diameter of less than 6 cm, for example less than 5 cm, for example less than 4 cm, for example less than 2 cm.

Relative humidity (RH).

In the context of the present invention, the term RH is intended to denote Relative Humidity and denotes the ratio of the partial pressure of water vapor to the equilibrium vapor pressure of water at a certain temperature.

Active components.

In the context of the present invention, the term active ingredient is defined as one or more compounds selected from the group of vitamins, minerals, antiseptics, preservatives, sunscreen agents, moisture-sensitizing agents or various other agents. The vitamins may be selected from vitamin A, for example retinol/retinyl esters, vitamin B, for example nicotinamide/niacin, vitamin D, for example vitamin D3, and/or vitamin E, for example various types of tocopherol, for example d-a-Tocopherol, d1-α- Tocopherol, γ-tocopherol or tocotrienols. Minerals can be selected from zinc and fluorine.

Antibacterial agents/antiseptics may be selected from salicylic acid, azelaic acid, benzoyl peroxide. Antiseptics for use according to the present invention should not kill the probiotic bacteria of the present invention or inhibit their growth when the probiotic bacteria are in a dry state, that is, have a water activity of no more than 0.3. Also, in the aqueous topical composition of the present invention, the antiseptics should not kill probiotic bacteria or inhibit their growth.

Preservatives may be selected from those well known in the art, such as sorbic acid, sodium sorbate, benzoic acid, sodium benzoate and hydroxybenzoate benzoates, lactic acid, propionic acid, sodium propionate and parabens, for example one or more than one of methylparaben, ethylparaben, propylparaben, butylparaben, heptylparaben, isobutylparaben, isopropylparaben, benzylparaben and their sodium salts. In one embodiment of the invention, the preferred preservative is selected from one or more of sorbic acid or benzoic acid. Preferably, the preservatives will be contained in the second compartment, that is, in the water-miscible solvent.

Sunscreen agents may be selected from para-aminobenzoic acid, Padimate O, phenylbenzimidazole sulfonic acid, Cinoxate, Dioxybenzone, Oxybenzone, Homosalate, Menthylan-19 045049 tranilate, Octocrylene, Octylmethoxycinnamate, Octyl salicylate, Sulisobenzone, Trolamine salicylate, Avobenzone, Ecamsule, titanium dioxide, zinc oxide , 4-methylbenzylidene camphor, Parsol Max,

Tinosorb M, Parsol Shield, Tinosorb S, Tinosorb A2B, Neo Heliopan AP, Mexoryl XL, Benzophenone-9, Uvinul

T 150, Uvinul A Plus, Uvasorb HEB, Parsol SLX, Amyloxate.

Various other agents may be selected from arginine, cranberry, tea tree oil, xylitol or extracts such as extracts from aloe vera, chamomile, milk thistle, walnut, witch hazel, dried burdock, dried licorice or sage.

The moisture-sensing/active water-sensing agents may be selected from proteinase/protease enzymes such as papain, bromelain and actinidia. Moisture-sensitive agents are defined as compounds that lose their activity upon contact with water.

Dehumidifier.

In the context of the present invention, the term desiccant refers to a hygroscopic substance that induces or maintains a state of dryness (dehydration) in its vicinity. Suitable desiccants are selected from one or more of activated carbon, calcium sulfate (drierite), zeolites, anhydrous calcium chloride, bentonite/clay, calcium oxide, calcium sulfate, molecular sieve and silica gel. Desiccants may be in the form of a sachet or porous pouch, and silica is a well-known type of desiccant that is suitable in the context of the present invention. The most preferred drying agent is a molecular sieve and/or silica gel. Other preferred desiccants are selected from one or more of anhydrous calcium chloride, bentonite/clay, calcium oxide, calcium sulfate, molecular sieve and silica gel.

In one embodiment of the invention, the desiccant is integrated into the first compartment, for example, as an integral part of the internal walls of the first compartment. Additionally, the first compartment may be a sachet having a desiccant coating on the inside. In an alternative embodiment, the desiccant is integrated into the first compartment by providing it with an integrated desiccant plastic sleeve. In another embodiment of the invention, the desiccant may be contained in a first compartment containing one or more types of probiotic bacteria or one or more moisture-sensing agents.

Hydrophobic compounds.

In the context of the present invention, the term hydrophobic compounds as used herein are compounds that are non-polar and thus prefer other naturally occurring neutral molecules and non-polar solvents.

Examples of hydrophobic compounds include alkanes, oils, fats and fatty substances in general.

Emulsifiers.

In the context of the present invention, an emulsifier is a compound capable of reducing the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. According to the present invention, an emulsifier is capable of reducing the surface tension between one or more water-miscible solvents of the invention and one or more hydrophobic compounds of the invention to provide an aqueous topical composition as defined herein. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents and dispersing agents, and are selected from one or more of alkyl (C ₁₂ . ₁₅ ) benzoate, capryl/capramidopropyl betaine, ceteareth-25, cetomacrogol, emulsifying wax , cetomacrogol-1000, cetostearyl alcohol, cetyl alcohol, cetyl ester wax, cetyl palmitate, cetyl PEG/PPG (polypropylene glycol)-10/1 dimethicone, emulsifying wax, ethyl oleate, monoglycerides, e.g. glycerol monostearate, glycerol monopalmitate, glycerol monooleate, di glycerides, for example glycerol dioleate, glycerol dipalmitate, glycerol distearate, mono and diglycerides, eg glyceryl behenate, glycerol stearate, glycerol laurate, glycerol palmitate, mono-, di- and triglycerides, eg glyceryl behenate, glycerol stearate, glycerol laurate, glycerol palmitate, hydrogenated cocoglycer ides, cetostearyl macrogol ester, macrogol glycerol ricinoleate, macrogol lauryl ether, macrogol oleyl ether, macrogol stearate, macrogol stearyl ether, methyl glucose isostearate, PEG-120 methyl glucose dioleate, PEG-100 stearate, polyglyceryl isostearate, polyglyceryl oleate, stearyl ether polyoxypropylene, polysorbate 20, polysorbate 60, polysorbate 80, propylene glycol dicaprylate/caprate, ricinolamidopropyl betaine, sorbitan isostearate, sorbitan laurate, sorbitan oleate, sorbitan sesquilate, sorbitan stearate, sorbitan trioleate, steareth-2, stearyl alcohol, sucrose stearate, sucrose palm itata, sucrose behenate, sucrose myristate, sucrose laurate, sucrose oleate, sucrose erucate, mixed fatty acid ester and sucrose, undecylenamidopropyl betaine, esters of mono- and diglycerides and citric acid, citrilated glycerol monooleate, citrilated glycerol monostearate, esters mono- and diglycerides and lactic acid, high molecular weight copolymers of acrylic acid, C ₁₀ -C ₃₀ -alkyl acrylate cross-linked with allyl pentaerythritol, glyceryl behenate and/or glycerol stearate.

- 20 045049

In a preferred embodiment, the emulsifier is selected from one or more of monoglycerides, diglycerides, triglycerides, esters of mono- and diglycerides and citric acid, glyceryl behenate/or glycerol stearate and, high molecular weight copolymers of acrylic acid, C ₁₀ -C ₃₀ -alkyl acrylate, cross-linked cross-linked with allylpentaerythritol. In an even more preferred embodiment, said emulsifier is selected from one or more of glyceryl behenate or glycerol stearate.

In a preferred embodiment, the emulsifier is present only in the water-miscible solvent.

Ledge-valley configuration.

In a protrusion-recess configuration, the recess usually receives and retains the protrusion. A portion bearing one or more than one projection or fitting within another is designated as a projection, while a portion containing corresponding recesses or fittings outside another is referred to as a recess.

A protrusion-valley configuration is a configuration where two mechanical elements, here a first compartment for retaining viable bacteria of at least one strain of probiotic bacteria and a second compartment for retaining a water-miscible solvent, are aligned such that at least a portion of one element, such as the first compartment, received part of another element, for example, a means for retaining the hydrophobic phase, such as in a lock/key or socket/plug configuration. In one embodiment, the projection-recess configuration is adapted in the form of a stick or bottle with a cap, preferably a reusable cap.

In the present invention, the first compartment may include both a recess portion and a projection portion. In the same way, the means for retaining the hydrophilic phase, that is, the second compartment can also comprise both a recessed part and a protruding part. The recess portion of the hydrophilic phase retaining means may be complementary to the protrusion portion of the first compartment, and the protrusion portion of the hydrophilic phase retaining means may be complementary to the recess portion of the first compartment.

Tight fit configuration.

In some embodiments, the lip-valley configuration of the first compartment and the means for retaining the hydrophilic phase, that is, the second compartment, constitute a tightly fitted configuration.

A tight fit is a fit of mechanical parts without play, such as the tightest fit that can be assembled for parts that are not expected to move relative to each other. The advantage of a tight fitting configuration is that it provides maximum leakage protection between surfaces that are in contact with each other.

Viable in an aqueous composition for topical use.

In the context of the present invention, the expression viable in an aqueous topical composition is intended to indicate the ability of probiotic bacteria to remain viable in an aqueous topical composition in the range of 1-106-10 ¹¹ CFU/g viable bacteria of at least one strain of probiotic bacteria for at least at least 5 minutes at 25°C/60% RH or 30°C/75% RH or 5°C, for example for at least 20 minutes at 25°C/60% RH or 30°C/75% RH or 5 °C, for example at least 1 hour at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 4 hours at 25°C/60% RH or 30°C/75 % RH or 5°C, for example at least 12 hours at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 24 hours at 25°C/60% RH or 30 °C/75% RH or 5°C, for example at least 2 days at

25°C/60% RH or 30°C/75% RH or 5°C, for example at least 3 days at 25°C/60% RH or

30°C/75% RH or 5°C, for example at least 4 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 5 days at 25°C/ 60% RH or 30°C/75% RH or 5°C, for example at least 6 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 7 days at

25°C/60% RH or 30°C/75% RH or 5°C, for example at least 8 days at 25°C/60% RH or

30°C/75% RH or 5°C, for example at least 9 days at 25°C/60% RH or 30°C/75% RH or 5°C, for example at least 10 days at 25°C/ 60% RH or 30°C/75% RH or 5°C, for example at least 10-14 days at 25°C/60% RH or 30°C/75% RH or 5°C.

Percent w/w or % (w/w).

In the context of the present invention, the expressions percentage wt./wt. or % (w/w) are used interchangeably and indicate the percentage of a component or ingredient relative to the total weight of the composition, that is, the weight of the final composition, unless otherwise indicated. In certain cases, these expressions may refer to the mass fraction relative to the intermediate composition.

Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Although any methods and materials equivalent or similar to those described herein can be used in practical

- 21 045049 application of the present invention, typical methods and materials are described. All of the methods described herein may be performed in any suitable order unless otherwise indicated herein or unless otherwise clearly inconsistent with the context.

Nouns used in the description of the invention (especially in the claims) should be considered to cover both the singular and the plural, unless otherwise indicated herein or unless clearly inconsistent with the context. The terms comprising, having, including and containing are to be understood as non-limiting terms (that is, including but not limited to) unless otherwise indicated. The listing of ranges herein is intended to serve solely as a shorthand for individual reference to each individual value falling within that range unless otherwise noted herein, and each individual value is included in the description as if it were individually listed herein. All of the methods described herein may be performed in any suitable order unless otherwise indicated herein or unless otherwise clearly inconsistent with the context.

The use of any and all examples, or introductory words before an example (for example, such as) used herein is intended solely to better explain the invention and does not limit the scope of the invention unless otherwise stated. The terminology of the specification and claims should not be construed as indicating any unclaimed element essential to the practice of the invention.

It should be noted that embodiments and features described in the context of one aspect of the present invention are also applicable to other aspects of the present invention.

The invention will now be described in more detail in the following non-limiting examples.

Examples

Review of examples.

Examples 1.1-1.3: Preparation and stability of aqueous gels containing probiotic bacteria.

Example 2: Preparation and stability of an aqueous gel lotion containing probiotic bacteria.

Example 3.1-3.2: Preparation and stability of dry compositions containing probiotic bacteria.

Example 4: Stability of a commercially available product containing bacteria.

Materials and methods:

An overview of the excipients and equipment used is presented in Table. 1, and the types of probiotic bacteria used are presented in Table. 2.

The probiotic bacteria used are Lactobacillus rhamnosus, Lactobacillus paracasei and Bifidobacterium animalis subsp lactis, Esse Sensitive Serum from Esse skincare was purchased online. Esse Probiotic Serum (Series 18ADF) from Esse skincare was purchased from a retail chain in Copenhagen.

- 22 045049

Table 1

Excipients and equipment

Trademark Type Type Company Pemulen™ TR-1 NF Carbomer high molecular weight cross-linked copolymers of acrylic acid and hydrophobic C10-30 alkyl acrylate comonomer Lubrizol Carbopol Ultrez® 10 NF Carbomer Cross-linked copolymer polyacrylic acid Lubrizol EMPROVE® exp Ph Eur Buffer Sodium bicarbonate Merck KGaA Esse Sensitive Serum Esse Sensitive Serum (Series 16AXCE, expiration date September 2018) Jojoba seed oil, sesame oil, hydrogenated vegetable oil, shea butter, isoamyl laurate, lactobacilli, tocopherol, sunflower oil, ascorbyl palmitate, vanillin, gamma-decalactone. Esse skincare Esse Probiotic Serum Esse Probiotic Serum (Series 18ADF, expiration date August 2019) Jojoba seed oil, sesame oil, shea butter, hydrogenated vegetable oil, isoamyl laurate, marula seed oil, lactobacilli, tocopherol, sunflower oil, ascorbyl palmitate, vanillin, gamma-decalactone Esse skincare Carbopol ETD 2020 NF Carbomer Cross-linked copolymer polyacrylic acid Lubrizol Trisodium citrate dihydrate Buffer Trisubstituted dihydrate sodium citrate Jungbunzla uer Paraffin oil Oil Paraffin oil Henry Lamotte High Oleic Sunflower Oil Oil High Oleic Sunflower Oil AAK Maximum Recovery Diluent (MRD) Thinner maximum output (Maximum Thinner maximum Maximum Recovery Diluent (MRD) output Oxoid Oxoid CM0733 Recovery Diluent (MRD) MRS Arap pH 6.2 (Oxoid CM0361) MRS Arap pH 6.2 MRS Arap pH 6.2 (Oxoid CM0361) Oxoid Smasher, bioMerieux Stomacher Stomacher (Smasher) bioMerieux Cysteine, HCl (Merck 102839) Cysteine, HCl Cysteine, HCl (Merck 102839) Merck MColorpHast™ pH- indicator strips 2.0 9.0 Merck KGaA

-23 045049

table 2

Strains

Type Trademark No in depository Company Lactobacillus rhamnosus LGG® ATCC 53103 Chr. Hansen Lactobacillus paracasei F19® LMG-P-17806 Chr. Hansen Bifidobacterium animalis subsp lactis BB-12® DSM 15954 Chr. Hansen

Example 1 - Preparation and stability of an aqueous gel containing probiotic bacteria.

Table 3

Composition of three different batches received

Example 1.1 1.2 1.3 Ingredients in mg or g per batch (%, g/100 g gel product) Lactobacillus rhamnosus 4072.9 mg (4.1%) - - Lactobacillus paracasei - 3.55 g (3.5%) Bifidobacterium animalis subsp. lactis - - 3.79 g (3.79%) Trisodium citrate dihydrate 749.9 mg (0.75%) 0.75 g (0.75%) 0.75 g (0.75%) Carbomer (Carbopol Ultrez® 10 NE) 1500.4 mg (1.5%) 1.51 g (1.5%) 1.51 g (1.5%) Demineralized water 93.64 g (93.7%) 94.5 g (94.2%) 93.87 (93.94%) Total weight of ingredients 99.96 g (100%) 100.3 g (100%) 99.92 g (100%)

Example 1.1 - Preparation and stability of an aqueous gel containing Lactobacillus rhamnosus.

Lyophilized Lactobacillus rhamnosus was ground and then sieved through a 200 μm sieve and stored at -20°C. The powder was left to acclimatize before weighing the ground material to be mixed with the aqueous phase. Demineralized water in a 250 ml glass beaker was heated to boiling and then cooled to room temperature. Carbopol Ultrez® 10 NF was weighed and slowly added to the water while stirring on a magnetic stirrer. The mixture was left for up to one hour to completely hydrate the carbomer. The pH when measured with strips was 2.5-3.0. The number of bacteria and ingredients used in Example 1.1 is presented in table. 3. Lactobacillus rhamnosus was added with vigorous stirring to ensure completely uniform distribution; then trisodium citrate dihydrate was added with vigorous stirring. The entire experimental batch was immediately divided into portions into 2 bottles with screw caps. One bottle was placed at 5°C±3°C. At time points day 0, 3, 7 and 14, the total number of Lactobacillus rhamnosus cells was determined as the number of colony forming units per g of product gel (CFU/g). The second bottle was placed at 25°C±3°C/60±5% RH. At time points day 0, 1, 2, 3 and 4, the total number of Lactobacillus rhamnosus cells was determined as the number of colony forming units per g of gel product (CFU/g). The total number of viable cells of Lactobacillus rhamnosus was determined as the number of colony-forming units per g of product (CFU/g) using the deep plate method in MRS pH 6.2 agar (Oxoid CM0361). Triplicates of 2 g of product were mixed with 198 g of Maximum Recovery Diluent (MRD) (eg Oxoid CM0733) for 2 minutes at 450-550 strokes per minute using a Stomacher (eg Smasher, bioMerieux) to obtain a 1:100 dilution. Serial dilutions in MRD were prepared for each weighing and two dilutions/volumes were used corresponding to 30-300 CFU/plate (2x2 plates for each weighing). The dishes were incubated under anaerobic conditions at 37°C for 3 days. Colonies were counted and the results were calculated as the average of three weighings in CFU/g. The results for the stability indicator are presented in table. 4 and fig. 1a.

- 24 045049

Table 4

Comparison of temperatures and CFU/g values obtained from a stability study of an aqueous gel containing ___________________________ containing Lactobacillus rhamnosus___________________________

Time, Day Number of colony forming units per g (CFU/g) 5°С 25°C/ 60% RH 0 1,ЗЭ+10 1,ЗЭ+1О 1 ND 9DE+09 2 ND 1.0E+10 3 1.2E+10 8.4E+09 4 ND 7DE+09 7 9.9E+09 - 14 7DE+09 -

ND: not available.

Conclusions from Example 1.1

The results presented in Fig. 1a show the stability of 4.1% Lactobacillus rhamnosus in the aqueous topical composition of the present invention at 5°C (circles, upper curve) and at 25°C/60% RH (squares, lower curve). It is evident that the stability of the aqueous topical composition of the present invention containing Lactobacillus rhamnosus at 25° C./60% RH is very good, since said Lactobacillus rhamnosus in an amount of at least 10 ⁹ CFU/g is viable on day 4. Also It is obvious that the stability of the aqueous topical composition of the present invention containing Lactobacillus rhamnosus at 5°C is even better, since said Lactobacillus rhamnosus in an amount of at least 10 ⁹ CFU/g is viable on day 14.

Therefore, the aqueous topical composition of the present invention has been shown to have the ability to keep the probiotic bacteria Lactobacillus rhamnosus viable in unexpectedly large numbers and for an unexpectedly long time in an aquatic environment, i.e. at least 4 days at 25°C/60 % RH and at least 14 days at 5°C. The limited reduction in the number of viable cells is unexpected and very promising for a commercial product containing the aqueous topical composition of the invention with a shelf life of at least 14 days at 5°C and at least 4 days at 25°C/60% RH.

Example 1.2 - Preparation and stability of an aqueous gel containing Lactobacillus paracasei.

Lyophilized Lactobacillus rhamnosus was ground and then sieved through a 200 μm sieve and stored at -20°C. The powder was left to acclimatize before weighing the ground material to be mixed with the aqueous phase. The demineralized water was autoclaved and then cooled to room temperature. The carbomer was weighed into the Blue Cap flask and water was added to the Blue Cap flask while stirring on a magnetic stirrer. The mixture was left for 2 days for complete hydration of the carbomer. The pH measured using strips was 2.5-3.0. The number of bacteria and ingredients used in Example 1.2 is presented in table. 3. Lactobacillus paracasei was added with vigorous stirring to ensure completely uniform distribution, then trisodium citrate dihydrate was added with vigorous stirring. The entire experimental batch was immediately divided into portions into 2 bottles with screw caps. One bottle was placed at 5°C±3°C. At time points day 0, 3, 7 and 14, the total number of Lactobacillus paracasei cells was determined as the number of colony forming units per g of gel product (CFU/g). The second bottle was placed at 25°C±3°C/60±5% RH. At time points day 0, 1, 2, 3, 4, and 7, the total number of Lactobacillus paracasei cells was determined as the number of colony forming units per g of gel product (CFU/g). The total number of viable cells of Lactobacillus paracasei was determined as the number of colony-forming units per g of product (CFU/g) using the deep plate method in MRS pH 6.2 agar (Oxoid CM0361). Triplicates of 2 g of product were mixed with 198 g of Maximum Recovery Diluent (MRD) (eg Oxoid CM0733) for 2 minutes at 450-550 strokes per minute using a Stomacher (eg Smasher, bioMerieux) to obtain a 1:100 dilution. Serial dilutions in MRD were prepared for each weighing and two dilutions/volumes were used corresponding to 30-300 CFU/plate (2x2 plates for each weighing). The dishes were incubated under anaerobic conditions at 37°C for 3 days. Colonies were counted and the result was calculated as the average of three weighings in CFU/g. The results for the stability indicator are presented in table. 5 and fig. 16.

- 25 045049

Table 5

Comparison of temperatures and CFU/g values obtained from a stability study of an aqueous gel containing ________________________containing Lactobacillus paracasei____________________

Time, Day Number of colony forming units per g (CFU/g) 5°С 25°C/ 60% RH 0 1.60E+10 1.60E+10 1 ND 1.10E+10 2 nd 7.70E+09 3 1.50E+10 7.9E+09 4 ND 6.6E+09 7 1,ЗЭ+10 4,ЗЭ+09 14 1.1E+10 ND

ND: not available.

Conclusions from example 1.2.

The results presented in Fig. 16 show the stability of 3.5% Lactobacillus paracasei in an aqueous topical composition of the present invention at 5°C (circles, upper curve) and at 25°C (squares, lower curve). It is evident that the stability of the aqueous topical composition of the present invention containing Lactobacillus paracasei at 25°C/60% RH is very good, since said Lactobacillus paracasei in an amount of at least 10 ⁹ CFU/g is viable on day 7.

It is also evident that the stability of the aqueous topical composition of the present invention containing Lactobacillus paracasei at 5° C. is even better, since said Lactobacillus paracasei in an amount of at least 10 ¹⁰ CFU/g is viable on day 14. Therefore, it has been shown that that the aqueous topical composition of the present invention has the ability to keep the probiotic bacteria Lactobacillus paracasei viable in unexpectedly large numbers and for an unexpectedly long time in an aquatic environment, i.e. at least 7 days at 25°C/60% RH or 30°C C/75% RH and at least 14 days at 5°C.

Example 1.3 - Preparation and stability of an aqueous gel containing Bifidobacterium animalis subsp lactis.

Lyophilized Bifidobacterium animalis subsp lactis was ground and then sieved through a 200 μm sieve and stored at -20°C. The powder was left to acclimatize before weighing the ground material to be mixed with the aqueous phase. Trisodium citrate dihydrate was stored at room temperature and did not require acclimation before weighing and mixing with the liquid phase. The demineralized water was autoclaved and then cooled to room temperature. The carbomer was weighed into the Blue Cap flask and water was added to the Blue Cap flask while stirring on a magnetic stirrer. The mixture was left for 2 days for complete hydration of the carbomer. The pH measured using strips was 2.5 - 3.0. The number of bacteria and ingredients used in Example 1.3 is presented in table. 3. Bifidobacterium animalis subsp lactis was added with vigorous stirring to ensure completely uniform distribution; then trisodium citrate dihydrate was added with vigorous stirring. The entire experimental batch was immediately divided into portions into 2 bottles with screw caps. One bottle was placed at 5°C±3°C. At time points day 0, 3, 7 and 14, the total number of Bifidobacterium animalis subsp lactis cells was determined as the number of colony forming units per g of gel product (CFU/g). The second bottle was placed at 25°C±3°C/60±5% RH. At time points day 0, 1, 2, 3, 4 and 7, the total number of Bifidobacterium animalis subsp lactis cells was determined as the number of colony forming units per g of gel product (CFU/g).

The total number of viable cells of Bifidobacterium animalis subsp lactis was determined as the number of colony-forming units per g of product (CFU/g) by subplating in MRS pH 6.2 agar (Oxoid CM0361) supplemented with 0.05% cysteine, HCl (Merck 102839). Triplicates of 2 g of product were mixed with 198 g of Maximum Recovery Diluent (MRD) (eg Oxoid CM0733) for 2 minutes at 450-550 strokes per minute using a Stomacher (eg Smasher, bioMerieux) to obtain a 1:100 dilution. Serial dilutions in MRD were prepared for each weighing and two dilutions/volumes were used corresponding to 30-300 CFU/plate (2x2 plates for each weighing). The dishes were incubated under anaerobic conditions at 37°C for 3 days. Colonies were counted and the result was calculated as the average of three weighings in CFU/g. The results for the stability indicator are presented in table. 6 and FIG. 2a.

- 26 045049

Table 6

Comparison of temperatures and CFU values obtained from a stability study of an aqueous gel containing ____________________containing Bifidobacterium animalis subsp lactis.___________________

Time, Day Number of colony forming units per g (CFU/g) 5°С 25°C/ 60% RH 0 2.8E+10 2.8E+10 1 ND 2.0E+10 2 ND 1.6E+10 3 1.8E+10 1.5E+10 4 ND 8.9E+09 7 1.6E+10 1.7E+09 14 6.5E+09 nd

ND: not available.

Conclusions from example 1.3.

The results are presented in Fig. 2a and show the stability of 3.79% Bifidobacterium animalis subsp lactis in the aqueous topical composition of the present invention at 5°C (circles, upper curve) and at 25°C (squares, lower curve). It is evident that the stability of the aqueous topical composition of the present invention containing Bifidobacterium animalis subsp lactis at 25° C./60% OB is very good, since said Bifidobacterium animalis subsp lactis in an amount of at least 10 ⁹ CFU/g is viable on day 7.

It is also evident that the stability of the aqueous topical composition of the present invention containing Bifidobacterium animalis subsp lactis at 5°C is even better, since said Bifidobacterium animalis subsp lactis in an amount of at least 10 ⁹ CFU/g is viable on day 14. Therefore , the aqueous topical composition of the present invention has been shown to have the ability to keep the probiotic bacteria Bifidobacterium animalis subsp lactis viable in unexpectedly large numbers and for an unexpectedly long time in an aquatic environment, i.e., at least 7 days at 25°C/ 60% RH or 30°C/75% RH and at least 14 days at 5°C.

Example 2 - Preparation and stability of an aqueous gel lotion containing Lactobacillus rhamnosus.

Table 7

Composition of the resulting aqueous gel lotion

Example 2 Ingredients in mg or g per batch (%, g/100 g of gel lotion product) Lactobacillus rhamnosus 4073 mg(4.1%) Sodium bicarbonate 600 mg (0.6%) Carbomer (Carbopol Ultrez® 10 NT) 0.99 g (1%) Carbomer (Pemulen) 250 mg (0.25%) Paraffin oil 15.02 g (15%) Demineralized water 79.16 g (79%) Total weight of ingredients 100 g

To obtain the mixture, autoclaved demineralized water at room temperature was mixed with carbomer powder, acrylic polymer, high molecular weight cross-linked copolymers of acrylic acid and hydrophobic C10-30 alkyl acrylate comonomer and paraffin oil, homogenized for 15-20 seconds using a homogenizer (increasing speed from approximately 3000 rpm /min to approximately 15,000 rpm and back to approximately 3,000 rpm). The pH measured using strips was 2.5-3.0.

Viscosity measured using a shear rate of 2.0 s ^-1 at 25° C. was approximately 200 mPa s.

Lyophilized Lactobacillus rhamnosus was weighed and added to the hydrated carbomer lotion and then shaken vigorously to ensure proper mixing. There was a noticeable increase in viscosity to some extent within 5-10 seconds after the addition of lyophilized bacteria, sodium bicarbonate was weighed and added to the mixture. The viscosity increased further within 27 045049 20-30 seconds.

Viscosity measured using a shear rate of 2.0 s ^-1 at 25°C was approximately

60000 mPa-s.

The resulting pH, measured using strips, was 4.0-4.5. The number of bacteria and ingredients used in Example 2 is presented in table. 7.

The entire experimental batch was immediately divided into portions into 2 bottles with screw caps. One bottle was placed at 5°C±3°C. At time points day 0, 3, 7 and 14, the total number of Lactobacillus rhamnosus cells was determined as the number of colony forming units per g of gel product (CFU/g). The second bottle was placed at 25°C±3°C/60±5% RH. At time points day 0, 1, 2, 3, 4, 7 and 14, the total number of Lactobacillus rhamnosus cells was determined as the number of colony forming units per g of product gel (CFU/g).

The total number of viable cells of Lactobacillus rhamnosus was determined as the number of colony-forming units per g of gel-lotion product (CFU/g) by deep plate method in MRS pH 6.2 agar (Oxoid CM0361). 2 g of product in triplicate were mixed with 198 g of pre-warmed (45°C) Maximum Recovery Diluent (MRD) (e.g. Oxoid CM0733) with 1% Tween 80 added for 2 minutes at 450-550 strokes per minute using a Stomacher ( for example Smasher, bioMerieux), obtaining a dilution of 1:100. Serial dilutions in MRD were prepared for each weighing and two dilutions/volumes were used corresponding to 30-300 CFU/per plate (2x2 plates for each weighing). The dishes were incubated under anaerobic conditions at 37°C for 3 days. Colonies were counted and the result was calculated as the average of three weighings in CFU/g. The results for the stability indicator are presented in table. 8 and FIG. 2b.

Table 8 Comparison of temperatures and CFU values obtained from a stability study of an aqueous lotion ________________________containing Lactobacillus rhamnosus________________________

Time, Day Number of colony forming units per g (CFU/g) 5°С 25°C/60% RH 0 1.5E+10 1.5E+10 1 ND 1.7E+10 2 ND 1.4E+10 3 1.6E+10 1.4E+10 4 ND 1,ЗЭ+10 7 1.5E+10 1.2E+10 14 1.4E+10 1.0E+10

ND: not available.

Conclusions from example 2.

The results are presented in Fig. 2b and in table. 8 and show the stability of 4.1% Lactobacillus rhamnosus in the aqueous topical composition of the present invention at 5°C (circles, upper curve) and at 25°C/60% RH (squares, lower curve).

It is apparent that the stability of the aqueous topical composition of the present invention containing Lactobacillus rhamnosus formulated as a gel lotion at 25° C./60% RH is surprisingly good since said Lactobacillus rhamnosus is at least 1 x 10 ¹⁰ CFU /g are viable on day 14.

It is also evident that the stability of the aqueous topical composition of the present invention containing Lactobacillus rhamnosus at 5° C. is equally good, since said Lactobacillus rhamnosus in an amount of at least 1x10 CFU/g, manufactured in the form of a gel lotion, is viable for a day 14.

Therefore, it has been shown that the aqueous topical composition of the present invention, formulated in the form of a gel lotion, has the ability to keep the probiotic bacteria Lactobacillus rhamnosus viable in unexpectedly large numbers and for an unexpectedly long time in the aqueous lotion environment, that is, at least 14 days at both 5°C and 25°C/60% RH.

Example 3 - Preparation and stability of a dry composition containing bacteria.

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Table 9

Composition of mixtures (i.e. dry compositions)

Example 3.1 3.2 Ingredients in mg per product (% of product) Lactobacillus rhamnosus 22.8 mg(11.4%) Bifidobacterium animalis subsp. Lactis 20 mg(10%) Sodium bicarbonate 10 mg (5%) 10 mg (5%) Inulin 50 mg (25%) 50 mg (25%) Maltitol 20 mg(10%) 20 mg(10%) Mannitol 97.2 mg (48.6%) 100 mg (50%) Total weight of ingredients 200 mg 200 mg

Example 3.1 - Stability of Lactobacillus rhamnosus in the first compartment.

A mixture consisting of Lactobacillus rhamnosus, sodium bicarbonate (buffer), inulin, maltitol and mannitol (excipients) was stirred. The number of bacteria and ingredients used in Example 3.1 is presented in table. 9.

200 mg of the mixture was then added to the first compartment for stability testing. The first compartments were placed at 25°C±3°C/60±5% RH. At time points 0, 1, 3, 6, 9, 12, 18, 24, 30, and 36 months, the total number of Lactobacillus rhamnosus cells was determined as the number of colony-forming units in the first compartment (CFU/first compartment). The total number of viable cells of Lactobacillus rhamnosus was determined as the number of colony-forming units in the first compartment (CFU/first compartment) by the deep plate method in MRS pH 6.2 agar (Oxoid CM0361). Triplicates of 200 mg of product were mixed with approximately 200 g of Maximum Recovery Diluent (MRD) (eg Oxoid CM0733) for 2 minutes at 450-550 strokes per minute using a Stomacher (eg Smasher, bioMerieux) to obtain a 1:1000 dilution. Serial dilutions in MRD were prepared for each weighing and two dilutions/volumes were used corresponding to 30-300 CFU/plate (2x2 plates for each weighing). The dishes were incubated under anaerobic conditions at 37°C for 3 days. Colonies were counted and the result was calculated as the average of three weighings in CFU/first compartment. The results for the stability indicator are presented in table. 10 and fig. 3a.

Table 10 Comparison of temperatures and CFU values obtained from a stability study of a mixture containing Lactobacillus rhamnosus at 25°C/60% RH or 30°C/75% RH. Comparison of temperatures and CFU values obtained from a stability study of a mixture containing Bifidobacterium animalis subsp lactis in the first compartment of the invention at 25°C/60% RH or 30°C/75% RH and 30°C/75% RH

Months Number of colony forming units per g (CFU/g) 25°C/60% RH 30°C±3°C/75±5% OB Lactobacillus rhamnosus Bifidobacterium animalis subsp lactis Bifidobacterium animalis subsp lactis 0 1,ЗЭ+10 1,ЗЭ+10 l,3E+10 1 9.0E+09 nd ND 3 9.6E+09 9DE+09 8.9E+09 6 9.9E+09 l.6E+10 l.5E+10 9 9.5E+09 ND ND 12 9,ZE+09 l,3E+10 8.0E+09 18 5.7E+09 8.3E+09 3.4E+09 24 4.1E+09 9.0E+09 ND thirty 4.2E+09 l,2E+10 4.5E+09 36 3.5E+09 1DE+10 ND

ND: not available.

Conclusions from example 3.1.

The results are presented in Fig. 3a and in table. 10 and show the stability of 11.4% Lactobacillus rhamnosus in the mixture of the present invention at 25°C/60% RH (squares).

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It is evident that the stability of the mixture in the first compartment according to the invention containing Lactobacillus rhamnosus at 25°C/60% RH is surprisingly good, since said Lactobacillus rhamnosus in an amount of at least 1x10 ⁹ CFU/g is viable for 36 months.

Therefore, it has been shown that the first compartment of the invention has the ability to keep the probiotic bacteria Lactobacillus rhamnosus viable in unexpectedly large numbers and for an unexpectedly long time in a mixture containing Lactobacillus rhamnosus, i.e. at least 36 months at 25°C/60% RH .

Example 3.2 - Stability of Bifidobacterium animalis subsp lactis in the first compartment A mixture consisting of Bifidobacterium animalis subsp lactis, sodium bicarbonate (buffer), inulin, maltitol and mannitol (excipients) was stirred. The number of bacteria and ingredients used in Example 3.2 is presented in table. 9.

200 mg of the mixture was then added to the first compartment for stability testing. The first compartments were placed at 25°C±3°C/60±5% RH and 30°C±3°C/75±5% RH. At time points 0, 3, 6, 12, 18, 24, 30 and 36 months (25°C/60% RH) and 0, 3, 6, 12, 18 and 30 months (30°C/75% RH) The total number of Bifidobacterium animalis subsp lactis cells was determined as the number of colony-forming units in the first compartment (CFU/first compartment). The total number of viable cells of Bifidobacterium animalis subsp lactis was determined as the number of colony-forming units in the first compartment (CFU/first compartment) by deep plate method in MRS pH 6.2 agar (Oxoid CM0361) supplemented with 0.05% cysteine, HCl (Merck 102839). . Triplicates of 200 mg of product were mixed with approximately 200 g of Maximum Recovery Diluent (MRD) (eg Oxoid CM0733) for 2 minutes at 450-550 strokes per minute using a Stomacher (eg Smasher, bioMerieux) to obtain a 1:1000 dilution. Serial dilutions in MRD were prepared for each weighing and two dilutions/volumes were used corresponding to 30-300 CFU/plate (2x2 plates for each weighing). The dishes were incubated under anaerobic conditions at 37°C for 3 days. Colonies were counted and the result was calculated as the average of three weighings in CFU/first compartment. The results for the stability indicator are presented in table. 10 and fig. 3b.

Conclusions from example 3.2.

The results are presented in Fig. 3b and in table. 10 and show the stability of 10.0% Bifidobacterium animalis subsp lactis in the mixture of the present invention at 25°C/60% RH (squares) and at 30°C/75% RH (circles).

It is obvious that the stability of the mixture in the first compartment according to the invention containing Bifidobacterium animalis subsp lactis at 25°C/60% RH is unexpectedly good, since said Bifidobacterium animalis subsp lactis in an amount of at least 1 x 10 CFU/g is viable for 36 months .

It is also evident that the stability of the mixture in the first compartment of the invention containing Bifidobacterium animalis subsp lactis at 30°C/75% RH is surprisingly good, since said Bifidobacterium animalis subsp lactis in an amount of at least 3x10 ⁹ CFU/g is viable for 30 months.

Therefore, it has been shown that the first compartment of the invention has the ability to keep the probiotic bacteria Bifidobacterium animalis subsp lactis viable in unexpectedly large numbers and for an unexpectedly long time in a mixture containing Bifidobacterium animalis subsp lactis, i.e. at least 36 months at 25°C /60% RH and at least 30 months at 30°C/75% RH.

Example 4 - Determination of the viability of Lactobacillus bacterial cells in a commercially available product, carried out at 45°C and 30°C (both aerobic and anaerobic conditions).

Commercially available products claiming viable Lactobacillus were also assessed for colony forming units per g. The commercially available Esse Sensitive Serum (Series 16AXE) from Esse skincare was purchased online and contained jojoba seed oil, sesame oil, hydrogenated vegetable oil , shea butter, isoamyl laurate, lactobacillus, tocopherol, sunflower oil, ascorbyl palmitate, vanillin and gamma-decalactone. Commercially available products were purchased, stored according to the manufacturer's recommendations prior to evaluation, and evaluated within the product's prescribed shelf life.

The total number of viable Lactobacillus cells was determined as the number of colony-forming units per g of product (CFU/g) using the deep plate method in MRS pH 6.2 agar (Oxoid CM0361). The total number of viable cells was determined as the number of colony-forming units per g of product (CFU/g) by surface plating on the surface of tryptone soy agar plates with sheep blood. 1.58 g of product was mixed with 160.43 g of pre-warmed (45°C) Maximum Recovery Diluent (MRD) (eg Oxoid CM0733) with 1% Tween 80 added for 2 minutes at 450-550 strokes per minute using a Stomacher ( for example Smasher, bioMerieux), obtaining a dilution of 1:100. Serial dilutions were prepared in MRD and two dilutions/volumes corresponding to 30-300 CFU/plate were used on 2 plates (3x2x2 plates). MRS pH 6.2 agar plates were incubated in anaerobic

- 30 045049 conditions at 37°C for 3 days. Sheep blood tryptone soy agar plates were incubated either aerobically or anaerobically at 37°C for 4 days. Colonies were counted and the result was calculated in CFU/g.

Another commercially available product with viable Lactobacillus claims was also assessed for colony forming units per g. The commercially available product Esse Probiotic Serum (Series 18ADF) from Esse skincare was purchased from a retailer in Copenhagen, and stored according to the manufacturer's recommendations until assessment. and evaluated within the prescribed shelf life of the product. Esse Probiotic Serum contains jojoba seed oil, sesame oil, hydrogenated vegetable oil, shea butter, isoamyl laurate, marula seed oil, lactobacillus, tocopherol, sunflower oil, ascorbyl palmitate, vanillin, gamma-decalactone, i.e. the same ingredients as Esse Sensitive Serum except marula seed oils.

The total number of viable Lactobacillus cells was determined as the number of colony-forming units per g of product (CFU/g) using the deep plate method in MRS pH 6.2 agar (Oxoid CM0361). 1.58 g of product was mixed with 160.43 g of pre-warmed (30°C) Maximum Recovery Diluent (MRD) (eg Oxoid CM0733) with 1% Tween 80 added for 2 minutes at 450-550 strokes per minute using a Stomacher ( for example Smasher, bioMerieux), obtaining a dilution of 1:100. Serial dilutions were prepared in MRD and two dilutions/volumes corresponding to 30-300 CFU/plate were used on 2 plates (3x2x2 plates). MRS Agar pH 6.2 plates were incubated under anaerobic conditions at 37°C for 3 days.

The total number of viable cells was also determined as the number of colony forming units per g of product (CFU/g) by surface plating on the surface of MRS agar plates. The product was applied directly to the surface of MRS agar plates and incubated to examine the presence of any colony-forming lactic acid bacteria. The product was not subjected to any sample preparation, i.e. heating and dilution. MRS agar plates were divided into two groups and incubated under aerobic or anaerobic conditions at 37°C for 3 days. Colonies were counted and the result was calculated in CFU/g.

No colonies were found on plates incubated aerobically under anaerobic conditions.

Table 11

Measured counts of viable Lactobacillus cells in commercially available products

Plate subculture method Number of colony forming units per g (CFU/g) Surface seeding method Number of colony forming units per g (CFU/g) Esse Sensitive Serum Series 16AXCE tested in May 2017 (product expiration date September 2018) < 1E+02 CFU/g Tested at 45°C < 1E+02 CFU/g Tested at 45°C Esse Probiotic Serum Series 18 ADF tested in 2019 (product expiration date August 2019) < 1E+02 CFU/g Tested at zo°c < 1E+02 CFU/g Tested at zo°c

Conclusions from example 4.

The results of assessing the viability of Lactobacillus are presented in table. 11. It is obvious that commercially available products do not show the presence of any viable Lactobacillus at all. It is therefore apparent that commercially available products that claim to contain cells of viable Lactobacillus bacteria are not at all stable upon opening the container containing the commercially available product. It should be noted that commercially available products were assessed well before the expiration date of the product. Additionally, it should be noted that the use of pre-warmed (45°C) Maximum Recovery Diluent (MRD) for deep plate methods was carried out in all examples of the present invention and when testing Esse Sensitive Serum. Additional tests were carried out using pre-warmed (30°C) Maximum Recovery Diluent (MRD), which demonstrate no difference, that is, Esse Sensitive Serum and Esse Probiotic Serum do not contain live bacteria, then

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Claims (5)

there are less than 1E+02 CFU/g Lactobacillus. CLAIM 1. A method for producing an aqueous composition for topical use, including: a) providing viable bacteria of at least one strain of probiotic bacteria in an amount of at least 5-10 ⁶ colony forming units (CFU)/g and at least one buffer, b) providing a mixture containing at least one thickener selected from one or more of carbomer, poloxamer, polyethylene glycol (PEG)/macrogol and/or carboxylic acid, and at least 60% (w/w) water , And c) adding said viable bacteria of at least one strain of probiotic bacteria from step a) to said mixture from step b) to obtain said aqueous composition for topical use having a pH of 4.0-6.7 and a viscosity of more than 2500 mPa-s , and the number of said viable bacteria of at least one strain of probiotic bacteria is 1-106-1011 CFU/g, and wherein said viable bacteria of at least one strain of probiotic bacteria are viable in an aqueous composition for topical use for up to 7 days at 25 °C/60% relative humidity (RH). 2. A method for obtaining an aqueous composition for topical use according to claim 1, including: a) providing a dry composition of viable bacteria of at least one strain of probiotic bacteria in an amount of at least 5-10 ⁶ CFU/g and at least one buffer, wherein said dry composition of viable bacteria of at least one strain of probiotic bacteria is ground into powder, having a water activity of no more than 0.30 and a particle size distribution in the region of less than 500 microns, b) providing a mixture containing at least one thickener selected from one or more of carbomer, poloxamer, PEG/macrogol and/or carboxylic acid, and water in an amount greater than 60% (w/w) to obtain the specified a mixture having a pH of 2-3 and a viscosity of less than 1000 mPa-s, and c) adding said dry composition from step a) to said mixture from step b) to obtain said aqueous composition for topical use having a pH of 4.0-6.7, and a viscosity of more than 2500 mPa-s, and the number of said viable bacteria according to at least one strain of probiotic bacteria in the range of 1-106-10 ¹¹ CFU/g, and wherein said viable bacteria of at least one strain of probiotic bacteria are viable in an aqueous topical composition for up to 7 days at 25°C/60 % RH. 3. A method for preparing an aqueous topical composition according to any one of claims 1 or 2, wherein said at least one thickening agent is one or more carbomers, and wherein said dry composition contains at least one buffer selected from one or more than one monovalent cation. 4. A method for obtaining an aqueous composition for topical use according to claim 1, including: a) providing a dry composition of viable bacteria of at least one strain of probiotic bacteria in an amount of at least 5-10 ⁶ CFU/g and at least one buffer, wherein said dry composition of viable bacteria of at least one strain of probiotic bacteria is ground into powder, having a water activity of no more than 0.30 and a particle size distribution in the region of less than 500 microns, b) providing a mixture containing at least one thickener selected from one or more poloxamers and at least 60% (w/w) water to obtain said mixture having a viscosity of about 100 mPa-s at a temperature not above 10°C and a viscosity of approximately 1000 mPa-s at temperatures above 25°C, and c) adding said dry composition from step a) to said mixture from step b) to obtain said aqueous composition for topical use having a viscosity of less than 500 mPa-s at a temperature not exceeding 10°C and a viscosity of more than 2500 mPa-s at a temperature above 30°C, and where the specified aqueous composition for topical application contains the specified viable bacteria of at least one strain of probiotic bacteria in an amount in the range of 1-106-10 ¹¹ CFU/g, and where the specified viable bacteria of at least one strain of probiotic bacteria are viable in an aqueous composition for topical use for up to 7 days at 25°C/60% RH. 5. An aqueous composition for topical use containing at least 60% (w/w) water and viable bacteria of at least one strain of probiotic bacteria, wherein said viable bacteria of at least one strain of probiotic bacteria are in an amount of 1-106- 10 ¹¹ CFU/g are viable in an aqueous topical composition for up to 7 days at 25°C/60% RH, and wherein said composition contains at least one thickener -