Freeze-dried compositions and methods for their preparation

Abstract

The present invention relates to a method for preparing a lyophilized composition comprising a non-pathogenic C. acnes strain, the resulting lyophilized composition, a topical composition comprising said lyophilized composition, and its use for treating or preventing acne in a human subject.

Description

[Technical field]

[0001] The present invention relates to a method for preparing a lyophilized composition comprising a non-pathogenic C. acnes strain, the resulting lyophilized composition, a topical composition comprising said lyophilized composition, and its use for treating or preventing acne in a human subject.

[0002] 2. Background of the Invention Acne (colloquially known as acne vulgaris) is a common chronic inflammatory skin disease that affects the pilosebaceous unit of the hair follicle. It is estimated that approximately 85% of adolescents and young adults suffer from the disease. Acne generally presents as inflammatory papules, pustules or nodules. Inflammation may also be accompanied by redness, swelling and tenderness.

[0003] Acne is highly correlated with the presence of certain (hereinafter referred to as pathogenic) strains of Cutibacterium acnes (C. acnes), whereas other strains of C. acnes, hereinafter referred to as non-pathogenic, have been found to have no adverse effects on the skin. Therefore, it has been recently proposed to replace pathogenic C. acnes strains present in acne skin with non-pathogenic C. acnes strains in order to reduce the degree and / or severity of acne (see A. Karoglan et al., Acta Derm Venereol 2019, 99:1253-1257). In this context, WO2016172196 proposes an approach to treat acne, which comprises the sequential steps of topically administering a bactericide or antibiotic to the skin of a subject with acne to significantly reduce the population of pathogenic C. acnes, followed by topically administering to said skin one or more live non-pathogenic C. acnes strains. This treatment allows the non-pathogenic C. acnes strains to become part of the natural skin microbiome, which can then be used to treat or prevent acne, as well as to maintain acne-free skin.

[0004] WO2018073651 discloses a composition for topical administration to the skin comprising two or more different live non-pathogenic C. acnes strains, where the biomass is collected by centrifugation and resuspended in soy peptone, and the suspension is then formulated as a gel.

[0005] However, product forms such as those outlined in WO2018083651 are not readily suitable for incorporation into a wide variety of common cosmetic product forms such as creams and lotions. Therefore, a high survival rate, i.e., at least 1×10 9 There is a continuing demand in the industry for alternative products and methods of preparing such product forms that exhibit viability of CFU / g yet are easy to handle, store and ship.

[0006] Surprisingly, it has been found that the viability of a freeze-dried composition containing a non-pathogenic C. acnes strain can be significantly improved when the biomass is separated by microfiltration. It has also been found that the viability can be further improved by a washing step. In addition, the washing step further surprisingly reduces the moisture content after freeze-drying and storage, which is beneficial.

[0007] Summary of the Invention The present invention provides a method for producing a lyophilized composition comprising a non-pathogenic C. acnes strain by the methods outlined herein, which provides a lyophilized composition comprising a non-pathogenic C. acnes strain with high viability and storage stability of the strain.

[0008] The present invention also provides a lyophilized composition comprising at least one non-pathogenic C. acnes strain produced by the method as outlined and defined herein.

[0009] The present invention further provides the use of the above lyophilized composition for treating or preventing acne in a human subject.

[0010] The present invention also provides compositions for topical administration comprising the lyophilized compositions produced by the methods outlined herein.

[0011] Detailed Description of the Invention The present invention provides a method for producing a lyophilized composition comprising a non-pathogenic C. acnes strain for preventing acne in a human subject.

[0012] In particular, the present invention relates to a method for producing a lyophilized composition comprising at least one non-pathogenic C. acnes strain, comprising: a) concentrating bacterial cells of at least one non-pathogenic C. acnes strain from a fermentation broth by microfiltration, preferably cross-flow microfiltration, to obtain a biomass concentrate comprising said cells; b) optionally washing the biomass concentrate to obtain a washed biomass concentrate; c) combining the concentrated or washed biomass concentrate with an aqueous solution comprising at least one cryoprotectant to obtain an optionally washed biomass blend; d) freeze-drying the biomass preparation to obtain a freeze-dried composition comprising at least one non-pathogenic C. acnes strain; The present invention provides a method comprising:

[0013] Surprisingly, the inventors of the present invention have discovered that concentration by cross-flow microfiltration in step a) preserves almost all of the viable non-pathogenic C. acnes strains in the fermentation broth and significantly increases their viability in the lyophilized composition upon storage. The washing step further improves the recovery of viable cells and reduces the moisture content of the resulting lyophilized composition.

[0014] In the present invention, the term "non-pathogenic C. acnes strain" refers to a strain that converts or degrades cis-9, cis-12 linoleic acid slowly or only negligibly in the medium, such as C. acnes strains type I, such as type IA (i.e., IA1 and IA2) and type IB, and type II. Examples of non-pathogenic C. acnes strains according to the present invention include, but are not limited to, C. acnes strains D1, A5, C3, H1, H2, H3, K1, K2, K4, K6, K8, K9, L1 and F4, as outlined and disclosed in WO2016172196. Preferably, the non-pathogenic C. acnes strain of the present invention is selected from the group consisting of C. acnes strains D1, A5, C3, H1, H2, H3, K1, K2, K4, K6, K8, K9, L1 and F4. More preferably, the non-pathogenic C. acnes strain of the present invention is selected from C. acnes strains C3 and / or K8.

[0015] In the present invention, the term "viability" refers to the concentration of viable cells in a sample, such as a lyophilized composition according to the present invention, and can be expressed as "CFU / g" or "CFU / ml" of sample.

[0016] In the present invention, the term "fermentation broth" refers to the mixture obtained after culturing a non-pathogenic C. acnes strain, as outlined, for example, in WO 2018073651. It is well understood by those skilled in the art that the fermentation broth further contains components typically present in a fermentation broth, such as water, nutrients such as amino acids, organic acids such as propionic acid, carbohydrates, various vitamins, growth factors, etc., in addition to the C. acnes strain, which are necessary for the strain to grow, and also products formed by the microorganism itself during cultivation.

[0017] The term "skin" as used herein is meant to include the outer surface of a mammal, particularly a human, including the skin and scalp. Preferred skin in all embodiments of the present invention is most preferably facial and body skin, such as facial skin.

[0018] As used herein, the term "prevention" refers to reducing the risk of developing acne.

[0019] The term "treatment" as used herein refers to improving the symptoms of acne, delaying the onset and / or shortening the duration. Treatment may be preventive (cosmetic) or therapeutic. Preferably, treatment is preventive.

[0020] In all embodiments of the invention, preferably each strain is cultivated separately and accordingly the lyophilized composition obtained by the method of the invention as outlined above preferably comprises only one C. acnes strain, in particular C. acnes strain C3 or C. acnes strain K8. However, according to the invention it is not excluded that the fermentation broth used in the lyophilization step d) comprises more than one C. acnes strain, for example by co-culture or by combination of two or more fermentation broths each comprising one (or more) non-pathogenic C. acnes strain, thereby obtaining a lyophilized product comprising more than one non-pathogenic C. acnes strain. It is further understood that it is also possible to obtain a freeze-dried composition according to the present invention comprising two or more non-pathogenic C. acnes strains by mixing two or more freeze-dried compositions obtained in step d), each of which comprises one non-pathogenic C. acnes strain.

[0021] In all embodiments of the present invention, the fermentation broth used in step a) according to the present invention preferably has a pH of about 10 8 ~10 12 CFU / g, preferably 10 9 ~10 11Contains CFU / g of a non-pathogenic C. acnes strain.

[0022] In all embodiments of the invention, it is further advantageous if the fermentation broth has a biomass volume (i.e. pellet volume obtained after centrifugation of the sample) selected in the range of 1-5% by volume, preferably in the range of 1.5-3% by volume, relative to the total volume of the fermentation broth.

[0023] After fermentation and prior to concentration, the fermentation broth is generally harvested by standard methods in the art, e.g., by transferring to a container. In particular, the fermentation broth of the present invention can be harvested at about 0-20° C., preferably 5-15° C., more preferably 6-12° C., e.g., 8° C., and then transferred, e.g., to a container tank for concentration.

[0024] In all embodiments of the present invention, the microfiltration in step a) is preferably cross-flow microfiltration, even more preferably a ceramic membrane filter is used. Even more preferably, said membrane filter has a retention (pore size) selected in the range of 0.1 to 10 μm, preferably in the range of 0.25 to 0.75 μm, most preferably in the range of 0.4 to 0.5 μm. Such membranes are well known in the art and are commercially available, for example from TAMI.

[0025] It is well known to those skilled in the art that the biomass concentrate obtained by said cross-flow microfiltration is generally characterized as a suspension of biomass in an aqueous medium.

[0026] According to the present invention, microfiltration can be carried out under a pressure of 0.1 to 10 bar, preferably 1 to 8 bar, more preferably 1.5 to 5 bar, for example 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 bar, at a temperature of 0 to 20°C, preferably 5 to 15°C, for example 8, 9, 10, 12 and 15°C.

[0027] In step a) of the present invention, the fermentation broth is preferably concentrated until a biomass concentrate of 5-50% by weight of the initial weight of the fermentation broth is obtained. More preferably, the fermentation broth is concentrated until a biomass concentrate of 10-35% by weight, more preferably 15-30% by weight of the weight of the initial fermentation broth is obtained. Thus, in a preferred embodiment, the biomass concentrate represents 5-50% by weight, more preferably 10-35% by weight, most preferably 15-30% by weight of the weight of the fermentation broth.

[0028] Compared to centrifugation, microfiltration in step a) of the present invention increases the viability of non-pathogenic C. acnes strains in the freeze-dried product obtained by the method of the present invention by at least 10%, 20%, 30%, 40% 50% or more after one month, preferably after three months, and also when stored at 40°C. Preferably, (cross-flow) microfiltration in step a) of the present invention increases the viability of non-pathogenic C. acnes strains in the freeze-dried product by at least 30% compared to centrifugation. Even more preferably, microfiltration in step a) of the present invention increases the viability of non-pathogenic C. acnes strains in the freeze-dried product by at least 40% compared to centrifugation. Most preferably, microfiltration in step a) of the present invention increases the viability of non-pathogenic C. acnes strains in the freeze-dried product by at least 50% compared to centrifugation.

[0029] Optionally, the concentrated biomass obtained in step a) is further treated by washing according to step b) of the method of the invention. Surprisingly, the inventors of the present invention have also found that said washing step significantly improves the recovery of viable cells and reduces the moisture content of the freeze-dried composition according to the invention, which is beneficial.

[0030] In step b) of the present invention, the biomass concentrate can be washed with an aqueous solution of any organic acid suitable for skin care, particularly for the treatment or prevention of acne.Examples of suitable organic acids include, but are not limited to, citric acid, retinoic acid, ferulic acid, glycolic acid, lactic acid, fruit acid, salicylic acid and hyaluronic acid.Preferably, the organic acid is citric acid.Optionally, the organic acid is used in the form of its buffer, such as a citrate buffer.

[0031] In all embodiments of the present invention, it is preferred to wash the concentrated biomass obtained in step a) with a 10-100 mM, preferably 20-80 mM, more preferably 30-70 mM, such as 40, 50 or even 60 mM aqueous citric acid solution or citrate buffer (comprising citric acid and sodium citrate).

[0032] Preferably, in step b) of the present invention, the aqueous (buffer) solution of organic acid is used in an amount of 1 to 10 times, preferably 2 to 8 times, more preferably 3 to 6 times, for example 3, 4, 5 or 6 times the weight of the concentrated biomass.

[0033] In step b) of the present invention, the concentrated biomass can be washed batchwise or continuously, for example during cross-flow microfiltration. In some embodiments, the concentrated biomass is washed batchwise with aqueous citric acid. In other embodiments, the concentrated biomass is washed continuously with aqueous citric acid. In the case of continuous washing, preferably aqueous citric acid is added to the biomass concentrate and again subjected to cross-flow microfiltration.

[0034] In all embodiments of the invention, washing is preferably continued until the biomass concentrate contains less than 10% by weight acetic acid, preferably less than 5% by weight, most preferably less than 3% by weight, e.g., less than 2, 1 or 0.5% by weight, based on the total weight of the biomass concentrate.

[0035] In the case of successive washings, in all embodiments of the invention, it is further preferred that an aqueous solution of an organic acid is added to the biomass concentrate obtained in step a) and again subjected to cross-flow microfiltration, more preferably this is achieved in such a way that the content of non-pathogenic C. acnes strains in the obtained washed biomass concentrate is (again) at least 1×10 10 CFU / g, preferably at least 1 × 10 11 CFU / g, most preferably until nearly all (greater than 90%, preferably greater than 95%, most preferably greater than 96%) viable cells are recovered.

[0036] Alternatively, in the case of successive washings, an aqueous solution of an organic acid is added to the biomass concentrate obtained in step a) (thereby obtaining a diluted biomass concentrate) which is then (again) subjected to cross-flow microfiltration until the weight of the washed biomass concentrate is less than 25% by weight, preferably less than 20% by weight, of the weight of the initial fermentation broth. Even more preferably, the weight of the washed biomass concentrate is between 10 and 20% by weight, even more preferably between 10 and 15% by weight, of the initial weight of the fermentation broth.

[0037] In step b) of the present invention, the biomass concentrate can be washed under a pressure of 0 to 10 bar, preferably 1 to 8 bar, more preferably 1.5 to 5 bar, such as 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 bar, at a temperature of 0 to 20°C, preferably 5 to 15°C, such as 8, 9, 10, 12 and 15°C.

[0038] In step c) of the present application, the concentrated biomass obtained in step a) or the washed biomass concentrate obtained in step b) is blended by admixing a cryoprotectant to the biomass concentrate to obtain a biomass blend.

[0039] In the present invention, the cryoprotectant may be selected from the group consisting of alcohols such as glycerol, propylene glycol, ethylene glycol, etc.; sugars such as glucose, galactose, lactose, sucrose, maltose, fructose, trehalose, raffinose, mannitol, sorbitol, etc.; polymers such as polyethylene glycol (PEG), polybipyridone (PVP), skim milk, gelatin, proteins and protein hydrolysates, peptides, yeast, broth, dextrin, maltodextrin, methylcellulose, povidone, serum, peptone, etc.; salts such as sodium sulfate, calcium lactate, sodium glutamate, sodium chloride, potassium chloride, sodium thiosulfate, trisodium citrate, ammonium acetate, ammonium chloride, etc.; acids such as citric acid, phosphoric acid, glutamic acid, tartaric acid, amino acids, ethylenediaminetetraacetic acid (EDTA), etc.; bases such as ammonium hydroxide, sodium hydroxide, sodium bicarbonate, etc.; and mixtures thereof.

[0040] Preferably, in all embodiments, the cryoprotectant is selected from the group consisting of glucose, galactose, lactose, sucrose, maltose, fructose, trehalose, raffinose, mannitol, sorbitol, polyethylene glycol (PEG), polypyrrolidinone (PVP), skim milk, gelatin, proteins and protein hydrolysates, peptides, yeast, broth, dextrin, maltodextrin, methylcellulose, povidone, serum, peptone, sodium sulfate, calcium lactate, sodium glutamate, sodium chloride, potassium chloride, sodium thiosulfate, trisodium citrate, ammonium acetate, ammonium chloride, citric acid, phosphoric acid, glutamic acid, tartaric acid, amino acids, ethylenediaminetetraacetic acid (EDTA) and ammonium hydroxide; and mixtures thereof.

[0041] More preferably, in all embodiments of the present invention, the cryoprotectant is selected from the group consisting of glucose, sucrose, trehalose, raffinose, mannitol, sorbitol, polyethylene glycol (PEG), dextrin, maltodextrin, peptone, monosodium glutamate, glutamic acid, sodium chloride, trisodium citrate and ammonium hydroxide; and mixtures thereof.

[0042] Most preferably, in all embodiments of the present invention, the cryoprotectant is selected from the group consisting of sucrose, trehalose, raffinose, mannitol, polyethylene glycol (PEG), maltodextrin, peptone, sodium glutamate, sodium chloride, trisodium citrate, glutamic acid and sodium bicarbonate; and mixtures thereof.

[0043] In one advantageous embodiment, the cryoprotectant is a combination of two or more of sucrose, maltodextrin, glutamic acid, sodium glutamate and trisodium citrate.

[0044] In one particularly advantageous embodiment, the cryoprotectant is a combination of sucrose, maltodextrin, monosodium glutamate and trisodium citrate.

[0045] In another particularly advantageous embodiment, the cryoprotectant is a combination of sucrose, maltodextrin and glutamic acid.

[0046] As would be expected by a person skilled in the art, an aqueous solution of a cryoprotectant is prepared prior to mixing with the biomass concentrate or washed biomass concentrate according to the invention. Preferably, said aqueous solution is clear and does not exhibit precipitation during preparation and storage. Preparation and mixing of such aqueous solutions can be carried out according to procedures known to those skilled in the art.

[0047] In all embodiments of the invention, the formulation obtained in step c) may contain 1 to 20% by weight, preferably 5 to 15% by weight, more preferably 6 to 12% by weight, such as 6, 7, 8, 9, 10, 11 and 12% by weight of cryoprotectant relative to the total weight of the formulation.

[0048] After the formulation has been prepared, it is freeze-dried in step d) according to the invention.

[0049] Freeze-drying is a well-established method for the archiving and long-term storage of bacterial strains. The approaches used vary widely, but all follow the standard methods associated with freeze-drying: freezing the sample, applying a high vacuum, warming the sample under vacuum to induce water sublimation, a drying phase to drive off excess water, and finally sealing the sample to prevent water uptake. The basic freeze-drying process can be divided into three stages: freezing, primary drying, and secondary drying.

[0050] frozen In the freezing step, the formulation of the present invention may be frozen at -10°C to -50°C, preferably -20°C to -40°C, for example -25°C, -30°C, -40°C.

[0051] In the present invention, the formulation can be flash frozen, for example, by placing the formulation in a tray that is pre-chilled at -20°C, -25°C or -40°C.

[0052] In the present invention, the formulation is preferably frozen gradually, for example by cooling at 1° C. / min to −20° C., −25° C. or −40° C.

[0053] In one embodiment of the invention, the formulation is rapidly frozen to -20°C and then cooled to -40°C at 1°C / min.

[0054] Primary drying Once the formulation is frozen, a vacuum is applied to remove the frozen water. At this stage, the formulation of the present invention may be subjected to a vacuum of 1 mbar or less, preferably 0.8 mbar or less, more preferably 0.6 mbar or less, most preferably 0.5 mbar or less, for example 0.1, 0.2, 0.3, 0.4 and 0.5 mbar.

[0055] The temperature for such primary drying can be -10°C to -50°C, preferably -20°C to -40°C, for example, -25°C, -30°C, or -40°C.

[0056] As will be appreciated by those skilled in the art, the length of time required for primary drying can be determined by comparative pressure measurements known in the art. In the present invention, primary drying takes between 30 and 80 hours, preferably between 40 and 60 hours.

[0057] Secondary drying After the primary drying described above, the formulation is further subjected to secondary drying to forcibly remove residual moisture, in which a vacuum is maintained while the temperature is increased.

[0058] As will be appreciated by those skilled in the art, it is important not to dry the formulation too much, as this may be detrimental, and for the same reason, the use of higher temperatures is not recommended. In the present invention, the temperature of the formulation is increased to more than 10° C., for example 20° C., 30° C., but less than 50° C. Preferably, the formulation is allowed to warm to ambient temperature.

[0059] As will also be appreciated by those skilled in the art, it is advantageous to gradually increase the temperature of the formulation of the present invention. Preferably, the temperature of the formulation of the present invention is increased to 20° C. in a period of 2 to 20 hours, preferably 5 to 15 hours, more preferably 6 to 10 hours, such as 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10 hours.

[0060] Preferably, the formulation of the present invention is maintained for an additional 1-10 hours after warming to the desired temperature, more preferably, the formulation of the present invention is maintained at 10°C, 20°C or 30°C for an additional 2-15 hours, even more preferably, for 3-10 hours, such as 3, 4, 5, 6, 7, 8, 9 and 10 hours.

[0061] For the above freeze-drying operation, standard freeze-drying equipment can be used, such as the Lyovac™ equipment from GEA (Berlin, Germany), the Gamma 2-20 Freeze dryer LCM-1 from Christ (Osterode am Harz, Germany), the Christ Martin™ Alpha 1-2 Lyophilisator from Fisher Scientific GmbH (Schwerte, Germany), and the Virtis Genesis 35L Pilot Freeze dryer from SP Scientific (PA, USA). A person skilled in the art can operate any of the equipment to perform the freeze-drying of the present invention according to the manufacturer's instructions.

[0062] After freeze-drying the formulation, a freeze-dried composition cake is obtained, which can be pulverized and stored in a sealed container such as an aluminum bag or bottle for further use.

[0063] In one embodiment, the method of the present invention does not include step b. In another embodiment, the method of the present invention includes step b).

[0064] In one preferred embodiment, the present invention provides a method for producing a lyophilized composition comprising at least one non-pathogenic C. acnes strain, comprising: a) concentrating bacterial cells of at least one non-pathogenic C. acnes strain from a fermentation broth by microfiltration to obtain a biomass concentrate; b) washing the biomass concentrate to obtain a washed biomass concentrate; c) combining the washed biomass concentrate with an aqueous solution comprising at least one cryoprotectant to obtain a biomass blend; d) freeze-drying the biomass formulation to obtain a freeze-dried composition. Including, The method is provided, wherein the concentration in step a) and the washing in step b) are performed by cross-flow microfiltration.

[0065] According to the method of the present invention, the obtained freeze-dried composition has a high viability of non-pathogenic C. acnes strains. In particular, the obtained freeze-dried composition has a high viability of at least 1×10 6 of non-pathogenic C. acnes strains. 9 CFU / g, preferably at least 1 × 10 10 CFU / g, more preferably at least 5×10 10 CFU / g, at least 6 × 10 10 CFU / g, at least 7 × 10 10 CFU / g, at least 8 × 10 10 CFU / g, at least 9 × 10 10 CFU / g, or at least 1 × 10 11 It has a viability of CFU / g.

[0066] Furthermore, the method of the present invention provides high stability (storage viability) of the bacterial strain in the resulting lyophilized composition. In particular, the lyophilized composition produced by the method of the present invention maintains at least 80%, preferably 85%, more preferably 90% viability of the bacterial strain even after storage at room temperature or even at 40° C. for 4 weeks.

[0067] As mentioned above, step b) is optional, but is preferably included in the method of the present invention. Step b) provides the method of the present invention with further advantages, for example, the viability of non-pathogenic C. acnes strains is better preserved compared to the method without step b). In addition, step b) provides low residual moisture content (less than 3% by weight, preferably less than 2% by weight, more preferably less than 1.5% by weight, even more preferably less than 1.2% by weight) in the freeze-dried composition of the present invention (measured using a Sartorius moisture analyzer (Sartorius AG, Germany)).

[0068] Thus, in a second aspect, the present invention relates to a freeze-dried composition having all the definitions and preferences given herein and obtainable (obtained) by the above-mentioned method according to the invention as outlined in the claims.

[0069] In particular, the present invention relates to a lyophilized composition comprising at least one non-pathogenic C. acnes strain, preferably one or more of type I, e.g., type IA (i.e., IA1 and IA2) and type IB, and type II, most preferably C. acnes strain C3 and / or C. acnes strain K8, wherein the viability of the at least one non-pathogenic C. acnes strain is at least 1×10 9 A lyophilized composition is provided that is a CFU / g composition.

[0070] Advantageously, in all embodiments of the invention, in the freeze-dried composition of the invention, the viability of the non-pathogenic C. acnes strain, preferably one or more of C. acnes strains type I, such as type IA (i.e. IA1 and IA2) and type IB and type II, most preferably C. acnes strain C3 and / or C. acnes strain K8, is at least 1×10 10 CFU / g composition, at least 5×10 10 CFU / g composition, at least 6×10 10 CFU / g composition, at least 7×10 10 CFU / g composition, at least 8×10 10 CFU / g composition, at least 9×10 10 CFU / g composition, and at least 1×10 11 CFU / g composition. More preferably, the viability of the non-pathogenic C. acnes strain in the freeze-dried composition of the present invention is 1×10 10 CFU / g composition ~1×10 13 CFU / g composition, more preferably 5×1010 CFU / g composition ~8×10 12 CFU / g composition, most preferably 1×10 11 CFU / g composition ~5×10 12 CFU / g composition, e.g., 3×10 11 CFU / g composition, 5×10 11 CFU / g composition, 6×10 11 CFU / g composition, 7×10 11 CFU / g composition, 8×10 11 CFU / g composition, 9×10 11 CFU / g composition, and 1×10 12 CFU / g composition. Preferably, the compositions of the present invention have at least 7×10 11 CFU / g of C. acnes strain C3 and / or at least 6 × 10 11 Contains CFU / g of C. acnes strain K8.

[0071] Advantageously, in the freeze-dried composition of the invention, the residual water content is less than 3% by weight, preferably less than 2% by weight, more preferably less than 1.5% by weight, most preferably less than 1.2% by weight.

[0072] Preferably, the freeze-dried composition of the invention maintains at least 80%, preferably 85%, more preferably 90% viability of the strains after storage for 4 weeks at room temperature, or even at 37 or 40°C.

[0073] The lyophilized composition of the present invention can be used to treat or prevent (prophylactic) acne in a human subject.

[0074] Thus, in a third aspect, the present invention provides the use of a lyophilized composition of the invention or a lyophilized composition prepared by a method according to the invention for treating or preventing acne in a human subject, and optionally the evaluation of the efficacy thereof.

[0075] The present invention also provides a method for treating or preventing acne in a human subject, comprising topically administering to the subject a topical composition comprising a lyophilized composition of the present invention.

[0076] Therefore, in a further aspect, the present invention also relates to a composition for topical administration comprising a lyophilized composition having all the preferences and definitions as outlined herein.

[0077] For use in topical compositions, the viability of the lyophilized compositions according to the invention is preferably reduced to about 1×10 by incorporation of maltodextrin (if necessary). 10 The CFU / g is adjusted (calibration lyophilized composition). Furthermore, preferably, one or more lyophilized compositions containing different C. acnes strains can be mixed, such as a lyophilized composition containing C. acnes C3 and a lyophilized composition containing C. acnes K8. The mixing can be performed either before or after the CFU / g adjustment.

[0078] Since the topical composition according to the invention is intended for topical application, it is well understood that it comprises a physiologically acceptable medium, i.e. a medium that is compatible with keratinous materials such as the skin, mucous membranes, keratinous fibers, etc. In particular, a physiologically acceptable medium is a cosmetically or dermatologically acceptable carrier.

[0079] The term "cosmetically / dermatologically acceptable carrier" (also referred to herein as carrier) refers to all vehicles / carriers conventionally used in cosmetic or dermatological compositions, i.e., suitable for topical application to keratinous tissue, having good aesthetic properties, compatible with the active agents present in the composition, and not raising unreasonable safety or toxicity concerns. Such carriers are well known to those skilled in the art.

[0080] The exact amount of carrier will depend on the actual level of the lyophilized composition and any other optional ingredients (eg, other active ingredients) that one of skill in the art would classify as different from a carrier.

[0081] In an advantageous embodiment, the composition for topical administration according to the present invention comprises from about 50% to about 99%, preferably from about 60% to about 98%, more preferably from about 70% to about 98%, for example in particular from about 80% to about 95%, of a carrier relative to the total weight of the cosmetic composition.

[0082] In certain advantageous embodiments, the carrier does not contain water, i.e. the topical composition according to the invention is an anhydrous topical composition.

[0083] In another particularly advantageous embodiment, the composition for topical administration according to the invention consists of a lyophilized composition according to the invention with all the definitions and preferences given herein, at least one oil and at least one oil-soluble antioxidant, such as, for example, tocopherol, the at least one oil being preferably chosen from fatty alcohols and / or fatty acid glycerides.

[0084] Particularly suitable compositions for topical administration according to the present invention are leave-on products, including any products that are applied to the human body.The compositions may be in the form of liquid, lotion, cream, foam, or may be applied by means of an implement, or through a face mask, pad or patch.Non-limiting examples of such compositions include leave-on skin lotions and creams, foundations, mascaras, sunless tanners and sunscreen lotions.

[0085] The topical compositions of the present invention (including the carrier) may contain conventional adjuvants and additives such as preservatives / antioxidants, fatty substances / oils, organic solvents, silicones, thickeners, emollients, emulsifiers, antifoaming agents, aesthetic ingredients such as fragrances, surfactants, fillers, anionic, cationic, nonionic or amphoteric polymers or mixtures thereof, propellants, acidifying or basifying agents, dyes, colorants / colorants, abrasives, absorbents, chelating and / or sequestering agents, essential oils, skin sensates, astringents, pigments, or other ingredients normally formulated in such compositions.

[0086] Examples of cosmetic excipients, diluents, adjuvants, additives, and active ingredients commonly used in the skin care industry that are suitable for use in the cosmetic compositions of the present invention are, for example, but not limited to, those listed in the International Cosmetic Ingredient Dictionary & Handbook by Personal Care Product Council (http: / / www.personalcarecouncil.org / ), accessible online at INFO BASE (http: / / online.personalcarecouncil.org / jsp / Home.jsp).

[0087] The required amounts of active ingredient and excipients, diluents, adjuvants, additives, etc. can be readily determined by one skilled in the art based on the desired product form and application. Additional ingredients may be added to either the oil phase, the aqueous phase, or separately if deemed appropriate.

[0088] The cosmetic actives useful herein may, in some cases, provide multiple benefits or operate via multiple modes of action.

[0089] Of course, the skilled artisan will take care to select any of the above-mentioned additional components, adjuvants, diluents and additives and / or their amounts such that the advantageous properties inherently associated with the combination according to the invention are not adversely affected or are not substantially affected by the envisaged addition or addition.

[0090] The amount of (preferably calibrated) lyophilized composition in the composition for topical administration according to the present invention may be adjusted by a person skilled in the art and is preferably selected in the range of 0.1 to 10% by weight, preferably 0.5 to 7.5% by weight, most preferably 0.75 to 5% by weight, for example 1 to 4% by weight, relative to the total weight of the composition.

[0091] In certain embodiments, the present invention also relates to: A freeze-dried composition (C) comprising at least one non-pathogenic C. acnes strain, wherein the viability of the non-pathogenic C. acnes strain in said composition is at least 1×10 9 CFU / g of the freeze-dried composition (C). Preferably, in the freeze-dried composition (C), the non-pathogenic C. acnes strain is selected from the group consisting of C. acnes strains type I, such as type IA (i.e., IA1 and IA2) and type IB, and type II, and mixtures thereof. More preferably, in the freeze-dried composition (A), the non-pathogenic C. acnes strain is selected from the group consisting of C. acnes strains D1, A5, C3, H1, H2, H3, K1, K2, K4, K6, K8, K9, L1 and F4, and mixtures thereof. Most preferably, in the freeze-dried composition (C), at least one non-pathogenic C. acnes strain is C. acnes strain C3 or C. acnes strain K8, and mixtures thereof. In all freeze-dried compositions (C), preferably the viability of at least one non-pathogenic C. acnes strain in said composition (C) is at least 1×10 10 CFU / g, e.g. 1×1010 CFU / g ~1×10 13 CFU / g, preferably 5×10 10 CFU / g ~8×10 12 CFU / g, more preferably 1×10 11 CFU / g ~5×10 12 CFU / g, e.g. 3×10 11 CFU / g, 5×10 11 CFU / g, 6×10 11 CFU / g, 7×10 11 CFU / g, 8×10 11 CFU / g, 9×10 11 CFU / g, and 1 × 10 12 In a preferred embodiment, the freeze-dried composition (C) has at least 7×10 CFU / g. 11 CFU / g of C. acnes strain C3 and / or at least 6 × 10 11 CFU / g of C. acnes strain K8. Advantageously, all freeze-dried compositions (C) exhibit a residual moisture content of less than 3% by weight, preferably less than 2% by weight, more preferably less than 1.5% by weight and most preferably less than 1.2% by weight relative to the total weight of composition (C).

[0092] A method (P) for producing a freeze-dried composition comprising at least one non-pathogenic C. acnes strain, comprising concentrating bacterial cells of at least one non-pathogenic C. acnes strain from the fermentation broth by microfiltration, preferably cross-flow microfiltration, to obtain a biomass concentrate comprising said cells; a) optionally washing the biomass concentrate to obtain a washed biomass concentrate; b) combining the concentrated or washed biomass concentrate with an aqueous solution comprising at least one cryoprotectant to obtain a biomass blend; c) freeze-drying the biomass preparation to obtain a freeze-dried composition comprising at least one non-pathogenic C. acnes strain; The method includes:

[0093] Preferably, the biomass concentrate is washed in step b) with an aqueous (buffered) solution of an organic acid selected from the group consisting of citric acid, retinoic acid, ferulic acid, glycolic acid, lactic acid, fruit acids, salicylic acid and hyaluronic acid and mixtures thereof, preferably citric acid.

[0094] More preferably, the biomass concentrate is washed with a 10 to 100 mM, preferably 20 to 80 mM, more preferably 30 to 70 mM, for example 40, 50 and 60 mM aqueous citric acid (buffer) solution.

[0095] The biomass concentrate or the washed biomass concentrate may be subjected in step c) to a process for isolating the biomass concentrate with alcohols such as glycerol, propylene glycol, ethylene glycol; sugars such as glucose, galactose, lactose, sucrose, maltose, fructose, trehalose, raffinose, mannitol, sorbitol; polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), skim milk, gelatin, proteins and protein hydrolysates, peptides, yeast, broth, dextrin, maltodextrin, methylcellulose, cellulose acetate, cellulose acetate esters ... It is further advantageous to prepare the composition by mixing with at least one cryoprotectant selected from the group consisting of polymers such as sucrose, povidone, serum, peptone, etc.; salts such as sodium sulfate, calcium lactate, sodium glutamate, sodium chloride, potassium chloride, sodium thiosulfate, trisodium citrate, ammonium acetate, ammonium chloride, etc.; acids such as citric acid, phosphoric acid, glutamic acid, tartaric acid, amino acids, ethylenediaminetetraacetic acid (EDTA), etc.; bases such as ammonium hydroxide, sodium hydroxide, sodium bicarbonate, etc.; and mixtures thereof. Preferably, the at least one cryoprotectant is selected from the group consisting of sucrose, maltodextrin, glutamic acid, sodium glutamate, and trisodium citrate, and mixtures thereof.

[0096] Advantageously, the formulation in step c) or method (P) comprises between 1 and 20% by weight, preferably between 5 and 15% by weight, more preferably between 6 and 12% by weight, such as 6, 7, 8, 9, 10, 11 and 12% by weight of cryoprotectant relative to the total weight of the biomass formulation having all the definitions and preferences given above.

[0097] A freeze-dried composition (C) or a freeze-dried composition produced by process (P) for use in the treatment or prevention of acne.

[0098] The following examples are provided to further illustrate the compositions and effects of the present invention. These examples are merely illustrative and are not intended to limit the scope of the present invention in any way.

[0099] Working Example CFU analysis CFU analysis was performed as follows: 100 μL samples were taken and diluted with saline at 10 -1 ~10 -9 Serial dilutions were prepared. -7 ~10 -9 100 μL of each dilution was plated onto TSA plates and incubated for at least 4 days in anaerobic jars at 37°C using the Anaerocult incubation system. Counts (plates with 30–300 colonies) were performed manually.

[0100] CFU analysis of the lyophilized composition obtained by the method of the present invention was performed as follows: 0.3 g of powder was resuspended in 9.7 mL of sterile physiological water (gravimetric dilution). Serial dilutions, plating and incubation were performed as described above.

[0101] Harvesting, Microfiltration and Washing Wash solution: 50 mM citrate buffer in water (pH 7.5) was prepared the day before and stored at 4° C. in a 20 L jerrican.

[0102] At the end of the fermentation, each fermentation broth, kept at 8°C, was collected and hygienically transferred to an inner container tank. The fermentation broth was then subjected to cross-flow microfiltration. This device was used for concentration and then washing with citrate buffer. For comparison, some batches were split and / or post-processed by centrifugation and resuspension / washing of the cell pellet in citrate buffer twice.

[0103] Biomass blending Aqueous cryoprotectant solutions were prepared on a weight basis as shown in Table 1 and made up to final volume with water after pH adjustment. Specifically, each component was added one at a time, waiting until the previous component was completely dissolved, and then the pH was adjusted to 6 with 50 mM citric acid solution. The prepared solutions were sterilized and stored at 4°C before use.

[0104] [Table 1]

[0105] A formulated biomass concentrate was prepared by adding 1 / 2 weight of cryoprotectant solution to the (washed) biomass concentrate (weight ratio of biomass / cryoprotectant solution=2:1).

[0106] Freeze drying Freeze drying of the (washed) biomass preparation was carried out in a Virtis Genesis 35L Pilot Freeze dryer from SP Scientific (PA, USA) as follows: Start: Store at 3℃ Freezing stage: 1℃ / min to -40℃ Primary drying stage: vacuum down to 0.1mbar, warm to -25°C in 15 minutes · Primary drying stage: -25℃ for 58 hours Secondary drying: Heat to 20℃ in 7.5 hours and hold at 20℃ for 4 hours.

[0107] After freeze drying, each tray was placed into a plastic zip-top bag. The bag was handled and slight pressure was applied to the bottom of the tray to remove the cake from the tray. The plastic bag was opened, the tray was removed and the cake was crushed by hand.

[0108] result Tables 2 and 3 provide an overview of the process parameters during the cross-flow microfiltration run of the C. acnes strain K8 and C. acnes strain C3 batches, respectively.

[0109] [Table 2]

[0110] [Table 3]

[0111] CFU analysis was performed on the biomass concentrate, the washed biomass concentrate, and the lyophilized compositions obtained from the washed and unwashed biomass concentrates, and showed that there was no recovery loss during microfiltration for both C. acnes strain K8 (Table 4) and C. acnes strain C3 (Table 5), and that the lyophilized compositions obtained from the washed biomass concentrate showed significantly improved recovery compared to the use of the unwashed biomass concentrate (Table 5).

[0112] [Table 4]

[0113] [Table 5]

[0114] Residual moisture content To investigate the storage stability, residual moisture content analysis was performed one month after freeze-drying of the C. acnes strain C3 batch using a Sartorius moisture analyzer (1 g / 100°C) (Sartorius AG, Germany). The results revealed that the washing process also reduced the residual moisture content of the freeze-dried composition (see Table 6), indicating improved stability.

[0115] [Table 6]

[0116] Comparative experiment between microfiltration and centrifugation After fermentation was completed and cooled, the broths of C. acnes C3 and C. acnes K8 were harvested and concentrated either by cross-flow microfiltration (two batches, referred to as Invention I and II in Table 9) or by centrifugation (referred to as Reference in Table 9), washed, formulated, and freeze-dried. The freeze-dried composition of C. acnes strain C3 was then blended with the freeze-dried composition of C. acnes strain K8, and the viability of the blend was approximately 1×10 10 (hereafter referred to as C. acnes blend). The viability of freeze-dried compositions prepared using either cross-flow microfiltration or centrifugation was examined over time during storage.

[0117] As can be seen from the data outlined in Table 7 below, cross-flow microfiltration provided a significant improvement in viability upon storage (accelerated stability study at 40° C.).

[0118] [Table 7]

[0119] [Table 8]

[0120] Preparation: Mix phase A and heat to 85°C under stirring. Homogenize at 14,000 rpm for 2 minutes. Allow to cool to room temperature. Weigh phase A into a sterile beaker. Add phase B to phase A and stir at 350 rpm for 5 minutes using an anchor stirrer.

Claims

1. 1. A method for producing a lyophilized composition comprising at least one non-pathogenic C. acnes strain, comprising: a) concentrating bacterial cells of at least one non-pathogenic C. acnes strain from a fermentation broth by cross-flow microfiltration to obtain a biomass concentrate comprising said cells; b) optionally washing the biomass concentrate to obtain a washed biomass concentrate; c) combining the concentrated or washed biomass concentrate with an aqueous solution comprising at least one cryoprotectant to obtain a biomass blend; d) freeze-drying the biomass preparation to obtain a freeze-dried composition comprising at least one non-pathogenic C. acnes strain; A method comprising:

2. 2. The method of claim 1, wherein the viability of the non-pathogenic C. acnes strain in the freeze-dried composition after 1 month, preferably after 3 months of storage, is increased by at least 30%, preferably at least 40%, most preferably at least 50% compared to concentration by centrifugation.

3. 2. The method of claim 1, wherein the biomass concentrate is washed in step b) with an aqueous (buffered) solution of an organic acid selected from the group consisting of citric acid, retinoic acid, ferulic acid, glycolic acid, lactic acid, fruit acids, salicylic acid and hyaluronic acid and mixtures thereof, preferably citric acid.

4. 4. The method of claim 3, wherein the biomass concentrate is washed with a 10-100 mM, preferably 20-80 mM, more preferably 30-70 mM, such as 40, 50 and 60 mM citric acid aqueous solution (buffer).

5. 10. The method of claim 1, wherein the concentrated biomass is washed during cross-flow microfiltration.

6. The aqueous solution of the organic acid is added to the biomass concentrate obtained in step a) and again subjected to cross-flow microfiltration, preferably in such a way that the content of the non-pathogenic C. acnes strain in the obtained washed biomass concentrate is at least 1 x 10 １０ CFU / g, more preferably at least 1 x 10 １１ 4. The method of claim 3, wherein the method is carried out until CFU / g is reached, most preferably until more than 90% viable cells are recovered.

7. In step c), the biomass concentrate is mixed with alcohols such as glycerol, propylene glycol, and ethylene glycol; sugars such as glucose, galactose, lactose, sucrose, maltose, fructose, trehalose, raffinose, mannitol, and sorbitol; polymers such as polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), skim milk, gelatin, proteins and protein hydrolysates, peptides, yeast, broth, dextrin, maltodextrin, methylcellulose, povidone, serum, and peptone; sulfuric acid.

10. The method of claim 1, wherein the biomass concentrate or the washed biomass concentrate is formulated by mixing with at least one cryoprotectant selected from the group consisting of salts such as sodium, calcium lactate, monosodium glutamate, sodium chloride, potassium chloride, sodium thiosulfate, trisodium citrate, ammonium acetate, ammonium chloride, etc.; acids such as citric acid, phosphoric acid, glutamic acid, tartaric acid, amino acids, ethylenediaminetetraacetic acid (EDTA), etc.; bases such as ammonium hydroxide, sodium hydroxide, sodium bicarbonate, etc.; and mixtures thereof.

8. 8. The method of claim 7, wherein the at least one cryoprotectant is selected from the group consisting of sucrose, maltodextrin, glutamic acid, sodium glutamate, and trisodium citrate, and mixtures thereof.

9. 2. The method of claim 1, wherein the formulation in step c) comprises 1 to 20 wt. %, preferably 5 to 15 wt. %, more preferably 6 to 12 wt. %, such as 6, 7, 8, 9, 10, 11 and 12 wt. % of cryoprotectant relative to the total weight of the biomass formulation.

10. The non-pathogenic C. acnes strain is a type I C. acnes strain, for example, IA (i.e., IA １ and I.A. ２ 2. The method of claim 1, wherein the C. acnes strain is selected from the group consisting of C. acnes strains D1, A5, C3, H1, H2, H3, K1, K2, K4, K6, K8, K9, L1 and F4, and mixtures thereof.

11. 2. The method of claim 1, wherein the at least one non-pathogenic C. acnes strain is C. acnes strain C3 or C. acnes strain K8, and mixtures thereof.

12. A freeze-dried composition comprising one or more freeze-dried compositions obtained by the method of any one of claims 1 to 11.

13. The freeze-dried composition has an initial viability of the non-pathogenic C. acnes strain of at least 1 x 10 viable cells per total weight of the composition. １０ The freeze-dried composition of claim 12, wherein the CFU / g.

14. 13. The freeze-dried composition of claim 12, wherein the residual moisture content of the composition is less than 3% by weight, preferably less than 2% by weight, more preferably less than 1.5% by weight, and most preferably less than 1.2% by weight, based on the total weight of the composition.

15. 13. The freeze-dried composition of claim 12, wherein the freeze-dried composition maintains at least 80%, preferably 85%, more preferably 90% viability of the bacterial strain after storage at RT, preferably 40°C, for 4 weeks.