Preservation of microorganisms

Abstract

The present invention relates to the field of microbial preservation, in particular to provide a two-chamber system comprising a first chamber comprising microcapsules comprising viable microorganisms in a non-aqueous composition of the microcapsule core and a second aqueous organic acid chamber. The present invention also provides methods for preserving microorganisms based on these systems and uses of these systems for preserving microorganisms.

Description

Invention Field

[0001] This invention relates to the field of microbial preservation, and in particular to a two-compartment system comprising a first compartment and a second aqueous organic acid compartment, wherein the first compartment comprises microcapsules containing live microorganisms in a non-aqueous composition of the microcapsule core. The invention also provides methods for preserving microorganisms based on these systems, and uses of these systems for preserving microorganisms. Background of the Invention

[0003] The use of beneficial microorganisms as probiotics in medicine has been developed over the past few decades. To ensure the stability of these probiotics, the bacteria need to be dried (e.g., freeze-dried or spray-dried) to achieve metabolic inactivation conditions. Therefore, they can survive for decades under proper storage conditions (such as humidity, temperature, etc.). By introducing water, the bacterial powder will be reactivated, and the bacteria will be able to perform their functions. To date, most probiotic preparations have been used for gastrointestinal applications. The most common formulations are capsules and tablets, which can be easily stored in anhydrous conditions and can be sealed under inert gases such as nitrogen or carbon dioxide to set a good relative humidity for maximum stability.

[0004] On the other hand, the use of probiotics in topical (or other types of aqueous) preparations may also hold great potential. One option might be to formulate these probiotics in anhydrous substances such as ointments or oil gels; however, patients generally do not accept those formulations. More acceptable formulations are in the form of gels / creams / foams / lotions. However, that is, in order to be properly formulated into gels, creams, foams, lotions, ointments, etc., these topical preparations themselves are highly aqueous. Obviously, the presence of such a high water content in these preparations poses a problem for the storage of probiotics under conditions of metabolic inactivation.

[0005] A second problem with these aqueous (e.g., topical) formulations is that they often contain agents incompatible with microbial survival; such as preservatives, surfactants, and emulsifiers, to prevent unwanted microbial growth and to form stable emulsions. However, these agents can also pose a major problem in formulations containing beneficial microorganisms.

[0006] Therefore, the object of the present invention is to provide a system that allows for the long-term storage of microorganisms without substantially harming them during use. Surprisingly, a two-compartment system has been found to provide a solution to the aforementioned problem, comprising a first compartment and a second compartment. The first compartment comprises microcapsules containing a water-insoluble and waterproof shell and microorganisms contained in a non-aqueous composition within the microcapsule core. The second compartment contains (or consists of) an aqueous composition comprising one or more organic acids (with a pH less than 7.0 and substantially free of buffers). In particular, this type of system has been found to enable the long-term storage of microorganisms because they are contained in the aqueous compartment during storage, preventing their exposure to water. Subsequently, upon combined use of the contents of both compartments, it has been surprisingly found that the organic acids used as a preservative do not immediately harm the released microorganisms, which are activated by the aqueous component of the second composition. This contrasts with other types of preservatives that have a very direct mechanism of action against microorganisms.

[0007] Furthermore, while the formulations of the present invention are particularly suitable for topical application of probiotics, the concept of the present invention can also be extended to other areas where problems arise in preserving / stabilizing microorganisms in aqueous environments. Therefore, these problems are solved by formulating them in a two-compartment system as defined in the present invention.

[0008] While the concept of formulating microorganisms in a two-compartment system has been disclosed, existing technologies do not offer a solution that protects the microorganisms when the contents of the two compartments are combined. In particular, the aqueous components required for microbial activation are often very harsh on microorganisms, primarily containing preservatives, surfactants, etc., incompatible with the long-term survival of such microorganisms. In contrast, by choosing organic acids as preservatives, we found that it is unnecessary to include additional preservatives, surfactants, or other harmful components, while maintaining the stability required for such formulations.

[0009] Antunes et al., 2013 (Food Science and Technology 54:125-131) describe a microencapsulation process in which a bacterial suspension is immobilized in cellulose acetate along with glycerol, maltodextrin, Tween, etc. During spray drying, the bacterial suspension is atomized using a single nozzle in a heated zone (100°C–200°C). This evaporates the water in the suspension, yielding a dried bacterial powder. The resulting powder resembles a matrix containing the microorganisms, rather than a microcapsule comprising a water-insoluble and impermeable shell and microorganisms contained in a non-aqueous composition of the microcapsule core.

[0010] US2012263826 describes an oral solution comprising at least one aqueous solution and a jelly-like capsule containing alginate, whey protein, and probiotics. Such jelly-like capsules are actually 1-fasic gel particles (i.e., a matrix containing probiotics), rather than a two-compartment system as disclosed herein, in which the probiotics are maintained in an anhydrous core of the capsule. Invention Overview

[0012] In a first aspect, the present invention provides a two-compartment system comprising:

[0013] - A first chamber comprising a microcapsule, the microcapsule comprising a water-insoluble and water-impermeable shell, and microorganisms in a non-aqueous composition of the microcapsule core; and

[0014] - A second chamber comprising an aqueous composition with a pH less than 7.0 and including one or more organic acids;

[0015] The second chamber contains virtually no buffer.

[0016] In a specific embodiment of the present invention, the second aqueous composition has a pH of less than 5.5, preferably less than 5.0, and more preferably less than 4.5.

[0017] In another specific embodiment, the one or more organic acids are selected from benzoic acid, sorbic acid, citric acid, acetic acid, lactic acid, oxalic acid, formic acid, dehydroacetic acid, fumaric acid, anisic acid, gluconic acid, malic acid, succinic acid, tartaric acid, phosphoric acid, and propionic acid and their derivatives.

[0018] In a further embodiment, the microorganism is a probiotic microorganism, more preferably selected from a list including Lactobacillus pentosus, Lactobacillus rhamnosus, and Lactobacillus plantarum.

[0019] In the specific implementation plan, the first chamber is (substantially) impermeable to water and oxygen.

[0020] In another specific embodiment, the water-insoluble and waterproof shell of the microcapsule of the present invention is composed of: alginate, xanthan gum, gum arabic, gellan gum, carrageenan, gelatin, cellulose or derivatives thereof; or polymers based on agar, protein, polyol, gelatin, PVA (polyvinyl alcohol), PLGA (poly(lactic-co-glycolic acid) copolymer), PLA (polylactic acid) and its derivatives, PCL, polyisohexyl cyanoacrylate, acrylate derivatives or starch, optionally combined with chitosan; or stearic acid.

[0021] In specific embodiments, the two-compartment system of the present invention is in the form of a gel, cream, foam, lotion, or ointment containing the microcapsules.

[0022] In a specific embodiment of the invention, the non-aqueous composition is selected from the following list: vegetable oils, mineral oils, silicone oils or hydrophilic polymers; particularly decanoic acid / caprylic acid triglycerides, liquid paraffin, polyethylene glycol, silicone or stearic acid.

[0023] In a specific embodiment of the invention, the one or more organic acids are used as preservatives, and the composition is substantially free of other preservatives.

[0024] In a further aspect, the present invention provides a method for preserving live microorganisms, comprising:

[0025] - Offers a two-room system;

[0026] - The live microorganisms are included in a first chamber, the first chamber comprising a microcapsule comprising a water-insoluble and water-impermeable shell and microorganisms contained in a non-aqueous composition of the microcapsule core;

[0027] - The aqueous composition of the second chamber of the two-chamber system includes one or more organic acids in an amount sufficient to achieve a pH of less than 7.0; wherein the second chamber is substantially free of buffers.

[0028] In a further aspect, the present invention provides the use of a combination of microcapsules and an aqueous composition, wherein the microcapsules comprise a non-aqueous composition containing live microorganisms; and the aqueous composition comprises one or more organic acids with a pH less than 7.0, which are used to preserve the aqueous composition without harming the microorganisms.

[0029] Brief description of the attached figures

[0030] Referring now specifically to the accompanying drawings, it is emphasized that the details shown are by way of example and are for the purpose of illustrative discussion of different embodiments of the invention only. They are provided to offer a description that is considered most useful and readily understood regarding the principles and concepts of the invention. In this regard, no attempt is made to show structural details of the invention more detailed than those necessary for a substantially clear understanding of the invention. The description with reference to the drawings is intended to guide those skilled in the art on how the invention can be practiced in several forms.

[0031] Figure 1 Stability of microorganisms in silicone; A) CF 1406, B) CF6570.

[0032] Figure 2 Stability of microorganisms in oil; A) Sunflower oil, B) Miglyol 812N.

[0033] Figure 3 Stability of microorganisms in polar media; A) PEG400, B) Glycerol.

[0034] Figure 4 Stability of microorganisms in hard fat; A) Whitepsol h15, B) Hydrobase 32 / 34. Invention Details

[0036] As detailed above, in a first aspect, the present invention provides a two-compartment system comprising:

[0037] - A first chamber comprising microcapsules, the microcapsules comprising a water-insoluble and water-impermeable shell and a non-aqueous composition of the microcapsule core containing microorganisms; and

[0038] - A second chamber comprising an aqueous composition with a pH less than 7.0 and including one or more organic acids;

[0039] The second chamber contains virtually no buffer.

[0040] More specifically, the present invention provides a two-compartment system as defined herein, comprising the following:

[0041] - Microcapsules having a water-insoluble and impermeable shell and microorganisms contained in a non-aqueous composition within the shell core; and

[0042] - An aqueous composition with a pH less than 7.0 and comprising one or more organic acids;

[0043] The aqueous composition therein is substantially free of buffers.

[0044] More specifically, the present invention provides a two-compartment system as defined herein, which comprises the following:

[0045] - Microcapsules capable of having a water-insoluble and impermeable shell and microorganisms contained in a non-aqueous composition within the shell core; and

[0046] - An aqueous composition with a pH less than 7.0 and comprising one or more organic acids;

[0047] The aqueous composition therein is substantially free of buffers.

[0048] In the context of this invention, the terms "insoluble in water" and "waterproof" for microcapsules are understood to mean waterproof. Specifically, a waterproof capsule can be described as one that does not degrade when suspended in a second water-containing chamber. However, microcapsules as used herein can (and generally will) degrade, for example, by losing their insolubility under certain stress conditions such as salt concentration or mechanical shear stress, thereby releasing their active contents, as described herein through a capsule release mechanism.

[0049] In the context of this invention, the term "aqueous composition" refers to a formulation containing water. Specifically, the formulation contains a significant amount of water, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% water. Therefore, the term "non-aqueous composition" refers to a formulation that does not contain a significant amount of water, for example, no more than 10%, preferably no more than 5%, even more preferably no more than 2%, and most preferably it contains no water at all, i.e., 0%.

[0050] In the context of this invention, the term "microorganism" refers to "living" microorganisms, meaning they are alive, not in their fragments, culture supernatant, or killed form. It is preferable to freeze-dry the live microorganisms to enhance their preservation.

[0051] In the context of this invention, the term "organic acid" refers to an organic compound with acidic properties. Obviously, in the context of this invention, any suitable organic acid can be used, provided it functions as a preservative.

[0052] These organic acids have been found to have a slow mechanism of action on microorganisms; when microorganisms come into contact with them, the organic acids do not immediately harm the (probiotic) microorganisms. After prolonged exposure to the environment, such as through the skin, the organic acids lose their activity, thus not harming the (probiotic) microorganisms for an even longer period. Although organic acids are preferred as preservatives in this invention (because they have been found to lose their activity after exposure to the environment), the invention can also be carried out using another preservative, as long as it has a slow mechanism of action, for example, requiring at least 24 hours to become active.

[0053] For the use of organic acids, formulations are preferably substantially free of buffers. The presence of buffers will keep the composition at a low pH for a longer period, thus requiring a longer time to inhibit the preservative effect of the organic acids, thereby increasing the risk of microbial damage upon contact with the organic acids. Although most components will have a small buffering effect, it is desirable to select components of the composition such that they do not substantially reduce or increase the time required to inactivate the organic acids.

[0054] Particularly suitable organic acids are selected from the list comprising: benzoic acid, sorbic acid, citric acid, acetic acid, lactic acid, anisic acid, oxalic acid, formic acid, dehydroacetic acid, fumaric acid, gluconic acid, malic acid, succinic acid, tartaric acid, phosphoric acid, and propionic acid and their derivatives. More specifically, the organic acids are selected from the list comprising: benzoic acid, sorbic acid, citric acid, acetic acid, lactic acid, oxalic acid, formic acid, dehydroacetic acid, fumaric acid, gluconic acid, malic acid, succinic acid, tartaric acid, phosphoric acid, and propionic acid and their derivatives; for example, sorbic acid.

[0055] In specific embodiments of the invention, the pH of the second aqueous composition according to the invention is less than 5.5, preferably less than 5.0, more preferably less than 4.5, less than 4.0, or less than 3.5. The pH of the formulation is highly relevant in the context of this invention. Generally, the lower the pH, the higher the preservative effect, thereby contributing to the long-term stability of the formulation of the invention. The desired pH is obtained by compounding with organic acids, thereby acting as preservatives in the formulation. Due to the presence of these organic acids, we have found that it is no longer necessary to include additional preservatives; therefore, the formulations of the invention are preferably substantially free of preservatives other than organic acids. Furthermore, the composition is also substantially free of other components harmful to microorganisms, such as phenoxyethanol, bronidox, isothiazolinone, and sodium lauryl sulfate, which are frequently used in topical formulations.

[0056] In a preferred embodiment, the live microorganism of the present invention is a live probiotic microorganism.

[0057] In the context of this invention, the term "probiotic" means including microorganisms that provide health benefits when used in the human or veterinary field. The formulations of this invention are highly suitable for any known probiotic microbial formulations, such as, but not limited to, *Lactobacillus*, and more particularly *Lactobacillus pentosus*, *Lactobacillus rhamnosus*, and / or *Lactobacillus plantarum*. Clearly, the formulations of this invention may contain only one species of probiotic microorganism or a combination thereof, depending on the intended use.

[0058] To increase the stability of microorganisms in the first chamber, the first chamber is preferably (substantially) impermeable to water and oxygen.

[0059] In the context of this invention, the term "microencapsulation" or "microcapsule" refers to a product obtained through a microencapsulation process. In this process, micron-sized (micrometer range) particles or droplets are surrounded by a coating to obtain small capsules with numerous useful properties. In the context of this invention, it is used to incorporate probiotic microorganisms. In a relatively simple form, a microcapsule is a small sphere surrounded by a uniform wall. The material inside the microcapsule is referred to as the core, inner phase, or filler; while the wall is referred to as the shell, coating, or membrane.

[0060] Those skilled in the art will readily recognize that several methods exist for manufacturing microcapsules, and this invention is not limited to any of these methods. Microencapsulation methods suitable for the context of this invention include, but are not limited to, pan coating, air suspension coating, centrifugal extrusion, vibrating nozzle, spray drying, ionogelation, condensed phase separation, interfacial polycondensation, interfacial crosslinking, in-situ polymerization, and matrix polymerization.

[0061] The initial purpose of microencapsulation was to separate the core from its surroundings, and in this invention, particularly to protect the encapsulated microorganisms from the preservative effects of surrounding organic acids and to prevent their activation in aqueous environments. Clearly, during use, the walls of the microcapsules need to rupture to release the microorganisms and allow them to function as needed. Such wall rupture is preferably achieved during use (e.g., when applying the formulation to the skin or other localized areas) through low pressure, friction, or shear stress.

[0062] In its simplest form, the microcapsules of the present invention comprise a water-insoluble and waterproof shell, wherein microorganisms are contained in a non-aqueous composition of the microcapsule core. However, where appropriate, the microcapsules may include additional layers, such as an extra coating layer, to increase protection of the contents.

[0063] In a specific embodiment of the invention, the microcapsule comprises a water-insoluble and waterproof shell; and the microorganism is contained in a non-aqueous composition of the microcapsule core. The presence of water is sufficient to reactivate freeze-dried bacteria, therefore it is important to formulate them in the absence of water, thereby allowing for long-term storage. The water-insoluble and waterproof shell may be composed of any suitable material, such as, but not limited to, alginate, xanthan gum, gum arabic, gellan gum, carrageenan, gelatin, cellulose or derivatives thereof; or based on agar, protein, polyol, gelatin, PVA (polyvinyl alcohol), PLGA (poly(lactic-co-glycolic acid) copolymer), PLA (polylactic acid) polymers and derivatives thereof, PCL, polyisohexyl cyanoacrylate, acrylate derivatives or starch, or hard fats such as witepsol or hydrobase.

[0064] While these components can be used on their own, they can also be combined with each other or cross-linked with polymers or chitosan. This further cross-linking can enhance the durability of the microcapsules. Furthermore, to further protect the contents of the microcapsules (i.e., microorganisms) from water and air, the capsules can optionally be coated with a suitable coating.

[0065] As described earlier, microorganisms should be stored under anhydrous conditions to ensure long-term storage. Therefore, the microcapsule core of the present invention is preferably an anhydrous medium, such as, but not limited to, vegetable oil, mineral oil, silicone oil, or hydrophilic polymers; particularly decanoic acid / caprylic acid triglycerides, liquid paraffin, polyethylene glycol, silicone, or hard fats such as Witepsol or Hydrobase.

[0066] Of course, the presence of preservatives is crucial for the long-term storage of formulations; however, it poses a serious problem for formulations containing microorganisms such as probiotics. As a solution, we have found that microencapsulation of (probiotic) microorganisms and the inclusion of organic acids within these microcapsules of the provided formulation are sufficient to ensure long-term storage without hindering microbial activity. Even when using the formulation and releasing the microorganisms, the latter are not hindered by the presence of compounded organic acids, as these organic acids rapidly lose their activity upon use, for example, due to the buffering capacity of the skin. Therefore, the organic acids of this invention are intended to be used as preservatives, and the formulations are substantially free of additional preservatives that could hinder microbial activity after release from the microcapsules.

[0067] Therefore, in a specific embodiment of the invention, the organic acid is intended to be used as a preservative, and the formulation is substantially free of other preservatives.

[0068] The formulations of the present invention are particularly suitable for topical application because such formulations typically have a high water content. Therefore, in specific embodiments, the aqueous formulations according to the present invention are topical aqueous formulations.

[0069] In the context of this invention, the term "local" means local delivery at a specific location on the body, particularly application to a specific part of the body or within the body. Specifically, it includes application to mucous membranes by means of aqueous, i.e., non-solid formulations such as creams, foams, gels, lotions, or ointments, or any other type of aqueous formulation. Clearly, the term "local" is not intended to include delivery in the form of solid dosage forms such as capsules or tablets.

[0070] The topical probiotic formulation of the present invention can be in any suitable form, such as, but not limited to, gels, creams, foams, lotions or ointments containing the microcapsules.

[0071] As detailed above, the discovery of this invention is that a microbial preparation that prevents these microorganisms from coming into contact with the water components in a two-chamber system and that uses organic acids is sufficient to allow the preparation to be stored for a long time without substantially harming the (probiotic) microorganisms.

[0072] Therefore, in a further aspect, the present invention provides a method for preserving live microorganisms, comprising:

[0073] - Offers a two-room system;

[0074] - The live microorganisms are included in a first chamber, the first chamber comprising a microcapsule comprising a water-insoluble and water-impermeable shell and microorganisms contained in a non-aqueous composition of the microcapsule core;

[0075] - The aqueous composition of the second chamber of the two-chamber system includes one or more organic acids in an amount sufficient to achieve a pH of less than 7.0; wherein the second chamber is substantially free of buffers.

[0076] In a final aspect, the present invention provides the use of a combination of one or more organic acids comprising microcapsules containing live (probiotic) microorganisms and an aqueous composition with a pH less than 7; more specifically, for preserving the aqueous composition without impairing the microorganisms in the microcapsules.

[0077] The formulations of the present invention are particularly well-suited for local administration, including direct application to the skin or mucous membranes such as the vagina.

[0078] The appropriate form of administration (which may be semi-solid or liquid, depending on the route of administration) and the methods and carriers, diluents and excipients used to prepare them will be clear to a person skilled in the art; see again for example US-A-6,372,778, US-A-6,369,086, US-A-6,369,087 and US-A-6,372,733, and the latest edition of standard manuals such as Remington's Pharmaceutical Sciences.

[0079] Some preferred but non-limiting examples of such preparations include elixirs, suspensions, emulsions, solutions, syrups, ointments, creams, and lotions, which can be formulated with carriers, excipients, and diluents that are themselves suitable for such preparations, such as lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, alginate, tragacanth gum, gelatin, polyethylene glycol, cellulose, (sterile) water, methylcellulose, edible oils, vegetable oils, and mineral oils, or suitable mixtures thereof.

[0080] The formulations of the present invention may optionally contain other ingredients, such as drugs and / or prebiotics, for example, to stimulate the growth of microorganisms.

[0081] The formulation may optionally contain other substances commonly used in pharmaceutical formulations, such as wetting agents, emulsifiers, suspending agents, and dispersants. However, it is very important that these additional substances do not substantially harm the probiotic organisms during the storage of the composition or during its use.

[0082] More specifically, the composition can be formulated in a pharmaceutical preparation comprising particles, which consists of a solid dispersion of the microorganisms of the present invention and one or more pharmaceutically acceptable water-soluble polymers.

[0083] The term "solid dispersion" defines a system in a solid state (not a liquid or gaseous state) containing at least two components, wherein one component is more or less uniformly dispersed in one or more other components.

[0084] Microorganisms can also be readily formulated in the form of microparticles, which have surface modifiers adsorbed on their surfaces in an amount sufficient to maintain an effective average particle size of approximately 1–5000 μm. Suitable surface modifiers can be preferably selected from known organic and inorganic pharmaceutical excipients. These excipients include various polymers, low molecular weight oligomers, natural products, and surfactants such as nonionic and anionic surfactants.

[0085] The invention will now be illustrated by the following synthetic and biological examples, which do not in any way limit the scope of the invention. Example

[0086] Example 1: Selection of a non-aqueous composition for the first chamber

[0087] The purpose of this embodiment is to select a suitable non-aqueous composition for including live microorganisms, which enables it to remain stable and viable for a long period of time.

[0088] Materials and methods

[0089] Stability Study

[0090] The three strains used in this stability study:

[0091] - Self-cultured Lactobacillus pentosus (i.e., lac4 or Lp)

[0092] - LGG players developed in-house (i.e., lac7 or LGG)

[0093] - Purchased freeze-dried LGG

[0094] Eight different anhydrous suspension media used for bacterial preservation were tested at room temperature (+ / -20°C). The suspension media used could be further subdivided into four types, with two subtypes in each group compared as follows:

[0095] ● Silicone:

[0096] ○ Cosmetic Liquid 1406 (Polydimethylsiloxane & Polydimethylsiloxane) – from Chemsil

[0097] ○ Cosmetic Liquid 6570 (Dimeric Siloxane, Trisiloxane, Cyclopentadimethylsiloxane & Dimethicone) – from Chemsil

[0098] ● Oil:

[0099] ○Sunflower Oil – From Everyday

[0100] ○Migyol 812N – from Hüls

[0101] ●Polar solvents:

[0102] ○PEG 400 – from Roth

[0103] ○ Glycerin - from Fagron

[0104] ● Hard fat:

[0105] ○Witepsol H15 - from VW chemicals

[0106] ○ Hydrobase 32 / 34 – from Prod’hyg laboratoires

[0107] Samples were obtained at 9 predetermined time points:

[0108] ●T0 = suspended before the suspension medium

[0109] ●T1 = 1 day after levitation

[0110] ●T2 = 1 week after suspension

[0111] ●T3 = 2 weeks after levitation

[0112] ●T4 = 1 month after levitation

[0113] ●T5 = 2 months after levitation

[0114] ●T6 = 6 months after levitation

[0115] ●T7 = 12 months after levitation

[0116] ●T8 = 24 months

[0117] All materials used are sterilized by autoclaving before use. Where possible, all ingredients and components are autoclaved; otherwise, they are filtered in a sterile environment.

[0118] T0 CFU assay

[0119] Lac 4 and Lac 7 were cultured in liquid MRS (de MAN, Rogosa & Sharpe) medium (37°C) until full growth was achieved. The medium was centrifuged at 2780G for 10 minutes. The supernatant was discarded, and the bacterial pellet was used for further processing.

[0120] Weigh 100 mg and dilute with physiological water (0.85% NaCl) to obtain a total volume of 10 ml. Prepare a series of dilutions (10-fold serial dilutions) using three strains (lac4, lac7, and LGG), and perform plate counting according to the plate spread method (derived from pharmacopoeia methods 2.6.12 and 2.6.13). Measurements were repeated three times. Results are expressed as CFU / g powder.

[0121] Suspension of bacteria in suspension medium

[0122] The obtained bacterial clumps were uniformly diluted 1 / 10 (m / m%) using different anhydrous suspension media. One gram of the suspension was weighed into a falcon tube and sealed in an aluminum bag (RH = 20%). Each sample contained 100 mg of bacteria for further testing. The aluminum bag was opened at regular intervals for survival testing.

[0123] Test after storage time

[0124] The sealed bag was opened at specific time points for stability testing. Centrifuge tubes containing 1 gram of anhydrous material were further processed by adding 1 gram of an emulsifier mixture (composed of polysorbate 80 and dehydrated sorbitan sesquioleate) and 8 grams of physiological water. This formed an emulsion through which bacteria could come into contact with water and be reactivated. Further sample preparation and plate counting were performed according to the methods described above, derived from Pharmacopoeia Methods 2.6.12: Microbiological Examination of Non-Sterile Products: Microbial Count Test and 2.6.13: Microbiological Examination of Non-Sterile Products: Test for Specified Microorganisms.

[0125] Results and discussion

[0126] The stability of the lyophilized powder was, as expected, far superior to that of the original strain. After just one month, there was an average reduction of 3 logs, while the lyophilized strain showed no detectable reduction. This demonstrates the importance of starting formulations with a suitable lyophilized (or other drying method) powder to ensure long-term stability of live microorganisms.

[0127] Therefore, the only results to be discussed further will be those of the freeze-dried LGG strain.

[0128] For silicon mixtures ( Figure 1 ),Oil( Figure 2 ) and hard fat ( Figure 3 The stability results are quite similar in hydrophilic media. Figure 3 In this study, it seems that only PEG 400 is suitable as a suspension medium, rather than glycerol, which has a negative impact on the stability of FDIGG.

[0129] For different anhydrous media, there is an average reduction of 1 logarithm after one year of stabilization. This leaves enough viable microorganisms for topical application and to produce beneficial effects on the skin.

[0130] This demonstrates that, by protecting suitable lyophilized powders suspended in an anhydrous medium from light, humidity, and preservatives, it is possible to obtain a phase / compartment containing stable probiotics. This compartment can be a two-phase system in which an aqueous and anhydrous phases are mixed in situ, or it can be a capsule suspended in a formulation.

[0131] Example 2: Selection of an aqueous composition for the second chamber

[0132] The aim is to create a topical formulation from live microorganisms that can be applied to the skin in which they will exert beneficial effects. Topical formulations such as creams typically contain oils, water, preservatives, and emulsifiers. The presence of emulsifiers and preservatives can negatively impact the stability of the lyophilized microorganisms. A method to ensure a certain shelf life is to suspend the lyophilized probiotics in an anhydrous medium (see Example 1). This anhydrous suspension will provide good stability for the lyophilized microorganisms. To prevent the negative impact of other components in the topical formulation, the anhydrous suspension is preferably separated from the rest of the formulation by physical phase separation or by encapsulation of the anhydrous suspension. Capsules can then be suspended in the topical formulation.

[0133] Once the phases are separated, a long-lasting topical product containing live microorganisms can be obtained. Problems can arise if the two phases are mixed before application to the skin. Freeze-dried probiotics are activated by absorbing water from the formulation, but they may also encounter emulsifiers and preservatives present, which can kill the activated microorganisms and prevent them from producing beneficial effects on the skin.

[0134] This experiment aims to screen and determine the short-term effects (10 minutes -> 1 day) of different formulations on the survival of freeze-dried probiotics.

[0135] 1. Method

[0136] 1.1 Experiment A

[0137] This experiment simulated lyophilized live microorganisms stably contained in an anhydrous suspension, then mixed with formulations containing different preservatives and emulsifiers. Three standard TMF formulations were incubated with 10% (m / m) lyophilized LGG. The TMF formulations are widely used (Belgium) and considered safe and stable for topical drug delivery. Samples were taken from the formulations after 10 minutes, and the number of viable cells was determined after 24 hours to determine the short-term antimicrobial effect of the formulations. Topical probiotic application is typically performed daily. Therefore, no sampling was performed after 24 hours. Sample preparation and CFU determination (plate count) were performed according to the plate-spreading method (derived from pharmacopoeia methods 2.6.12 and 2.6.13). Measurements were repeated three times. Results are expressed as cfu / g powder.

[0138] The ingredients of the tested cream are as follows:

[0139]

[0140]

[0141]

[0142] 1.2 Experiment B

[0143] To expand the screening platform of Experiment A, Experiment A was repeated using commercially available formulations containing different preservatives and emulsifiers. The experiments were conducted as described in 2.1. Cream A from Experiment A was used as a reference. Other pharmaceutical cream bases (1-5) were screened and compared with commercially available cosmetic formulations (6-11), as detailed in the table below:

[0144]

[0145] 2. Results

[0146] 2.1 Experiment A

[0147]

[0148] The survival of lyophilized LGG after suspension in the above formulations for 10 minutes appeared to have only a minor effect. After 24 hours, Cream A showed almost no significant reduction. A slight reduction was noted in Cream C (±0.5 logarithms), while Cream B showed a significant reduction (±3 logarithms). Creams A and C both contain the organic acid sorbic acid as a preservative, while Cream B contains parabens as a preservative and sodium lauryl sulfate (a known anionic emulsifier with antibacterial properties).

[0149] In conclusion, organic acids, with their slow-acting mechanism (onset time of 1-3 days (data not shown)), can be considered safe preservatives for use in topical formulations in combination with lyophilized microorganisms. Formulation A is recommended for further testing in conjunction with lyophilized probiotics in topical formulations.

[0150] 2.2 Experiment B

[0151]

[0152] *The method used has four log-decreasing detection limits. A CFU count of 0 means that less than 1.0E+07 CFU / gram of powder has been detected.

[0153] First, it was noted that the pharmaceutical cream base (1-5) appeared to be less antibacterial than the tested cosmetic formulations (6-10) in the short term. The cosmetic formulations were extremely antibacterial (greater than 4 log reductions after 10 minutes), and their components (emulsifiers and preservatives) were deemed unsuitable for the formulation purpose. The main problematic components were identified as phenoxyethanol, bronidox, isothiazolinone, and sodium lauryl sulfate. Other components with antibacterial properties were also present, but of lesser importance.

[0154] The best-performing formulation (after 24 hours) was again a buffered polycetol cream, with sorbic acid as the sole preservative (±0.15 log reduction after 24 hours). The second-best-performing formulation was Nourivan pharmaceutical matrix, containing only sorbic acid as a preservative. The third-best formulation was carbomeergel containing parabens, and the fourth-best formulation was pentravan cream containing both sorbic acid and benzoic acid as preservatives. Lanette cream contains both parabens and sorbic acid as preservatives.

[0155] It is clear that organic acids (and parabens) are slow-acting preservatives, unlike those commonly found in cosmetic formulations, and therefore do not significantly harm probiotics after application to the skin. Formulations 1, 4, and 5, containing only organic acid preservatives, showed minimal reduction after 10 minutes. Although parabens may also be suitable as preservatives, they are known sensitizers and are therefore preferred not to be used in the cosmetic industry. Therefore, it concludes that combining organic acid preservatives with probiotics is a suitable option.

[0156] Example 3: Stability Analysis

[0157] Our capsules have a uniform wall (shell) surrounding the probiotic-containing core. This shell protects the bacteria, ensuring that they are not immediately released when our capsules are suspended in an aqueous environment.

[0158] As is evident in the table below, we are able to provide good stability at both refrigeration and room temperature:

[0159]

[0160] Example 4: Survival determination in creams containing organic acids as preservatives

[0161] The objective of this embodiment is to determine the efficacy of probiotic capsules suspended in oil / water creams in the presence of different concentrations of sorbic acid preservative. A secondary objective is to demonstrate that the selected preservative is suitable for protecting the cream. This was demonstrated through challenge tests on creams (without probiotic capsules) with different preservative concentrations, according to the European Pharmacopoeia (5.1.3).

[0162] Materials and methods:

[0163] The composition used

[0164] A preservative- and fragrance-free basic cream was used as the basis for further testing. The preservative tested was sorbic acid (2,4-hexadienoic acid), an organic acid with a pKa of 4.76 at 25°C at different concentrations. The pH of the creams under all test conditions was adjusted to 4.5 using HCl / NaOH.

[0165] Attack test & efficacy of organic acid preservative concentration

[0166] Attack tests were used to determine the efficacy of the antimicrobial preservation system of dermatological products. The aim was to improve the safety and durability of the products. The concentrations of sorbic acid tested ranged from 0.5% to 0.3% to 0.1% to 0% (control).

[0167] The microorganisms attacked were Escherichia coli (DSM 1576), Pseudomonas aeruginosa (DSM 1128), Candida albicans (DSM 1386), Staphylococcus aureus (DSM 799), and Aspergillus niger (DSM 1988).

[0168] The addition of the attacked microorganisms at a rate of 10 6 The test was performed at CFU / ML (in 100g of product). Samples were taken at 6 hours, 24 hours, 7–14 days, and 28 days later. For bacteria, a challenge test was considered successful if at least a 3-log reduction was observed after 7 days; for yeast / mold, a 2-log reduction was observed after 14 days, and a 3-log reduction after 4 weeks. Results are shown 4 weeks after sampling.

[0169] Survival of probiotic capsules in cream bases containing different preservatives

[0170] Probiotic capsules were suspended at 15% (m / ) in cream matrices with different preservative concentrations. To simulate in vivo release of probiotics, citrate buffer was used in vitro. Citric acid dissolves the capsule membrane, thereby releasing the probiotics within the capsule. CA buffer was prepared by dissolving 31.2 g Na₂HPO₄ and 1.25 g citric acid in 971.54 g sterile water. The final solution was an isotonic solution with a pH of 7.4 ± 0.2.

[0171] The sample cream was taken in duplicate and dissolved in CA buffer. A 1 / 10 dilution series was then prepared, with 0.1 ml spread onto MRS agar plates using the "copacabana plating method" with 5 glass beads (4 mm diameter). The results (after 3 days of incubation at 37°C) were calculated as CFU / g of cream. Storage temperatures were 4°C and 25°C. Samples were taken after 1–2–3 months. The results shown indicate the stability of the capsules after 3 months of refrigerated storage.

[0172] result

[0173] Attack test results

[0174] Results of the attack test on the base cream (excluding probiotic capsules) after 4 weeks.

[0175]

[0176] *An attack test is successful if a log reduction greater than 3 is achieved. The numbers must be interpreted as follows: 3 = less than 1 log reduction compared to the initial bacterial load; 2 = between 1 and 2 log reductions; 1 = between 2 and 3 log reductions; 0 = greater than 3 log reductions.

[0177] Survival of probiotic capsules in creams containing different concentrations of sorbic acid

[0178] Survival of probiotic capsules after 3 months in basic creams containing different concentrations of sorbic acid preservative.

[0179] blank 0 9.E+07 1.E+07 100 Sorbic acid 0.3 7.E+07 1.E+07 76 Sorbic acid 0.1 7.E+07 2.E+07 79 Sorbic acid 0.03 6.E+07 4.E+07 68

[0180] in conclusion

[0181] Organic acids (such as sorbic acid) are valuable preservatives for dermatological preparations. Sorbic acid exhibits good preservative effects at concentrations as low as 0.1%. The absence of sorbic acid leads to the failure of attack tests because it appears unable to kill all tested microorganisms within a foreseeable timeframe. Therefore, using organic acids as the sole preservative in a formulation, when present at appropriate concentrations, appears suitable for preventing spoilage by bacteria, yeast, or mold.

[0182] Suspending probiotic capsules (15% m / m) in the same base cream used for the challenge test showed that the probiotic capsules were unaffected by different preservative concentrations. The bacterial CFU load of creams containing different preservatives changed by no more than 32% (less than 0.3 log reduction) compared to the control. This is well within the margin of error and not statistically significant.

[0183] This means that the probiotic capsules provide good protection against the organic acids tested in the trial against the cream formulation, while the preservatives prevent the growth of exogenous organisms in the cream.

Claims

1. A two-compartment system for use as a formulation applied to the skin, comprising the following: - A first chamber comprising a microcapsule, the microcapsule comprising a water-insoluble and water-impermeable shell and a live microorganism contained in a non-aqueous composition of the core of the microcapsule; as well as - The second chamber comprises an aqueous composition with a pH less than 7.0 and including one or more organic acids; The organic acid is used as a preservative, and the composition does not contain any other preservatives.

2. The two-compartment system according to claim 1, wherein the aqueous composition in the second compartment has a pH of less than 5.

5.

3. The two-chamber system according to claim 1, wherein the aqueous composition in the second chamber has a pH of less than 5.

0.

4. The two-chamber system of claim 1, wherein the aqueous composition in the second chamber has a pH of less than 4.

5.

5. The two-compartment system according to any one of claims 1 to 4, wherein the one or more organic acids are selected from the list comprising: benzoic acid, sorbic acid, citric acid, acetic acid, lactic acid, anisic acid, oxalic acid, formic acid, dehydroacetic acid, fumaric acid, gluconic acid, malic acid, succinic acid, tartaric acid, and propionic acid and their derivatives.

6. The two-compartment system according to any one of claims 1 to 4, wherein the live microorganism is a live probiotic microorganism selected from Lactobacillus pentosus (… Lactobacillus pentosus Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) and Lactobacillus plantarum ( Lactobacillus plantarum A list of ).

7. The two-chamber system according to any one of claims 1 to 4, wherein the first chamber containing microorganisms is impermeable to water and oxygen.

8. The two-chamber system according to any one of claims 1 to 4, wherein the water-insoluble and waterproof shell is composed of: alginate, xanthan gum, gum arabic, gellan gum, carrageenan, gelatin, cellulose or derivatives thereof; or a polymer based on agar, protein, polyol, gelatin, polyvinyl alcohol, polylactic acid-glycolic acid copolymer, polylactic acid and derivatives thereof, PCL, polyisohexyl cyanoacrylate, acrylate derivatives or starch, optionally in combination with chitosan; or stearic acid.

9. The two-compartment system according to any one of claims 1 to 4, wherein it is in the form of a gel, cream, foam, lotion, or ointment containing the microcapsules.

10. The two-compartment system according to any one of claims 1 to 4, wherein the non-aqueous composition is selected from the list comprising: vegetable oils, mineral oils, silicone oils or hydrophilic polymers; caprylic / caprylic triglycerides, liquid paraffin, polyethylene glycol, silicone or stearic acid.

11. A method for preserving live microorganisms, comprising: - Provide a two-compartment system according to any one of claims 1 to 10; - The live microorganisms are included in a first chamber, the first chamber comprising a microcapsule, the microcapsule comprising a water-insoluble and water-impermeable shell and a live microorganism contained in a non-aqueous composition of the core of the microcapsule; - The aqueous composition of the second chamber of the two-chamber system includes one or more organic acids in an amount sufficient to achieve a pH of less than 7.0; The second chamber does not contain a buffer.

12. Use of the two-compartment system according to any one of claims 1 to 10, for preserving an aqueous composition without impairing the microorganisms in the microcapsules.

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