Method for loading microorganisms into multiphase biomaterials

Abstract

The present invention provides - a method for loading microorganisms or parts thereof onto and / or into a pre-synthesized multiphase biomaterial made of nanocellulose, the method comprising the steps of resuspending the microorganisms in a buffer or medium and then loading them onto and / or into the multiphase biomaterial; - the use of such loaded multiphase biomaterials in nutritional, food, pharmaceutical, medical, cosmetic, in particular oral, mucosal, cutaneous and transdermal, ophthalmic, dermatological or women's health applications; Regarding.

Description

[Technical Field]

[0001] The present invention - A method for loading a microorganism or a part thereof onto and / or into a pre-synthesized multiphase biomaterial containing nanocellulose, the method comprising the step of resuspending the microorganism in a buffer or culture medium and then loading it onto and / or into the multiphase biomaterial, - Use of such pre-filled multiphase biomaterials in nutrition, food, pharmaceutical, medical, and cosmetic applications, particularly in oral, mucous membrane, skin and transdermal, ocular, dermatochemical, or women's health applications, Regarding. [Background technology]

[0002] Probiotics are live microorganisms that, when administered in appropriate amounts, provide health benefits to the host (Non-Patent Literature 1). The most commonly studied and commercially available probiotics are primarily from the genera Lactobacillus and Bifidobacterium. In addition, other microorganisms such as Propionibacterium, Streptococcus, Bacillus, Enterococcus, Escherichia coli, and yeast are also used. Probiotic / synbiotic formulations (e.g., supplements / cosmetics / biomedicinal care products) are systems "designed to provide physiological benefits and / or reduce the risk of chronic disease beyond basic nutritional function."

[0003] So-called prebiotics are defined as selectively fermented components that bring about specific changes in the composition and / or activity of gut bacteria, resulting in health benefits for the host. Prebiotics often function as a capture matrix during passage through the gastrointestinal tract, further releasing microorganisms in the gut and functioning as a fermentable substrate (Non-Patent Literature 2). Most prebiotics are complex carbohydrates of plant origin. Prebiotics and probiotics can be combined to support the survival and metabolic activity of probiotics, and the resulting product belongs to the synbiotic group. Synbiotics refer to food components or nutritional supplements that combine probiotics and prebiotics in a synergistic, i.e., symbiotic way (Non-Patent Literature 3). According to the present invention, the term "synbiotics" also includes synergistic combinations of probiotics and components ("prebiotics") that produce metabolites with health benefits through selective component metabolism by added microorganisms.

[0004] In this regard, probiotic bacteria arise as active ingredients in dietary supplements, functional foods, and topical applications, which are suggested to support health and well-being. These are (mostly) living microorganisms and are said to provide beneficial health effects to the host by supplementing the natural microbiota, or by exhibiting regulatory properties (by reducing pathogens through competition for survival or by producing active metabolites in various locations (e.g., intestines, skin, oral cavity, vaginal canal)). However, probiotic bacteria at the time of application are very often inactivated by conditions (e.g., highly acidic stomach, bile acids, or local environmental factors), and therefore the effectiveness of probiotic bacteria largely depends on the number of living cells that can reach the site of action. Thus, the development of rapid delivery systems for cosmetic, biomedical, or food applications that can capture, protect, transport, and appropriately deliver active substances is important, not only for food applications but especially from the basic perspective of topical applications.

[0005] Probiotics / synbiotics are well known for their beneficial effects in promoting health in many parts of mammals / humans (e.g., gastrointestinal tract, skin, mucosa). One challenge is to provide beneficial probiotics / synbiotics to their sites of action in the active state, in the amounts necessary to exert an effect, and over the necessary time. The latter aspect is particularly important for topical application to the skin and mucosa (e.g., the mucosa inside and outside the vagina or oral cavity). In many cases, in addition to acting beneficially and specifically, auxiliary factors, nutrients or starting materials, and environmental requirements (such as humidity) are required to survive the probiotics / synbiotics during the storage period until use. Furthermore, when combined with specific components / raw materials, it may bring about a symbiotic effect with respect to the application. This poses an additional challenge for probiotic / synbiotic formulations for topical application. In the form of a cream preparation for topical application, bioactives can only be utilized temporarily and often have limited viability.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Disclosure of the Invention

Problems to be Solved by the Invention

[0007] Therefore, the challenge to be addressed is to provide a simple and rapid loading technology for probiotic microorganisms, and the resulting product, which has the following capabilities. - Protecting (often delicate) probiotics / bioactive substances, - Reaching a sufficient number of doses in the appropriate area (e.g., local, gastrointestinal, vagina) to obtain a beneficial effect. - To capture / load a bioactive substance so that it is positioned at the site of action and causes sustained release of the cell or the active substance / metabolite (long-term action), - When combined with other ingredients, it enables the in-situ generation of active ingredients by probiotics, resulting in synbiotics. - Maintain probiotics / synbiotics in a viable state throughout storage until application. - Absorption of environmental fluids (e.g., tampons, oral administration), and - To provide masking for anticipated malodors.

[0008] The delivery system for biomedical applications must address the characteristics of the captured biological formulation as well as the characteristics of the user. In the best-case scenario, the delivery system would be a low-toxicity, highly water / liquid-absorbing bacterial nanocellulose, which itself provides a supporting effect. The present invention provides, as a solution, a method leading to the formulation of bacterial / microbial (nano)cellulose that protects probiotics and / or other bioactive components for topical application.

[0009] Nanocellulose is a term referring to cellulose in a nanostructure. This includes cellulose nanofiber (CNF), also known as cellulose nanocrystals (CNC or NCC), microfibrillated cellulose (MFC), or bacterial nanocellulose (BNC), which refers to nanostructured cellulose produced by bacteria. BNC is a nanofibril polymer produced by strains such as Komagataeibacter xylinus, one of the best bacterial species that exhibits the highest efficiency in cellulose production. BNC is a biomaterial with unique properties such as chemical purity, excellent mechanical strength, high flexibility, high absorbency, and exceptional moldability and flexibility, allowing it to be molded into any shape and size. Furthermore, the material is vegetarian and vegan, and has a high moisture content.

[0010] BNC production is gaining increasing popularity due to its environmentally friendly properties. Many types of BNCs have been developed for a variety of applications, including tissue regeneration, drug delivery systems, artificial blood vessels, and scaffolds for biomedical engineering in vitro and in vivo (Zizaja et al., Biomacromolecules 2007 / 1 / 8(1):1-12; De Azeredo, Trends Food Sci Technol 2013 / 30:56-69; Almeida et al., Eur J Pharm Biopharm 2014 / 86:332-336; Oliveira Baldo et al., Carbohydr Polym 2015 / 128:41-51; Martinez Sanz et al., J Appl Polym Sci 2016 / 133). Depending on the application, BNC can provide improved mechanical quality to biomaterials due to its biocompatibility, biofunctionality, non-toxicity, and ease of sterilization (Klemme et al., Angew Chem Int Ed Engl 2011 50:5438-5466).

[0011] Various formulations exist that use microbial cellulose to deliver probiotics, with differences in the use of additional polymers, immobilization / capture methods, resulting probiotic loading, viability, efficacy / type of bacterial cells, and general advantages (e.g., handling, human intestinal tolerance), but they are not suitable for topical application. The survival period of probiotic bacteria must be within a specific limited period, not just while incorporated into the formulation. Known systems differ not only in terms of protecting probiotic bacteria but also in terms of dosage form and viability at the start of application. Actual loading techniques are mainly carried out by time-consuming adsorption during microbial cellulose production or capture of microbial cells. Long-term culture has the disadvantage that the microorganisms may grow further during culture, making it difficult to accurately determine the final loading concentration.

[0012] The viability of probiotic lactic acid bacteria immobilized on various forms of BNC in artificial gastric juice and bile saline solutions has been analyzed. Microbial immobilization was performed by adsorption of bacterial cells onto the surface of synthetic BNC and co-culture of probiotic bacteria with cellulose-synthetic gluconoacetobacter xylinus (G. xylinus) (Zivicka et al., Food Science and Technology 68, 2016, pp. 322-328).

[0013] A comparative evaluation of bacterial cellulose (Nata) as an antifreeze and carrier adjuvant in the freezing process of probiotic lactic acid bacteria is described in a study. In this study, bacterial cellulose produced by Acetobacter xylinum was compared with other established antifreezes (e.g., 10% skim milk, calcium alginate encapsulation, or 0.85% saline, distilled water) for its antifreeze properties and carrier adjuvant capacity for probiotic lactic acid bacteria. Individual lactic acid bacteria were cultured in MRS broth in the presence of nata cubes or powdered bacterial cellulose (PBC), 10% skim milk, saline, or distilled water, and then freeze-dried. As a result, colony-forming units decreased by 3.0 log cycles in all lactic acid bacteria compared to the original cell suspension (Bawa et al., Food Science and Technology 43, 2010, pp. 1197-1203).

[0014] Polish Patent Publication No. 415670 discloses a method for immobilizing microorganisms on and / or within bacterial cellulose, characterized by adding wet or dry bacterial cellulose to a suspension of Lactobacillus spp. at a McFarland standard turbidity of 1°, and culturing the suspension at room temperature (25°C) for 24 hours while shaking at 180 rpm. To immobilize Lactobacillus spp., bacterial cellulose in the form of membranes or beads obtained as a result of culturing for 6 days under steady conditions or under shaking at 180 rpm may be used. In the immobilization method described in Polish Patent Publication No. 415670, approximately 400 × 10¹⁶ units per gram of wet cellulose are used. 5 Probiotic bacteria were immobilized on cells, and the survival rate of bacteria immobilized on wet cellulose exceeded 50% in the presence of artificial stomach acid and exceeded 90% in the presence of artificial bile salts. Furthermore, approximately 30 × 10⁶ bacteria were found per gram of dry cellulose. 5By immobilizing probiotic bacteria into cells and immobilizing them on dry cellulose, the viability of the bacteria can exceed 50% in the presence of artificial stomach acid and exceed 90% in the presence of artificial bile salts. However, the method described in Polish Patent Publication No. 415670 requires the immobilized bacteria to be pre-cultured for a long period of time, and the bacterial cellulose material must be cultured in a bacterial suspension for 24 hours, which is, in short, a time-consuming method.

[0015] Chinese Patent Publication No. 109528691A1 describes the production of microcapsules containing cellulose nanofibers (CNF) and probiotics. Nanoparticles are prepared by mixing Lactobacillus plantarum with a nanofiber-containing solution. This document reports a microencapsulation technique in which the transport system (CNF) is formed during the loading process (formation and loading are performed in a single step). Thus, the prepared CNF is blended with probiotics in a liquid and dropped into a CaCl2 solution, which is a crosslinking agent, to form a probiotic-cellulose nanofiber core. Next, a layer-by-layer method is applied to coat the resulting core with alginate and chitosan.

[0016] The disadvantages of known techniques include the lengthy loading procedures (primarily due to the length of adsorption or co-culture time), which can also lead to undesirable and uncontrolled replication of probiotics. These lengthy culture times further lead to the uncontrolled reduction of other components, resulting in a heterogeneous distribution of probiotics within the BNC network. Techniques known from the prior art are not fast or flexible depending on the type of immobilized microorganism.

[0017] The advantages of the present invention in light of prior art are that the proposed method is a fast, simple, flexible / adaptive, and cost-effective method for loading bacterial cellulose material. The loading technique is very fast and controllable. It provides a sustainable resource-saving method using a natural, biocompatible, semi-inert carrier. The resulting fleece structure has not only high resistance but even a three-dimensional structure, improving the short lifespan of probiotics by resulting in sustained release and / or in-situ generation of active ingredients. According to the present invention, very flat, uniform, or transparent structures, or even specially molded shapes, are possible.

[0018] Furthermore, since the probiotics / synbiotics remain viable throughout storage until application to the skin (especially while remaining on the skin), the present invention is very suitable for topical or intestinal application (oral). Products according to the present invention provide high liquid absorption capacity to absorb (environmental) fluids (for example, tampons, pantry slips, or oral application). Semi-dry systems are also suitable for the present invention. Moreover, any expected odors can be masked by products according to the present invention. [Means for solving the problem]

[0019] The present invention relates to a method for loading microorganisms or a portion thereof onto and / or into a pre-synthesized multiphase biomaterial made of nanocellulose. - Synthesize a bacterial-synthesized nanocellulose (BNC) multiphase biomaterial, - Resuspend the microorganism in a buffer or culture medium, - a) A method of mixing the multiphase biomaterial and the microorganism at a temperature of 37°C or lower, preferably 10 to 37°C, for 1 to 60 minutes, preferably 5 to 10 minutes, at a speed of 300 rpm or higher, preferably 500 rpm to 3,500 rpm. b) A method of injecting the microorganism into the multiphase biomaterial and culturing it at a temperature of 37°C or lower, preferably 4 to 37°C, for a maximum of 72 hours, preferably a maximum of 1 hour, or c) A method of culturing the multiphase biomaterial in a buffer or culture medium containing the resuspended microorganisms at a temperature of 37°C or lower for 60 minutes or less, preferably within 10 minutes. The process of loading the microorganism onto and / or into the BNC material by any of the following methods. Regarding methods including

[0020] The incubation time in step a) is 1 to 60 minutes, preferably 5 to 10 minutes. The method in step a) is also called the "high-speed method" in this invention and is performed using a vortex. In a preferred configuration, the BNC nonwoven fabric is vortexed with the bacterial suspension at a vortex strength of 10.5 (approximately 3300 rpm) for 10 minutes at room temperature (Vortex Genie 2). The loaded suspension is then removed and the BNC nonwoven fabric is washed under vortex for 10 seconds.

[0021] In step b), the culture time is up to 72 hours, preferably up to 1 hour. Generally, the injection method in step b) does not require a long culture time to load the microorganisms. Therefore, in a preferred embodiment, the culture time is 1 second to 1 hour, preferably 1 second to 10 minutes, more preferably 1 second to 60 seconds.

[0022] A method for synthesizing a bacterially synthesized nanocellulose (BNC) multiphase biomaterial is disclosed in U.S. Patent Publication No. 2015 / 0225486.

[0023] For example, it is preferable to use a nonwoven BNC material such as that described in International Publication No. 2018 / 215598A1. According to the present invention, the nonwoven BNC material is a nonwoven fabric of BNC fibers in particular. The terms "nonwoven BNW" and "BNC fleece" may be used interchangeably in accordance with the present invention.

[0024] In a preferred embodiment, microorganisms are sprayed onto a multiphase biomaterial. In a preferred embodiment, a bacterial suspension or bacterial powder is sprayed in a preferred configuration. It is preferable to spray the bacterial suspension onto the BNC nonwoven fabric within 1 minute.

[0025] In an advantageous configuration of a method for loading microorganisms or a portion thereof onto and / or into a pre-synthesized multiphase biomaterial made of nanocellulose, loading into and / or onto the BNC material is carried out by one of the following: a) A method of mixing the multiphase biomaterial and the microorganisms at a temperature of 37°C or lower, preferably 10 to 37°C, for 1 to 60 minutes, preferably 5 to 10 minutes, at a speed of 300 rpm or higher, preferably 500 rpm to 3,500 rpm. b) A method of injecting the microorganism into the multiphase biomaterial and culturing it at a temperature of 37°C or lower, preferably 4 to 37°C, for a maximum of 72 hours, preferably a maximum of 1 hour.

[0026] Using options a) and b), microorganisms are better dispersed among the multiphase biomaterials and adhere more completely to the multiphase biomaterials.

[0027] This invention relates to a method for loading bacteria using bacterial cellulose, a carrier component for temporarily immobilizing bacteria, where the temporarily immobilized bacteria provide beneficial effects in topical application (e.g., skin or mucous membranes). It is a method for obtaining formulations in which probiotics / symbiotics are incorporated / captured / temporarily immobilized / loaded into bacterial cellulose for topical application in cosmetics, biomedical care, or personal care, providing a method for obtaining transdermal effects, anti-inflammatory effects, sedative effects, anti-wrinkle / anti-aging effects, pathogen inhibition or modulation effects, acidification effects, anti-redness effects, or other appearance-enhancing effects. Examples include, among others, probiotic / symbiotic masks, patches, sanitary napkins, tampons, etc.

[0028] The carrier serves as a habitat for probiotics / synbiotics. The immobilized biological agents are used to induce the biosynthesis and release of metabolites and enzymes, or the release of bacterial cells themselves, to have beneficial effects on each local environment (e.g., skin, oral cavity, vagina).

[0029] Bacterial cellulose is a three-dimensional network and a carrier for immobilizing and capturing microorganisms and other substances. Immobilized biological preparations (including microorganisms) are used for the biosynthesis of bioactive metabolites (e.g., antimicrobial agents, metabolic bioactive substances) in situ / in vivo, for inducing the release of microorganisms and bioactive substances, and / or as immobilized microfactories during fermentation processes.

[0030] The application areas may include not only cosmetics (improving appearance such as redness from rosacea or acne), but also medical applications (disruption of the vaginal microbiome), feminine hygiene products, or other consumer products.

[0031] In preferred embodiments, the microorganisms are loaded as vegetative cells or in a dormant state, preferably as bacterial spores or cell extracts. In the advantageous configuration of the present invention, the microorganisms are dried, preferably by spray drying or freeze-drying, and used in powder form.

[0032] Many bacteria can withstand adverse conditions such as temperature, drought, and antibiotics through endospores, exospores (microbial cysts), conidia, or by reducing metabolic activity and lacking specialized cellular structures. Up to 80% of bacteria in wild samples appear metabolically inactive, but many can be revived. The high diversity levels of most natural ecosystems are due to such dormancy. Endospores are robust, non-reproductive structures produced by certain bacteria in the phylum Firmicutes during a dormant state. Endospore formation is usually triggered by a lack of nutrients and typically occurs in Gram-positive bacteria. In endospore formation, bacteria divide within their cell wall, with one side incorporating the other. Endospores can keep bacteria dormant for centuries. When the environment becomes more favorable, endospores can reactivate themselves into a vegetative state. Most types of bacteria cannot transform into endospores. Examples of bacteria capable of forming endospores include the genera Bacillus and Clostridium. Endospores consist of bacterial DNA, ribosomes, a large amount of dipicolinic acid, and spore-specific chemicals that help maintain the dormant state of the endospore, accounting for up to 10% of the spore's dry weight.

[0033] In another configuration of the present invention, the microorganisms are wet or dry, and / or pre-cultured or not pre-cultured. The multiphase biomaterial is wet, dry, partially dry, or reswells in buffer.

[0034] It is preferable that the nanocellulose is derived from plants, algae, or microorganisms, preferably Komagataeibacter, more preferably Komagataeibacter xylinus. Komagataeibacter xylinus is one of the bacteria best known for its ability to produce cellulose. It is also known by several other names, mainly Acetobacter xylinum and Gluconacetobacter xylinus. In 2012, a new genus, Komagataeibacter, was established and given its current name.

[0035] In this invention, it is preferable to use a BNC nonwoven fabric with an average thickness of at least 0.5 mm for loading microorganisms. It is particularly preferable that the average thickness of the BNC nonwoven fabric be 1 mm to 5 mm, more preferably 2 mm to 3 mm. It has been shown that when the average thickness of the BNC nonwoven fabric is 2 mm to 3 mm, better re-swellability of the loaded BNC nonwoven fabric can be achieved.

[0036] In certain embodiments of the present invention, the nanocellulose is bacterial-synthesized nanocellulose (BNC) having a layered structure, and is preferably selected from the following: - BNC containing a network of cellulose fibers or nanowhiskers, - BNCs containing two or more different layers of cellulose fibril, where each layer is composed of BNCs derived from different microorganisms or microorganisms cultured under different conditions. - A BNC containing at least two different cellulose networks, or - BNC composite material further containing polymers.

[0037] Cellulose nanowhiskers (NW), also known as cellulose nanocrystals or nanocrystalline cellulose, offer an important nanoscale material with great potential for various applications (Lambie et al., Acta Chem Scand 3, pp. 649-650, 1949). NW is a result of the incomplete decomposition of cellulose (Pletzinger et al., Cellulose 25, pp. 1939-1960, 2018).

[0038] In advantageous embodiments of the present invention, at least two different bacterial cellulose networks are preferably prepared in combination with further polymers, either as a composite homogeneous phase system or as a layered phase system consisting of at least one composite homogeneous phase and at least one single phase.

[0039] A preferred method is described in European Patent Publication No. 2547372. Particularly preferred is the preparation of at least two different cellulose-producing bacterial strains together or separately and their synthesis in a common medium to obtain several different bacterial cellulose networks, the BNC structure and properties of the multiphase biomaterial being influenced by the selection of at least two different bacterial strains, their preparation and inoculation, and the influence on the synthesis conditions, and the bacterial cellulose network being synthesized as a composite homogeneous phase system or as a layered phase system consisting of at least one composite homogeneous phase and at least one single phase. Furthermore, it is preferred that at least two different bacterial cellulose networks are prepared independently of each other, then combined and synthesized together. In the advantageous configuration of co-synthesis, at least two different bacterial cellulose networks are already combined before inoculation.

[0040] In a preferred configuration of the present invention, additional substances are added during the bacterial synthesis of BNC to control the resulting pore size / mesh size, which are preferably selected from polyethylene glycol (PEG), β-cyclodextrin, carboxymethylcellulose (CMC), methylcellulose (MC), and cationic starch, preferably 2-hydroxy-3-trimethylammoniumpropyl starch chloride and TMAP starch.

[0041] This modification allows for the specific tuning of BNC to suit the microorganism to be loaded. A notable advantage of bacterial cellulose is that the property-controlled fiber network and pore system formed by the self-assembly of cellulose molecules can be modified in situ during biosynthesis using additives. This allows the pore size to be matched to the size of the microorganism to be loaded. Adding polyethylene glycol (PEG) 4000 reduces the pore size. The presence of β-cyclodextrin or PEG400 significantly increases the pore size. Surprisingly, these co-substrates function as removable additives that are not incorporated into the BC sample. In contrast, carboxymethylcellulose and methylcellulose as additives result in structurally altered composite materials. By using cationic starch (2-hydroxy-3-trimethylammoniumpropyl starch chloride, TMAP starch) and incorporating the starch derivative into the BC prepolymer, a double-network BC composite was obtained (Hessler & Klem, Cellulose 16(5):899-910, 2009).

[0042] In an advantageous configuration of the present invention, in a subsequent step, the loaded multiphase biomaterial is cultured with a drying water binder, the water binder being an osmotically and / or hygroscopically effective solution, preferably consisting of a single sugar, salt, sugar-containing or sugar-like substance, polyethylene oxide, a combination of various representatives from this group of water-binding substances, and / or a combination of one and / or more representatives from this group of water-binding substances with one or more surfactants and / or one or more preservatives.

[0043] The water binder is added for drying purposes and to maintain swelling by almost completely reconstructing the cellulose structure. The adsorption effect of the water binder affects the viscosity (consistency), and thereafter, the material exposed to the adsorbent dries regardless of structural changes in the material. The drying method is described in International Publication No. 2013 / 060321A2.

[0044] As a water-binding agent, an osmotically and / or hygroscopically effective solution is used, which preferably consists of a single sugar, salt, sugar-containing or sugar-like substance, polyethylene oxide, a combination of various representatives from this group of water-binding substances, and / or a combination of one and / or more representatives from this group of water-binding substances with one or more surfactants and / or one or more preservatives. Preferred water-binding agents to be used are glucose, magnesium chloride, and sugars. In a preferred configuration, a surfactant and / or preservative-containing solution is used in addition to the water-binding agent to further modify the re-swelling behavior.

[0045] The water-bound solution may contain an osmotically active substance and / or a hygroscopic substance at a concentration of 0.01% to the saturation limit, preferably 5% to 20%. It is preferable to use a surfactant and / or preservative in combination with the osmotically and / or hygroscopically effective solution at a concentration of 0.01% to the saturation limit, preferably 0.01% to 10%.

[0046] Cellulose or cellulose-containing materials being treated with a moisture binder may be air-dried or vacuum-dried.

[0047] In a preferred configuration, the cellulose or cellulose-containing material to be subjected to the adsorption effect of the water-binding solution is immersed in the water-binding solution. In an alternative configuration, the water-binding solution is sprayed, dropped, brushed, or cast onto the cellulose or cellulose-containing material to be subjected to the adsorption effect of the water-binding solution. Alternatively, the water-binding agent may already be added to the cellulose in addition to the cellulose culture step for the purpose of exposure to the adsorbent.

[0048] In preferred embodiments, the microorganisms include the genera Bifidobacterium, Carnobacterium, Corynebacterium, Cutibacterium, Lactobacillus, Lactococcus, Leuconostoc, Microbacterium, Oenococcus, Pasturia, Pediococcus, Propionibacterium, Streptococcus, Bacillus, and Geobacillus. bacillus), Gluconobacter, Xanthonomas, Candida, Debaryomyces, Hanseniaspora, Kluyveromyces, Komagataella, Lindnera, Ogataea, Saccharomyces, Schizosaccharomyces, Wickerhamomyces, Xanthophyllomyces, and Yarrowia, preferably Cutibacterium acnes acnes), Lactococcus lactis, Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus gasseri, Bacillus subtilis, Bacillus megaterium, Micrococcus luteusMicrococcus luteus, Micrococcus lylae, Micrococcus antarcticus, Micrococcus endophyticus, Micrococcus flavus, Micrococcus terreus, Micrococcus yunnanensis, Arthrobacter agilis, Nesterenkonia halobia, Kocuria kristinae, Kocuria rosea, Kocuria varians, Kytococcus cedentarius It is a probiotic bacterial or yeast strain selected from *Dermacoccus sedentarius*, *Dermacoccus nishinomiyaensis*, or a mixture thereof.

[0049] Staphylococcus epidermidis, Lactobacillus fermentum, Lactobacillus rhamnosus strain DSM32609, Lactobacillus plantarum strain DSM32758, Lactobacillus delbrueckii susp. bulgaricus strain DSM32749, Lactobacillus plantarum LN5 strain DSM33370, Lactobacillus brevis LN32 strain DSM33377 LN32), DSM33368 L. plantarum S3, DSM33369 L. plantarum S11, DSM33376 L. paracasei S20, DSM33375 L. paracasei S23, DSM33374 L. reuteri F12, DSM33367 L. plantarum F8 F8), DSM33366 L. plantarum S4, DSM33364 L. plantarum S28, DSM33363 L. plantarum S27, DSM33373 L. paracasei S18a, DSM33365 L. plantarum S18b, DSM33362 L. plantarum S13It is even more preferable to use Lactococcus plantarum S13), Lactococcus lactis sups. lactis (DSM 32767 strain), Lactobacillus fermentum (DSM 32750 strain), Propionibacterium acnes, and Cutibacterium acnes.

[0050] In a more preferred embodiment of the present invention, the additional step is performed before, after, or in parallel with loading the microorganisms into a multiphase biomaterial, which is loaded with further components and / or nutrients selected from amino acids, fatty acid salts, anthocyanins, monosaccharides, and extracts, preferably lysine salts of DHA and EPA, rhamnose, and tryptophan. These additional components can provide metabolites with health benefits resulting from microbial metabolism or can be selectively fermented by the microorganisms and can be classified as prebiotics. Such a composition comprising a probiotic microorganism as defined above and one or more components / prebiotics can be named a synbiotic.

[0051] A further aspect of the present invention relates to a nonwoven multiphase biomaterial containing nanocellulose, which includes at least two different bacterial cellulose networks containing at least one living microorganism, obtained by a method according to the present invention.

[0052] In a favorable configuration, the multiphase biomaterial contains at least one living microorganism at a rate of at least 3.00 × 10¹⁶ per gram of cellulose. 7 It contains at the cell concentration.

[0053] The present invention also relates to the use of nonwoven multiphase biomaterials according to the present invention in applications relating to food, oral cavity, mucous membranes, skin and transdermal, ophthalmology, nutrition, cosmetics, dermatochemistry, oral health, or women's health.

[0054] Such cellulose products are relevant to applications in medicine (Zimmer Biomet implant materials, wound dressings, skin substitutes), pharmaceuticals (e.g., drug delivery systems), and technology (e.g., filter systems and membrane systems).

[0055] Therefore, cellulose and cellulose-containing materials can be provided with minimal time and technical and economic costs, without stressing the cellulose and without impairing the stability and effectiveness of the loaded material (e.g., drugs, probiotics, additives, etc.). [Brief explanation of the drawing]

[0056] [Figure 1] Figure 1 is a schematic diagram of the loading capacity measurement using the high-speed method (vortex) and the core-shell method (injection). [Figure 2] Figure 2 is a SEM micrograph of a BNC fleece (top left) loaded with L. lactis, prepared using the vortex method. [Figure 3] Figure 3 shows the release profiles of BNC fleece loaded with L. lactis (left) and BNC fleece loaded with Bacillus subtilis (right) in MRS broth medium. [Figure 4] Figure 4 shows the quantitative analysis of Bacillus subtilis in freeze-dried BNC fleece cultures after loading with Bacillus subtilis using the vortex method (top) and the injection method (bottom). [Figure 5] Figure 5 shows the quantitative analysis of lyophilized BNC fleece cultures after loading with B. megatherium using the vortex method (top) and the injection method (bottom). [Figure 6] Figure 6 shows the quantitative analysis of lyophilized BNC fleece cultures after loading L. lactis using vortex (top) and injection (bottom) methods. [Figure 7] Figure 7 shows the quantitative analysis of L. lactis-loaded BNC fleece cultures prepared by the vortex method using suspensions of L. lactis powder suspended in MRS broth medium (left) and physiological saline isotonic solution (right). [Figure 8] Figure 8 shows the quantitative analysis of probiotics loaded into modified BNC fleece compared to standard fleece after enzymatic digestion using cellulose. [Examples]

[0057] Experimental Example 1: Incorporation of probiotics without the use of additional polymers (after pre-culture) A) Characterization and sterilization of pre-loading BNCs in terms of size (surface area, volume, thickness, weight) All BNC fleeces were stored at 4°C (or room temperature if packaged) and equilibrated at room temperature for 30 minutes. The diameter and height were measured at three different points within the fleece using a vernier caliper scale. The mean and standard deviation of the diameter, height, and volume (V) of the BNC fleeces were calculated using Equation 1 below. K=πr 2 h (1) (In the formula, π = 3.14, r: radius, h: height.) Furthermore, the surface area (A) of each BNC fleece was obtained by applying Equation 2. A = 2πrh + 2πr 2 (2) (In the formula, π = 3.14, r: radius, h: height.) The data was expressed as the mean ± standard deviation of all measured values. The size characteristics of BNC fleece were evaluated against standard BNC fleece synthesized according to the standard methods of the local laboratory. The BNC fleece measured weight: 1.16 ± 0.06 g, diameter: 1.6 ± 0.07 cm, and height: 0.5 ± 0.04 cm. The surface area of ​​each BNC fleece was 7.24 ± 0.27 cm². 2 The volume is 1.2 ± 0.1 cm³. 3 That was the case. Thin BNC fleece for use as a mask or patch, or for use in a rolled form, is characterized by a thickness of 1-4 mm, preferably 2-3 mm, to ensure optimal re-swelling properties.

[0058] B) Loading of the probiotic suspension onto the BNC fleece Preparation of probiotic cultures and suspensions (L. lactis, Bacillus subtilis) Under sterile conditions, two sterilized 100 mL volumetric glass Erlenmeyer flasks were filled with 20 mL of sterilized MRS broth with a pH of 6.2 ± 0.2 for L. lactis and 20 mL of sterilized TSB medium with a pH of 7 - 7.2 for Bacillus subtilis. Approximately 2 mg of L. lactis powder was added to the MRS medium and mixed. One flask was prepared with the probiotic strain and the other with the MRS blank. Subsequently, 5 μL of the Bacillus subtilis frozen suspension was added to the TSB medium and mixed. One flask was prepared with the probiotic strain and the other with the TSB blank. The probiotic cultures were prepared under sterile conditions and cultured at 37 °C with shaking at 100 rpm for 8 hours. The control MRS medium was cultured under the same conditions. After 8 hours, the cultures were transferred from the incubator to a laminar air flow bench, mixed, and each culture (500 μL) was collected into a sterilized 2 mL volume Eppendorf cup using a sterilized 1 mL pipette. The optical density (OD 600 ) of the collected samples was measured three times each at a wavelength of 600 nm using a UV cuvette and an optical density spectrophotometer (bio - photometer) in comparison with the blank MRS or TBS medium. 0.5 OD 600 = 10 8 The volume required to prepare a 10 mL probiotic suspension at a concentration of cells / mL (loading ratio = 1 g BNC: 10 mL loading solution) was calculated, and the finally calculated volume was placed into a 50 mL volumetric sterilized tube. The total volume was made up to a maximum of 10 mL using the corresponding medium (MRS for L. lactis and TSB for Bacillus subtilis) or 0.9% NaCl physiological saline, and then mixed.

[0059] I. Loading of the probiotic suspension onto the BNC fleece by the high - speed method (vortex) BNC fleece was added to a probiotic suspension (L. lactis, Bacillus subtilis) in a 50 mL tubing. Control BNC fleece was added to sterile culture medium or physiological saline. The tubing was vortexed at room temperature for 10 minutes at a vortex strength of 10.5 (approximately 3300 rpm) (Vortex Genie 2). The loaded suspension was removed, and the BNC fleece was washed with 10 mL of physiological saline under vortex for 10 seconds.

[0060] II. Loading probiotic suspension into BNC fleece by injection method BNC fleece was prepared as described above. The probiotic suspension was divided into 10 8 The solution was prepared at a concentration of 125 μL per cell. The injection needle was inserted into the center of the BNC fleece, and the volume was injected (5 units).

[0061] III. Loading pre-cultured and uncultured probiotic suspensions into BNC fleece by spraying. The BNC fleece was prepared as described above. 10 mL probiotic suspensions (pre-cultured) in physiological saline were cultured at 0.5 OD from L. lactis and B. megatherium, respectively. 600 =10 8 The solution was prepared at a concentration of cells / mL. Using a sterile glass reagent spray (Sterile Glass Reagent Spray ArtNr: 11526914, Fischer Scientific, Germany), a 5 mL probiotic suspension was uniformly sprayed onto a BNC (mask patch or other form). The procedure for loading powdered probiotics is as follows: Under sterile conditions in a lamina airflow bench (Heraeus HS 18 / 2), 100 mg of probiotic powder (e.g., L. lactis) was weighed into a sterile 2 mL Eppendorf balance using a balance (Sartorius H95 Basic). The L. lactis powder was sprayed directly onto the BNC fleece using compressed air. Alternatively, the L. lactis powder was added to an MRS (35 mL) in a 50 mL centrifuge tube and mixed under sterile conditions in a lamina airflow bench.600 A powdered probiotic suspension (e.g., L. lactis) was prepared by suspending L. lactis in MRS broth medium and physiological saline at a concentration of 1 McFarland unit. Next, the L. lactis powder suspension was sprayed onto BNC fleece using a sterile glass reagent spray (Sterile glass reagent spray ArtNr:11526914, Fischer Scientific, Germany).

[0062] Figure 1 shows an overview of various loading techniques: schematic diagrams of loading volume determination using the high-speed method (vortex) and the core-shell method (injection). Probiotic culture (P) is centrifuged and 0.5 OD 600 Then resuspend in 0.9% physiological saline (NaCl) (Step 1), and administer by high-speed method (HS) (3300 rpm, 10 min, 22°C) or direct injection (I) (125 μL, 0.5 OD) 600 The BNCs were loaded using either of the following methods (Step 2). The loaded BNCs and control probiotics were re-cultured at 37°C and 100 rpm for 18 hours (Step 3), followed by OD 600 The following was measured (step 4).

[0063] Experimental Example 2: Loading volume of BNC fleece loaded with (pre-cultured) probiotic suspension (L. lactis, Bacillus subtilis) using vortex and injection loading methods. A) Evaluation of loading characteristics: Loading capacity, position, and uniformity of distribution The probiotic culture was incubated at 37°C for 8 hours with shaking at 100 rpm. Then, it was centrifuged at 4000 rpm for 10 minutes and resuspended in 0.9% saline (NaCl). 600 0.5 (=10 8 The solution was prepared to the concentration of cells / mL of physiological saline. Probiotic cultures were loaded into BNCs by vortex or injection as described in BI and BII of Experimental Example 1. Schematic diagrams of the loading volume determination using the rapid method (vortex) and core-shell method (injection) are shown in Figure 1. In the BNC fleece, OD 600 that is 0.5 MacFarrand units (approximately 10 8Probiotics (equivalent to cells / mL) were loaded and then re-cultured in growth medium at 37°C and 100 rpm for 18 hours. OD of standard probiotic cultures. 600 In comparison, the OD of BNC after re-culture 600 The loading volume was measured by measuring the OD. For standard probiotic cultures, 600 that is 0.5 MacFarrand units (approximately 10 8 The solution was prepared by adding an equal amount of probiotics (equivalent to cells / mL) to the growth medium. In microbiology, the McFarland standard is used as a reference value to adjust the turbidity of bacterial suspensions in order to standardize microbiological tests by keeping the bacterial count within a predetermined range.

[0064] The amount of probiotics loaded is a crucial factor in measuring the performance of the developed form and defining the activity of the probiotics to a similar degree. The number of loaded probiotics was investigated to evaluate the loading capacity of the procedure used, and the number of probiotics released from the loaded BNC fleece was measured. To inhibit the growth of probiotics during the experiment, the loading process was performed in an isotonic solution. The BNC fleece loaded with probiotics were re-cultured in a suitable medium and compared with free probiotics cultured under the same conditions and concentrations.

[0065] The loading capacity of Bacillus subtilis was measured using the rapid method (vortex) and the core-shell method (injection). The BNC fleece was used with OD 600 that is 0.5 MacFarrand units (approximately 10 8 After loading probiotics (equivalent to cells / mL), the cultures were re-cultured in TSB growth medium at 37°C and 100 rpm for 8 hours. OD of standard Bacillus subtilis cultures. 600 In comparison, the OD of BNC after re-culture 600 The loading capacity was measured by measuring the OD. For standard Bacillus subtilis cultures, 600 that is 0.5 MacFarrand units (approximately 10 8The culture medium was prepared by adding an equal volume (equivalent to cells / mL) to the growth medium. The turbidity of the vials containing BNC fleece loaded with probiotics was evident, indicating the release and growth of probiotics from the BNC fleece into the medium. For both probiotics, the loading volume was higher using the injection method compared to the rapid method. For L. lactis, the loading volume using the injection method was 36.2% ± 4.7%, compared to 10.1% ± 2.2% using the vortex method. For Bacillus subtilis, the loading volume using the vortex method was 22.14% ± 3.1%, compared to 42.85% ± 5.4% using the injection method.

[0066] B) Detection of loading position by microbial autofluorescence Preparation of staining solution for determining viability The Live / Dead BacLight Bacterial viability kit L7012 was prepared according to the manufacturer's instructions. For L. lactis and Bacillus subtilis, 0.5 OD 600 The amount of probiotic culture needed to prepare a 50 mL suspension was calculated, and the final volume was centrifuged at 4000 rpm at room temperature for 15 minutes. The pellet was resuspended in 1 mL of purified water, 1 mL of the last prepared viability test stain was added, mixed, and incubated in the dark at room temperature for 15 minutes. After 15 minutes, the stained probiotics were centrifuged at 4000 rpm at room temperature for 10 minutes. The stain was removed, and the stained probiotics were resuspended in 30 mL of sterile saline, vortexed for 10 seconds, and washed. The resuspended probiotics were centrifuged at 4000 rpm at room temperature for 10 minutes and resuspended in 50 mL of sterile saline.

[0067] Visualization of probiotic distribution in BNC fleece BNC fleece was transferred to a 50 mL volume tube, 5 mL of 1% methylene blue staining solution was added, and the mixture was maintained at room temperature for 10 minutes. The methylene blue solution was removed, and the BNC fleece was washed three times under vortexing with 30 mL of physiological saline each time. Subsequently, by vortexing, viability-determining stained probiotics were loaded into methylene blue-stained BNC fleece in physiological saline. As a control, methylene blue-stained BNC fleece was immersed in 10 mL of physiological saline and mixed under the same conditions. The loaded suspension was removed, and the BNC fleece was washed under vortexing with 10 mL of physiological saline. Light was shone from above onto the fleece, and cross-sections and photographs were taken using a molecular light.

[0068] The distribution of probiotics in BNC fleece was detected by applying a fluorescence staining method. BNCs were stained with methylene blue to prevent autofluorescence. Next, the viability-determining stained probiotics were incorporated into BNC fleece by vortex and injection methods and detected using a fluorescence detection camera. Top and cross-sectional photographs show that L. lactis is uniformly distributed throughout the cross-section and has only a slight tendency to incorporate into the prepolymer, which allows for the incorporation of more material due to its large pore size and loose structure. Bacillus subtilis shows a strong tendency to incorporate into the prepolymer, which may be related to its large embryo size.

[0069] Evaluation of loading uniformity and distribution using a scanning electron microscope (SEM) After fixing and drying the loaded BNC fleece using critical point drying, it was sputter coated and observed with a scanning electron microscope (SEM). The subsequent procedure was as follows: The BNC fleece was fixed at room temperature for 10 hours in 3 mL / well of fixative solution consisting of 2.5% glutaraldehyde and 4% formaldehyde in sodium cacodylate buffer M (pH 7.4). After removing the fixative solution, the BNC fleece was washed three times with physiological saline, and then dehydration was completed in an ethanol-based system for 15 minutes each time, increasing in concentration (30%, 50%, 70%, 80%, 90%, 100%, and 100%). The BNC fleece was dried by critical point drying using an automatic critical point dryer Leica EM CPD300 (Leica). Next, the BNC pieces were mounted in an SEM sample holder and sputter-coated with gold (layer thickness 30 nm) in a sputter coater (Sputter Coater BAL-TEC SCD005) under vacuum using an inert gas (argon). Subsequently, they were analyzed and microscopically imaged using a Sigma-VP scanning electron microscope (Carl Zeiss, Germany) operating at 5 kV with an in-lens detector.

[0070] The distribution of probiotics in BNC fleece loaded using the vortex method was measured by scanning electron microscopy (SEM) in various sections, compared to unloaded, natural BNC fleece. Both the unloaded and probiotic-loaded BNC fleece were immobilized in a mixture of glutaraldehyde and formaldehyde to stabilize the final form and maintain the position of the loaded probiotics before drying and SEM imaging were completed. Microscopic analysis of the BNC fleece, as shown in Figure 2, revealed that the loaded probiotics were widely distributed across the surface of the BNC fleece. Furthermore, the loaded probiotics were uniformly localized in both the transverse and perpendicular sections, confirming the uniformity of loading within the BNC fleece to which the vortex method was applied.

[0071] Figure 2 shows SEM micrographs of BNC fleece loaded with L. lactis (top, left), prepared by the vortex method. The L. lactis-loaded BNC fleece was examined at various locations: surface (top, right), cross-section (bottom, right), and vertical section (bottom, left). Micrographs were taken at 5kV using an in-lens detector.

[0072] Experimental Example 3: Loading of pure L. lactis powder (not pre-cultured) into BNC fleece using the vortex method. L. lactis powder is added to 35 mL of MRS or physiological saline in a 50 mL centrifuge tube and mixed, and under sterile conditions in a lamina airflow bench, OD 600 L. lactis suspensions were prepared by suspending L. lactis in MRS broth medium and physiological saline at a concentration of 1 McFarland unit. Each suspension was divided into three 50 mL centrifuge tubes, 10 ml each, without pre-culturing. Sterile BNC fleece was then added to the tubes and loaded by vortexing as described above. The loaded BNC fleece was washed with physiological saline and transferred to MRS (10 mL) in a 30 mL clear glass bottle. As a control, OD 600 L. lactis cultures were prepared by adding a L. lactis suspension (5 μL) containing 1 MacFarland unit to MRS (10 mL) in a 30 mL clear glass bottle. The bottle was photographed (Canon PowerShot SX620HS) and incubated in an Inforrs HT Multitron Standard at 37°C and 100 rpm for 24 hours. After 24 hours, the bottle was transferred to a laminar bench, photographed (Canon PowerShot SX620HS), and the optical density (OD) was measured as described above. 600 The following measurements were taken: 25 μL taken from the vial was spread onto the surface of an MRS agar plate using a sterile glass spreader and incubated at 37°C for 48 hours (Heraeus 6000). Photographs of the colonies grown on the agar plate were taken (Canon PowerShot SX620HS).

[0073] In previous experiments, the probiotics applied were always cultured in broth medium until the late logarithmic growth phase before being loaded into BNCs for use in subsequent experiments. This experiment was designed to investigate the viability and survival rate of probiotics loaded directly into BNC fleece from powder without prior culture in broth medium. OD 600 L. lactis suspensions were prepared by adding L. lactis powder to MRS broth medium and physiological saline (0.9% NaCl), an isotonic solution, so that each suspension amounted to 1 MacFarland unit. These suspensions were then loaded into BNC fleece using the vortex method.

[0074] Visual controls of BNC fleece loaded with cultured L. lactis showed clear turbidity in the culture bottles, indicating cell proliferation. L. lactis loaded from both MRS and saline suspensions showed measured OD 600 As confirmed by the above, after 24 hours of incubation, considerable viability and survival rate were maintained, and proliferation was observed. L. lactis loaded from MRS and saline suspensions, after incubation under standard conditions, showed OD values ​​of 1.71 ± 0.15 MacFarland units and 1.6 ± 0.13 MacFarland units, respectively. 600 These results were consistent with observations of MRS agar plates showing typical growth of L. lactis colonies. Similar results were obtained when L. lactis powder was sprayed directly onto BNC fleece using compressed air.

[0075] Experimental Example 4: Loading Bacillus subtilis spore powder into BNC fleece using three different methods (vortexing, injection, and spraying). Under sterile conditions in a lamina airflow bench, using an optical density spectrophotometer (biophotometer), OD was measured in sterile 0.9% NaCl. 600 A 50 mL Bacillus subtilis spore suspension with a concentration of 0.5 MacFarland units was prepared. The Bacillus subtilis spore suspension was loaded into a BNC fleece using the vortex method as described above. 600A Bacillus subtilis spore suspension was loaded into another BNC fleece by injection at the specified concentration. Bacillus subtilis spores were then loaded into another BNC fleece by spraying as described above. Since uniform distribution of bacterial cells was confirmed by SEM microscopy, all three different loading techniques were applicable to spore-forming Bacillus. Resaturations of bacterial spores loaded using the three different techniques showed viability on both culture media and agar plates.

[0076] Experimental Example 5: Loading of Lactobacillus species and mixtures thereof The following strains were used: Lactobacillus fermentum (ID 51611), Lactobacillus rhamnosus (DSM 32609), and Lactobacillus plantarum (DSM 32758). Under aerobic standard conditions of 37°C and 100 rpm, the bacterial strains were cultured in MRS broth medium, then suspended in Tris-magnesium buffer pH 7.4 + 50% glycerin, packed into cryopreservation tubes, and stored at -80°C until use. Next, the aerobically cultured strains, and some of their mixtures, were identified on MRS agar plates, fixed as described above, dried in a critical point dryer, and then microscopically characterized by SEM. Under sterile conditions in a Lamina Airflow Bench (Heraeus HS 18 / 2), four sterile 100 mL Erlenmeyer glass flasks were filled with 20 mL of sterile MRS broth medium at pH 6.2 ± 0.2. 5 μL of each Lactobacillus strain was added to one flask, and one flask was retained as an MRS blank. These flasks were then incubated in an orbital shaker incubator (Infors HT Multitron Standard) at 37°C and 100 rpm for 8 hours. The flasks were then transferred to the Lamina Bench (Heraeus HS 18 / 2), and the concentration of each strain was measured in 0.1 MacFarland units (OD) using a biophotometer with sterile isotonic saline (0.9% NaCl). 600 The mixture was then prepared. Finally, 15 μL of the prepared bacterial suspension was added to 15 mL of MRS broth medium in a 30 ml sterile clear glass bottle, and three bottles containing each Lactobacillus strain were prepared. In another 15 ml of MRS broth medium in a 30 ml sterile glass bottle, 5 μL of each different Lactobacillus strain was mixed, and three bottles were prepared for each mixture as follows. - Lactobacillus fermentum + Lactobacillus rhamnosus - Lactobacillus fermentum + Lactobacillus plantarum - Lactobacillus rhamnosus + Lactobacillus plantarum - Lactobacillus fermentum + Lactobacillus rhamnosus + Lactobacillus plantarum

[0077] The pH values ​​of the prepared single cultures and mixed cultures were measured before incubation. 5 mL from each vial was transferred to a 20 mL glass beaker, and the pH value was detected using a pH meter (Mettler Toledo 1140). All vials were simultaneously incubated in an orbital shaker incubator (Infors HT Multitron Standard) at 37°C and 100 rpm for 8 hours. After 8 hours of incubation, the vials were transferred to a lamina bench (Heraeus HS 18 / 2), and the pH value of each culture was remeasured as described above (Mettler Toledo 1140).

[0078] Loading of various mixtures of Lactobacillus strains (L. fermentum, L. rhamnosus, L. plantarum) into BNC fleece, and evaluation of pH changes in the cultured BNC. Prepare cultures of each Lactobacillus strain, and use physiological saline to concentrate 90 mL of each culture as described above, and then add 0.5 MacFarland units of OD. 600 The cultures were adjusted as described above. Different Lactobacillus strains were mixed together in separate 50 mL volume tubes as described above. Each Lactobacillus strain, and their mixtures, were loaded into three BNC fleeces using the vortex method (and spray loading as described above). Under sterile conditions in a lamina airflow bench (Heraeus HS 18 / 2), each loaded BNC was added to 15 mL of MRS broth medium in a 30 mL volume sterile clear glass bottle, and the pH value was measured as described above before incubation (pH meter, Mettler Toledo 1140). All bottles were incubated in an orbital shaker incubator (Infors HT Multitron Standard) at 37 °C and 100 rpm for 8 hours. After 8 hours of incubation, the bottles were transferred to a lamina bench (Heraeus HS 18 / 2) and the pH value was remeasured (Mettler Toledo 1140).

[0079] BNC fleece loaded with Lactobacillus using vortex and spray methods was fixed as described above, dried, and observed with a scanning electron microscope (SEM). The growth behavior of Lactobacillus strains was investigated under typical culture conditions (37°C, 100 rpm shaking) and an aerobic environment on selected MRS broth medium and MRS agar plates. All Lactobacillus strains (L. fermentum, L. rhamnosus, and L. plantarum) grew on broth medium and exhibited spherical colonies of varying growth confluence on MRS agar. The colonies were white and had smooth surfaces. SEM images of L. fermentum grown on MRS agar showed a typical elongated basil morphology with a size range of 1.5–3 μm and a cell width of 0.5–0.7 μm, appearing as single cells or clustered short chains in pairs. Similarly, L. rhamnosus exhibited a basil-like morphology with a length of 1.0–2.7 μm and a width of 0.4–0.8 μm, while L. plantarum showed elongated rods with rounded tips, measuring 2.5–5.5 μm in length and 0.6–0.9 μm in width. Furthermore, various mixtures of Lactobacillus strains were co-cultured in broth medium, and the grown colonies were observed optically and microscopically using SEM on agar-MRS. After culturing under standard conditions for 8 hours, the effect of Lactobacillus growth on the pH value of the culture medium was investigated. In particular, all single strains and mixtures essentially lowered the pH value of the culture medium, as shown in Table 1.

[0080] [Table 1]

[0081] The pH values ​​of L. fermentum, L. rhamnosus, and L. plantarum decreased significantly from 6.0±0.03 before culture to 4.57±0.01, 4.21±0.09, and 4.35±0.15, respectively, after 8 hours of culture (P≦0.002). Furthermore, mixtures of all Lactobacillus strains (L. fermentum + L. rhamnosus, L. fermentum + L. plantarum, L. rhamnosus + L. plantarum, and L. fermentum + L. rhamnosus + L. plantarum) also showed significant decreases in pH values ​​to 4.35±0.07, 4.37±0.03, 4.1±0.08, and 4.4±0.1, respectively (P≦0.001). The reported decreases in pH values ​​of the mixed cultures were statistically significant compared to the cultures of each single strain (P<0.05). Only the L. rhamnosus + L. plantarum mixture did not show a significant difference in pH value compared to each of the single cultures (P>0.05).

[0082] Furthermore, single Lactobacillus strains and several mixtures thereof were loaded into BNC fleeces and observed by SEM. The loaded BNC fleeces were then cultured in MRS medium, and the change in pH was measured. A significant decrease in pH was detected in all loaded BNC cultures (P<0.001, Table 2). The pH values ​​of the medium for BNCs loaded with single Lactobacillus strains (BNCs loaded with L. fermentum, BNCs loaded with L. rhamnosus, and BNCs loaded with L. plantarum) decreased from 6.0±0.01 before culturing to 4.59±0.02, 4.13±0.03, and 4.05±0.06 after 8 hours of culturing, respectively. Furthermore, BNCs loaded with Lactobacillus mixtures (BNCs loaded with L. fermentum + L. rhamnosus, BNCs loaded with L. fermentum + L. plantarum, BNCs loaded with L. rhamnosus + L. plantarum, and BNCs loaded with L. fermentum + L. rhamnosus + L. plantarum) showed a significant decrease in pH values ​​of 4.28±0.05, 4.38±0.01, 4.09±0.04, and 4.33±0.02, respectively.

[0083] Furthermore, loading a single Lactobacillus strain or a mixture of Lactobacillus strains into BNCs did not significantly affect the pH value compared to unloaded cultures (P>0.05). Both loaded and unloaded Lactobacillus strains showed a similar decrease in the pH value of the culture medium after 8 hours of incubation under standard aerobic conditions, suggesting that loading probiotics into BNC fleece does not affect their behavior.

[0084] [Table 2]

[0085] Similar results were obtained when Lactobacillus strains were loaded using the spraying technique described above (see Table 3).

[0086] [Table 3]

[0087] Vortex and spraying methods yielded similar results regarding the effect on pH values, particularly pathogen inhibition, when using L. delbrueckii strain DSM32749 alone, and in combination with L. delbrueckii strain DSM32609 and L. rhamnosus strain DSM32758. Since L. delbrueckii has weak growth capacity under aerobic conditions and prefers anaerobic conditions, pre-culture and pH-reduced culture were performed under anaerobic conditions.

[0088] Experimental Example 6: Preparation of room-temperature storable products by spraying and vortexing techniques (B. megatherium) BNC preparation and sterilization, probiotic loading In two 500 mL glass vials under sterile conditions on a laminar airflow bench (Heraeus HS 18 / 2), BNCs (masks, patches, or other forms) were immersed in either 50 mL of MRS and TSB broth medium or an isotonic mixture of 0.9% NaCl + 5% glucose. The BNCs were autoclaved in medium (Varioklav® 85T horizontal platform) at 121°C and 1 bar for 15 minutes. The BNC vials were transferred to the laminar airflow bench (Heraeus HS 18 / 2), the BNCs were removed from the medium, wrapped directly in aluminum composite foil, and the foil was sealed with welded seams (Famos F108). The BNCs loaded with medium or NaCl / glucose were then E-beam sterilized and sterile-packed.

[0089] To load 10 mL of probiotics, use 0.5 OD for both L. lactis and B. megatherium. 600 (=10 8 A bacterial suspension was prepared by suspending the bacteria in physiological saline at a concentration of (cells / mL). 5 mL of the probiotic suspension was sprayed into a BNC using a sterile glass reagent sprayer. Loading was also performed using the vortex method as described above.

[0090] The loaded BNCs were freeze-dried for 1 to 6 days, preferably 3 to 5 days, using a freeze-dryer (Epsilon 2-4 LSC, Martin Christ GmbH, Osterode, Germany) until the residual moisture content was 3% to 14% (moisture analyzer: Ohaus MB45, Ohaus Corporation, USA). To ensure flatness during the drying process, the BNC fleece was placed between two foils. The measured residual moisture content was 13.92% ± 0.85%.

[0091] For long-term storage (at room temperature, 4°C, or above 30°C) to ensure reswellability (and stability), freeze-dried loaded BNCs should be wrapped in a material that is nearly impermeable to water / humidity (e.g., dry loaded masks wrapped in mask pack packaging material (film composition; PET / PE- / ALU / PE=12 / 15 / 9 / 50μm)) and thermally sealed using weld seams (Famos) or internal packaging foil and mask pack packaging material.

[0092] Re-culturing of loaded BNC The loaded BNCs were transferred to broth medium in 30 ml sterile glass bottles (MRS for L. lactis spray mask slices, TSB for B. megatherium spray mask slices) and re-cultured in an orbital shaker incubator (Infors HT Multitron Standard) at 37°C and 100 rpm for 8 hours; blanks of MRS and TSB were cultured under the same conditions. After 8 hours, the cultures were transferred from the incubator to a lamina airflow bench (Heraeus HS 18 / 2), photographed, mixed, and 500 μL of each culture was collected into 2 mL sterile Eppendorf cups using a sterile 1 mL pipette. Optical density (OD) of the collected samples. 600 The wavelength (nm) was measured three times at 600 nm using a UV cuvette and optical density spectrophotometer (biopphotometer) compared to blank MRS or TSB medium. Using a loop, slice cultures were spread onto agar plates (MRS-agar for L. lactis spray vortex mask slice suspensions, and TSB-agar for B. megatherium spray vortex mask slice suspensions), incubated on the plates at 37°C for 24 hours (incubator Heraeus 6000), and then photographed.

[0093] Re-swelling of loaded BNC One lyophilized BNC mask was immersed in water (or a solution containing another active ingredient) in a glass beaker and re-swelled at room temperature for 10 minutes, and the rolling ability of the re-swelled mask was evaluated. Another lyophilized BNC mask was rolled, and the rolled BNC was then immersed in water in a 250 mL glass beaker for 10 minutes. A third lyophilized BNC was rolled, transferred to a 50 mL tube, then 20 mL of water was added to the tube, and it was held at room temperature for 10 minutes.

[0094] The efficiency of loading lip masks with probiotics was investigated for both L. lactis and B. megatherium. Masks were autoclaved with the corresponding broth medium, followed by electron beam sterilization, and then a probiotic suspension was sprayed onto their surface. The probiotic-sprayed masks were then lyophilized to maintain the stability of the probiotics and BNC material. The lyophilized probiotic-loaded BNCs were re-cultured in broth medium. Optical observation of the culture vials revealed turbidity due to the growth of the loaded probiotics. After 8 hours of incubation, the lyophilized L. lactis-loaded BNCs showed an OD of 0.66 ± 0.03 McFarland units. 600 This showed the reported OD. 600 is 1cm 2 This represents the amount of L. lactis present on the mask surface. Furthermore, the photograph of the suspension spread on the MRS agar plate shows the measured OD 600 The image shows typical white, spherical colonies characteristic of L. lactis, which proliferated confluently in correlation with the target organism. This result confirms the survival ability of the loaded L. lactis and its ability to proliferate after being released from the mask.

[0095] Freeze-dried B. megatherium loaded slices are placed 1 cm above the mask surface. 2 In OD 600 The turbidity was higher, at 1.65 ± 0.02 MacFarland units. Colonies grown on TSB agar showed large, smooth, irregular, milky-white colonies, consistent with B. megatherium, supporting the stability and viability of the loaded B. megatherium.

[0096] The re-swelling ability of BNC masks loaded with isotonic mixtures was investigated in water at room temperature using several methods and configurations. First, lyophilized loaded BNC masks were re-swelled in 100 mL of water in a glass beaker until the mask was completely re-swelled. In all methods, the BNCs re-swelled normally within 10 minutes at room temperature. Similar results were obtained for loading via vortex.

[0097] Experiment Example 7: Release of loaded probiotics The release of loaded probiotics from the BNC carrier is essential for efficient biological activity at the effect site. Therefore, probiotic-loaded BNC fleeces prepared by vortex and injection methods were cultured in corresponding broth media, and the release and growth profiles at specific time points up to 48 hours were evaluated. The results, as shown in Figure 3, indicate that the release and growth of loaded probiotics leads to a constant increase in the number of probiotics in the medium.

[0098] Figure 3 shows the release profiles of L. lactis-loaded BNC fleece in MRS broth medium (left) and B. bacillus-loaded BNC fleece in TSB broth medium (right), using both vortex (top) and injection (bottom) loading methods. Results are shown as the average of three independent measurements and displayed for up to 8 hours for visualization purposes.

[0099] As shown in Figure 3 (top), both L. lactis and Bacillus subtilis loaded by the vortex method were already detectable in broth medium after 1 hour, and then grew rapidly up to 5 hours, after which they grew steadily. In contrast, the injection method presented the possibility of a slower release of loaded probiotics (detected at 3 hours, as shown in the bottom of Figure 3) and subsequent regular growth. Notably, Bacillus subtilis loaded by both the vortex and injection methods showed an OD of 8 hours. 600These values ​​were 2.5 ± 0.1 MacFarland units and 2.4 ± 0.2 MacFarland units, respectively, for L. lactis loaded by the vortex method and injection method. 600 The reported amounts were higher compared to (0.44 ± 0.2 McFarland units and 0.38 ± 0.2 McFarland units, respectively). These results clearly demonstrate the efficiency of BNC as a suitable carrier for transporting probiotics.

[0100] Experimental Example 8: Re-culturing of probiotics from freeze-dried BNC The stability of lyophilized probiotic-loaded BNC fleece (loaded with L. lactis, Bacillus subtilis, and B. megatherium by vortex, injection, and spray methods) was evaluated by re-culturing after various incubation periods (1 day, 1 week, 1 month, 3 months, and 6 months). Lyophilized control and probiotic-loaded BNC fleece were cultured in broth medium (MRS for BNC loaded with L. lactis, and TSB for BNC loaded with Bacillus subtilis). The cultures were incubated in an orbital shaker at 37°C for 8 hours with shaking at 100 rpm, and the optical density (OD) was compared with that of the control medium. 600 The following measurements were taken. Figure 4 shows the quantitative analysis of Bacillus subtilis in cultures obtained by loading Bacillus subtilis by the vortex method (top) and injection method (bottom) onto freeze-dried BNC fleece, culturing it in TSB for 8 hours, and then storing it for 1 day, 1 week, and 1 month. The results are shown as the mean ± standard deviation of three independent measurements for each sample.

[0101] Figure 5 summarizes the results for B. megatherium stored for 6 months. Figure 5 shows the quantitative analysis of cultures of BNC fleece loaded with B. megatherium using the vortex method (top) and injection method (bottom), followed by lyophilization and storage at room temperature for 6 months. The results are shown as the mean ± standard deviation of three independent measurements. The results for B. Megatherium are summarized in Table 4.

[0102] [Table 4]

[0103] Figure 6 shows the quantitative analysis of cultures of L. lactis loaded into lyophilized BNC fleece using the vortex method (top) and injection method (bottom), stored at room temperature for 6 months. The results are shown as the mean ± standard deviation of three independent measurements.

[0104] Figure 7 shows the quantitative analysis of L. lactis-loaded BNC fleece cultures prepared by the vortex method using suspensions of L. lactis powder suspended in MRS broth medium and physiological saline isotonic solution without prior culture. The results are shown as the mean ± standard deviation of three independent measurements for each sample. The results are summarized in Table 5.

[0105] [Table 5]

[0106] Furthermore, the probiotic (L. lactis and Bacillus subtilis) loading capacity of modified BNC fleece was compared and evaluated with that of standard BNC fleece. Figure 8 shows the quantitative analysis of probiotics loaded into modified BNC fleece compared to standard fleece after enzymatic digestion using cellulose. Results are presented as the mean ± standard deviation of three independent measurements for each sample. Similar results were obtained for loading by spraying.

[0107] Experimental Example 9: Manufacturing Method and Bacterial Cellulose Products Including Probiotics / Synbiotics for Topical Application Products that may be suitable for topical application include thin masks, patches, three-dimensional (3D) BNC products: face masks and lip masks, and sanitary products (e.g.) (Panty liners, tampons, sanitary napkins) are available. Pre-synthesized BNCs (as masks, patches, or other 3D products such as tamponades) were prepared by loading them with culture medium or NaCl / glucose solution, and in combination with loading nutrients and technical aids. The BNCs (e.g., masks) were immersed in glass vials under sterile conditions in a lamina airflow bench (in 50 mL of culture medium such as Heraeus HS 18 / 2, MRS, or TSB). Alternatively, the BNC masks were immersed in an isotonic mixture of 0.9% NaCl + 5% glucose, and the loaded masks were freeze-dried and sterilized as described in Experimental Example 6. The prepared BNCs were then loaded with probiotics and active nutrients using various techniques.

[0108] Loading of BNC masks by spraying Probiotics (e.g., L. lactis and B. megatherium) suspended in physiological saline at a concentration of 0.5 OD. 600 A probiotic suspension (10 mL) was prepared. Using a sterile glass reagent sprayer, 5 mL of the probiotic suspension was uniformly sprayed onto a BNC (e.g., a mask).

[0109] Loading the BNC mask using Vortex BNC fleece was added to the probiotic suspension in 50 mL volume tubes, and three tubes were prepared for each probiotic strain. BNC fleece was added to sterile medium or physiological saline. Using a multi-tube holder (SI-V506 vertical 50 mL tube holder), the tubes were vortexed at a vortex strength of 10.5 for 10 minutes at room temperature (Vortexer Genie 2). The loaded suspension was removed, and the BNCs were washed with 10 mL of physiological saline under vortexing for 10 seconds.

[0110] Drying of loaded BNC masks The BNC masks loaded with probiotics were dried using a freeze-drying oven (Epsilon 2-4 lsc Christ). Freeze-drying together ensures a 3D structure for re-swelling ability. To ensure optimal flatness of the BNC fleece after drying, the masks / patches were placed between the bottom and top of the foil during freeze-drying. The loaded BNCs were freeze-dried for 1 to 6 days, preferably 3 to 5 days, using a freeze-dryer (Epsilon 2-4 LSC, Martin Christ GmbH, Osterode, Germany) until the residual moisture content was 3% to 14% (moisture analyzer: Ohaus MB45, Ohaus Corporation, USA). If the BNCs do not reach the specified maximum residual moisture content of 14% during drying, it may adversely affect their re-swelling ability and reduce their stability.

[0111] packaging For long-term storage (at room temperature, 4°C, or above 30°C) to ensure reswellability (and stability), freeze-dried loaded BNCs are wrapped in a material that is nearly impermeable to water / humidity. The packaging material for the packaging foil is an aluminum composite foil consisting of polyethylene terephthalate (PET), aluminum (Al), and polyethylene (PE). For example, a dry loaded mask is wrapped in mask pack packaging material (e.g., PET / PE-ws / ALU / PE=12 / 15 / 9 / 50μm) and thermally sealed using a welded seam (Famos) or an inner packaging foil (PET, 50μL) and mask pack packaging material. The packaging material for the packaging foil is an aluminum composite foil consisting of polyethylene terephthalate (PET), aluminum (Al), and polyethylene (PE) (Tesseraux GmbH, Würstadt, Germany, or Gruber Folien GmbH, Straubing, Germany).

[0112] Product Usage Before using a BNC mask, remove it from its packaging and re-swell it with water, for example, to soften the BNC material and reactivate the probiotics, or (in the case of an anti-inflammatory mask) re-swell it with a liquid containing the active ingredients to soften the BNC mask, reactivate the probiotics, and activate the probiotics.

[0113] Experimental Example 10: Anti-inflammatory Mask Product: BNC loaded with B. megatherium (by spraying and vortexing) for anti-inflammatory topical use. material: For anti-inflammatory topical application, strains of the genus B. megatherium, particularly strains DSM32963, DSM33300, and DSM33336, were used. Furthermore, BNCs were loaded with an anti-inflammatory omega-3 lysine salt (AvailOm®) containing approximately 32% by weight of L-lysine and approximately 65% ​​by weight of polyunsaturated fatty acids, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

[0114] Before loading the isotonic mixture of 0.9% NaCl and 5% glucose, the BNC masks were synthesized, washed, and sterilized. Under sterile conditions in a lamina airflow bench (Heraeus HS 18 / 2), 25 μL of B. megatherium cryopreserved suspension was added to 150 mL of TSB broth medium in a sterile 250 mL Erlenmeyer flask. The flask was corked and incubated in an orbital shaker incubator (Infors HT Multitron Standard) at 37°C and 100 rpm for 8 hours. After 8 hours, the culture was transferred to a lamina airflow bench (Heraeus HS 18 / 2), distributed into three 50 mL centrifuge tubes, and centrifuged at room temperature and 4000 rpm for 20 minutes using a tubular centrifuge (Eppendorf centrifuge 5804R). The supernatant was removed, and the precipitate was resuspended in pre-warmed (37°C) sterile isotonic saline (0.9% NaCl). Using an optical density spectrophotometer (biophotometer), the optical density of the B. megatherium suspension was measured to 0.5 OD in physiological saline.600 I adjusted it to that.

[0115] Under sterile conditions in a lamina airflow bench (Heraeus HS 18 / 2), each mask was transferred to an internal packaging foil (PET, 50 μm) using plastic tweezers. The suspension was loaded into each mask by uniformly spraying 5 mL of B. megatherium suspension suspended in physiological saline onto the mask surface using a sterile glass reagent spray. The loaded masks were then covered with a second internal packaging foil (PET, 50 μm) and freeze-dried for 5 days in a freeze-dryer (Sublimator 3×4×5, Zirbus technology GmbH, Germany) until the maximum residual moisture content reached 14%. After freeze-drying, the masks were wrapped in mask packing material, thermally sealed using welded seams (Famos), and the packaged products were stored. Storage stability tests were conducted at 4°C, room temperature, 30°C, and 40°C.

[0116] To analyze the re-swelling ability and stability of lip masks loaded with isotonic mixtures and B. megatherium, and then freeze-dried, masks were loaded with an isotonic mixture of 0.9% NaCl + 5% glucose and the probiotic B. megatherium as described above, then freeze-dried, wrapped in aluminum composite foil, and stored at 4°C. The re-swelling ability and B. megatherium stability were evaluated as described in Experimental Example 6.

[0117] To evaluate the re-swelling ability of BNC lip masks and the viability of loaded B. megatherium, the masks were stored at 30°C and 40°C for two months, then re-swelled with 20 mL of water at room temperature for 10 minutes. Under sterile conditions in a lamina airflow bench (Heraeus HS 18 / 2), the masks were removed, and three slices (1 × 1 cm) taken from each mask were incubated in an orbital shaker incubator (Infors HT Multitron Standard) in 10 mL of TSB broth medium at 37°C and 100 rpm for 8 hours. Subsequently, the optical density of the resulting cultures was measured for quantitative analysis, and they were spread on TSB agar plates for qualitative observation.

[0118] After freeze-drying and storage at 4°C for 6 months or at room temperature for 5 months, the re-swelling ability of BNC masks loaded with an isotonic mixture and BNC masks loaded with B. megatherium was examined. The results showed that the mask slices maintained high re-swelling ability and exhibited a significant increase in volume. The mask slices rapidly returned to their initial shape within 7–10 minutes, showing a substantial weight increase (P = 0.001 to 0.019 ± 0.001 g to 0.27 g). The re-swelling ability of the prepared BNC masks during storage at 0.4°C for a given time was confirmed, with a value of ±0.019 g.

[0119] Table 6 summarizes the reswelling ability of lyophilized lip masks loaded with isotonic mixtures and B. megatherium after storage at 4°C for 6 months. The dried slices maintained their reswelling ability after storage at 4°C for the aforementioned period, showing a significant weight increase (P<0.05) within 7–10 minutes in water at room temperature. All observed variations in weight increase between time intervals were not statistically significant (P>0.05). The stability and viability of B. megatherium loaded into the BNC masks were also evaluated after a 6-month storage period. Cultured slices from loaded BNC masks showed significant turbidity and remarkable growth under standard culture conditions.

[0120] Table 7 summarizes the quantitative analysis of isotonic mixtures and lip mask slices loaded with B. megatherium and then freeze-dried, after storage at 4°C for 6 months. The culture slices also showed remarkable viability and activity of the loaded B. megatherium, and OD 600 A significant growth was reported at 1.48 ± 0.24 McFarland units. A notable increase in the measured growth at P=0.035 was detected after a 3-month storage period, and this increase may be related to an increased number of B. megatherium loaded or to uneven spraying of the probiotic suspension onto the BNC mask surface.

[0121] [Table 6]

[0122] The re-swelling ability and stability were assessed by testing the viability after re-culturing. Fungal growth could be detected if the masks were not sufficiently dried during packaging. This was not the case when the masks were completely dried to a maximum residual moisture content of 14% after the freeze-drying procedure.

[0123] [Table 7]

[0124] Similar results were obtained regarding re-swelling ability and viability when stored at room temperature, 30°C, and 40°C after proper freeze-drying and packaging. Foil packaging was appropriate, but better results were obtained by using two inner foils (as described above) before wrapping in a sealed outer foil.

[0125] To measure specialized pro-resolving mediators (SPMs) and their precursors, samples were prepared from BNC lip masks loaded with isotonic mixtures and B. megatherium. Two BNC lip masks were loaded with the isotonic mixture and B. megatherium, then lyophilized and re-swelled. The first lyophilized BNC mask was loaded with a 0.01% liposome AvailOm® aqueous suspension (1), and the second mask was loaded with a 0.01% powdered AvailOm® aqueous solution (2). Slices from the re-swelled masks were then cultured under standard conditions in TSB broth medium and TSB agar plates. Alternatively, the two BNC lip masks were first loaded with the isotonic mixture. Then, B. megatherium was added in 0.5 MacFarland units of OD. 600The following concentrations were added to (1) a 0.01% aqueous suspension of liposome AvailOm® and (2) a 0.01% aqueous solution of powdered AvailOm®. The B. megatherium-AvailOm® mixture was then sprayed onto a mask, freeze-dried, and re-swelled in water. Slices from the re-swelled mask were cultured on TSB broth medium and TSB agar plates as described above.

[0126] Slices from unloaded BNC masks were cultured in broth TSB and TSB agar as controls. Both the slices cultured in broth TSB and the slices cultured in TSB agar were then prepared for SPM measurement and its precursor. Broth medium was diluted 2:1 (V / V) with methanol in a 50 mL volume tube. Agar containing cultured slices (2 × 2 cm) was transferred to another 50 mL volume tube, 8 mL of methanol was added, and both the broth medium and agar samples were cooled at -20°C for 60 minutes and centrifuged at 4500 rpm for 10 minutes. Finally, the supernatant was collected in separate tubes for quantitative and qualitative analysis of SPM, compared to cultures from unloaded BNC mask slices prepared using the same procedure as controls.

[0127] The generation of specific anti-inflammatory mediators (SPMs) and their precursors from a B. megatherium-AvailOm® mixture loaded into BNC lip masks was investigated using broth and agar plates. Both liposomal formulations and powders of AvailOm®, along with B. megatherium, were loaded into BNC masks using two sequential pathways. In the first method (A), the lyophilized B. megatherium-loaded BNC masks were re-swelled using a liposomal AvailOm® suspension or powder AvailOm® solution. In the second method (B), the AvailOm® suspension / solution was mixed with B. megatherium, sprayed onto the BNC mask before lyophilization, and then re-swelled with water. Next, slices of BNC lip masks loaded with B. megatherium and AvailOm® and re-swollen were cultured in TSB broth medium and TSB agar plates, and SPM production was measured compared to unloaded BNC mask slices cultured in TSB medium and TSB agar as a control. As a result, several lipid mediators produced by lipoxygenase, cytoplasmic phospholipase A2, and cyclooxygenase 1 or 2 were measured by ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS).

[0128] SPM is known for its natural anti-inflammatory properties. Therefore, the anti-inflammatory masks / patches resulting from the above are for topical anti-inflammatory treatment of the skin or mucous membranes. The following SPMs were most notably produced: 17-HDHA (17-hydroxydocosahexaenoic acid), 14-HDHA (14-hydroxydocosahexaenoic acid), 13-HDHA (13-hydroxydocosahexaenoic acid), 7-HDHA (7-hydroxydocosahexaenoic acid), 4-HDHA (4-hydroxydocosahexaenoic acid), 15-HEPE (15-hydroxyeicosapentaenoic acid), 12-HEPE (12-hydroxyeicosa 11-HEPE (11-hydroxyeicosapentaenoic acid), 5-HEPE (5-hydroxyeicosapentaenoic acid), 15-HETE (15-hydroxyeicosatetraenoic acid), 12-HETE (12-hydroxyeicosatetraenoic acid), 11-HETE (11-hydroxyeicosatetraenoic acid), 8-HETE (8-hydroxyeicosatetraenoic acid), 5-HETE (5-hydroxyeicosatetraenoic acid), AA (arachidonic acid), EPA (eicosapentaenoic acid), DHA (docosahexaonic acid), PD1 (protectin D1), AT-PD1 (aspirin-induced protectin D1), PDX (protectin DX), RvD5 (resolvin D5), MaR1 (malecin 1), MaR2 (malecin 2), t-LTB4 (trans-leukotriene B4), LTB4 ( Leukotriene B4), 20-OH-LTB4 (20-hydroxyleukotriene B4), PGE2 (prostaglandin E2), PGF2a (prostaglandin F2 alpha), TXB2 (thromboxane B2), LXA4 (lipoxin A4), AT-LXA4 (aspirin-induced lipoxin A4), LXA5 (lipoxin A5), RvD1 (resolvin D1), RvD4 (resolvin D4).

[0129] Experimental Example 11: Bacillus subtilis-containing BNC patch / mask for Staphylococcus aureus inhibition The loading was performed using the three different methods mentioned above (vortex, spray, and injection). To prepare the supernatant, 35 mL of each of the last cultured bacterial suspensions of B. megatherium strain DSM32963 and Bacillus subtilis strain DSM33561 were centrifuged in a 50 mL volume centrifuge tube at 4500 rpm for 30 minutes at 4°C using a tubular centrifuge (EEppendorf centrifuge 5804R). The supernatant was collected in a 50 mL syringe and filtered into another 50 mL centrifuge tube using a 0.2 μm syringe filter. Under sterile conditions in a Lamina Airflow Bench (Heraeus HS 18 / 2), Staphylococcus aureus was subjected to OD (Oxygen-Drug) treatment. 600 At a concentration of 0.1 McFarland units, 10 mL of probiotic-free supernatant, free of both B. megatherium and Bacillus subtilis, was added to a 30 mL sterile glass bottle. 5 mL of Staphylococcus aureus was added to OD. 600 The solution was added at a concentration of 0.1 McFarland units to 5 mL of B. megatherium or Bacillus subtilis suspension in a 30 mL sterile glass bottle. Gentamicin at a concentration of 300 μg / mL was added to TSB medium, followed by OD. 600 A positive control was prepared by adding Staphylococcus aureus at a concentration of 0.1 MacFarland units. The bottles were incubated in an orbital shaker incubator (Infors HT Multitron Standard) at 37°C and 100 rpm for 18 hours. After 18 hours, the bottles were transferred to a lamina airflow bench (Heraeus HS 18 / 2) and photographed. Using a loop, 5 μL of each bottle was spread onto TSB agar, and the agar plates were incubated at 37°C for 24 hours (Heraeus 6000 incubator), and photographs of the agar plates were taken.

[0130] For agar diffusion tests, OD of B. megatherium and Bacillus subtilis 600 This was adjusted to 0.1 McFarland units using sterile physiological saline (NaCl 0.9%). (Drug adsorption of Staphylococcus aureus) 600The solution was adjusted to 0.5 McFarland units using sterile saline NaCl 0.9%. 20 μL of Staphylococcus aureus was spread on the surface of Mueller-Hinton agar using a sterile glass spreader. The wells were dissolved on the agar plate using the back of a 1 mL pipette tip. A small amount of Mueller-Hinton agar was dissolved in a boiling water bath, and 100 μL of this was used to seal the bottom of each prepared well. After the agar solidified at the bottom of the wells, the wells were filled with sterile saline NaCl 0.9% as a negative control, and with gentamicin 300 μg / mL as a positive control, either with supernatant free of B. megatherium or Bacillus subtilis, or with a suspension of B. megatherium or Bacillus subtilis. The agar plates were incubated at 37°C for 24 hours (in an incubator Heraeus 6000), after which photographs were taken to identify the inhibition zones.

[0131] The antimicrobial activity of BNCs loaded with Bacillus subtilis and B. megatherium against Gram-positive Staphylococcus aureus was evaluated by agar diffusion tests. Accordingly, bacterial suspensions of Bacillus subtilis and Staphylococcus aureus were prepared in TSB broth medium as described above. A supernatant without Bacillus subtilis was prepared, and Bacillus subtilis was loaded into BNC fleeces using the vortex method. Three BNC fleeces were loaded with a suspension of Bacillus subtilis in TSB medium, and three more BNC fleeces were loaded with a suspension of Bacillus subtilis in physiological saline. Furthermore, three BNC fleeces were loaded with supernatant without Bacillus subtilis, three BNC fleeces were loaded with gentamicin as a positive control, and three BNC fleeces were loaded with isotonic saline as a negative control. The OD of Staphylococcus aureus was also evaluated. 600The solution was adjusted to 0.5 MacFarland units using sterile saline (NaCl 0.9%) and measured using an optical density spectrophotometer (biophotometer). 20 μL of Staphylococcus aureus was spread onto the surface of a Mueller-Hinton agar plate using a sterile glass spreader. The final control and loaded BNC fleece were added to the surface of the Mueller-Hinton agar plates: 1. Negative control (positive control): BNC loaded with saline, 2. Positive control (negative control): BNC loaded with gentamicin, 3. BNC loaded with Bacillus subtilis in TSB medium, 4. BNC loaded with Bacillus subtilis in saline, 5. BNC loaded with supernatant without Bacillus subtilis. The agar plates were incubated at 37°C for 24 hours (Heraeus 6000 incubator), then photographs were taken to identify the inhibition zone.

[0132] The inhibitory activity of each probiotic (B. megatherium and Bacillus subtilis) against Gram-positive Staphylococcus aureus was tested before loading into BNCs. Only Bacillus subtilis was effective in inhibiting Staphylococcus aureus. Staphylococcus aureus was cultured with each of the following: probiotic suspensions prepared by culturing for more than 24 hours and supernatants without probiotics. Results from the co-culture test showed that the prepared cultures were turbid. To classify the grown strains and detect inhibitory effects, the turbid suspensions were spread on agar plates with each control (control) of probiotics and Staphylococcus aureus strains. Photographs of the agar plates showed that B. megatherium had no inhibitory effect on Staphylococcus aureus. Neither the B. megatherium suspension nor the supernatant without B. megatherium showed any inhibitory effect on Staphylococcus aureus. On the other hand, significant inhibition of Staphylococcus aureus by Bacillus subtilis strain DSM33561 was detected. Bacillus subtilis colonies were observed only on the surface of the test plates, and no growth of Staphylococcus aureus colonies was detected in either the Bacillus subtilis suspension plate or the Bacillus subtilis-free supernatant plate. These results were further reinforced by the agar well diffusion test. The B. megatherium plate showed an inhibition zone in the gentamicin well, but no inhibition zone was detected in the B. megatherium suspension or the B. megatherium-free supernatant. The Bacillus subtilis suspension showed an inhibition zone with a radius of 0.5 ± 0.1 mm in the well, which is associated with the growth of Bacillus subtilis colonies. However, in contrast to the results of the co-culture test, the Bacillus subtilis-free supernatant did not show a sufficient inhibition zone, which may be related to the low concentration of the active molecule in the amount of supernatant used. With a different Bacillus subtilis strain, the results obtained from the co-culture test were further reinforced by the standard agar well diffusion test. A significant inhibition zone could be detected around both the Bacillus subtilis-free supernatant and the Bacillus subtilis cell-containing wells, which is associated with significant growth around the well edges.

[0133] After loading bacterial cultures into BNCs, the antimicrobial activity of Bacillus subtilis against Gram-positive Staphylococcus aureus was demonstrated using two standard tests (co-culture test and agar well diffusion test). Probiotics (Bacillus subtilis, B. megatherium) were loaded into BNCs using TSB broth medium and isotonic saline as loading solutions, via vortex, spray, and injection methods. The antimicrobial activity of Bacillus subtilis in the BNC fleece against Staphylococcus aureus was represented by a marked inhibition zone around the BNC fleece. The BNC fleece used TSB broth medium and physiological saline showed an inhibition zone of 3-4 mm with the vortex method, a 5 mm inhibition zone with the spray method, and no inhibition zone with the injection method. No inhibition zone was observed for B. megatherium with any loading method. Simultaneously, Bacillus subtilis colonies proliferated near the BNCs. Inhibition zones were also detected around BNCs loaded with supernatant that did not contain Bacillus subtilis. The vortex method detected an inhibition zone of 1–3 mm, while the spray method detected an inhibition zone exceeding 2 mm. Surprisingly, regarding inhibition by Bacillus subtilis on BNCs, when loading was performed by vortex or spray, the cell-free extract of loaded Bacillus subtilis strain DSM33561 was effective, in contrast to the pure cell-free extract. The results are summarized in Table 8.

[0134] [Table 8]

[0135] Similar inhibitory results were detected for other Bacillus subtilis strains, namely Bacillus subtilis DSM33353 and DSM33298.

[0136] Experimental Example 12: Probiotics in BNC for women's / vaginal health products using Lactobacillus or Lactococcus species Considering the re-swelling ability of the BNC and its ability to carry / load probiotics, a single probiotic or a mixture of probiotics is loaded into the BNC (thin layer or 3D structure), for example, as a layer of a panty liner, a layer of a sanitary napkin, or a layer rolled up as a tampon, or as a three-dimensional structure as a tampon or tamponade. The loaded probiotics help maintain the vaginal environment by lowering pH, generating H2O2, or inhibiting genitourinary pathogens. The following strains were used for these applications: Lactobacillus rhamnosus (DSM 32609), Lactobacillus fermentum, Lactobacillus plantarum (DSM 32758), and Lactobacillus delbrueckii susp. bulgaricus (DSM 32749).

[0137] Evaluation of the reswelling ability of flat BNCs and rolled BNCs in water. For tampon or layer-type panty liner products, four BNC fleece sheets (10 x 10 cm) were immersed in 400 mL of an isotonic mixture of 0.9% NaCl + 5% glucose, then autoclaved and lyophilized as described above. The lyophilized BNC fleece was immersed in 100 mL of water in a 250 mL glass beaker and re-swelled at room temperature for 10 minutes, after which the rolling ability of the re-swelled mask was evaluated. The second lyophilized BNC mask was rolled up and immersed in 100 mL of water in a 250 mL glass beaker for 10 minutes. The third lyophilized BNC fleece was rolled up and transferred to a 50 mL tube, then 20 mL of water was added to the tube and it was held at room temperature for 10 minutes. The fourth lyophilized BNC fleece was rolled up and transferred to a 50 mL tube, the tube was inverted into a Petri dish, then 20 mL of water was added to the Petri dish and it was held at room temperature for 10 minutes.

[0138] The re-swelling ability of BNC fleece loaded with isotonic mixtures was investigated in water at room temperature using several methods and configurations. First, lyophilized loaded BNC masks were re-swelled in 100 mL of water in a glass beaker. After 10 minutes, the masks were fully re-swelled, and their flexibility and rolling ability after re-swelling were observed.

[0139] Next, a freeze-dried loaded mask was rolled up and fully re-swelled in room temperature water in a glass beaker for 10 minutes. During re-swelling, the mask unrolled and returned to its original flat shape in the water after 10 minutes. Furthermore, a third freeze-dried loaded BNC fleece was rolled up and re-swelled in water using a tube similar to a vaginal cavity. The fleece fully re-swelled and filled the entire tube, whereas when the fleece was placed in an inverted tube in a Petri dish filled with water, it slowly re-swelled from the bottom of the fleece in contact with the fluid without unrolling. Therefore, flat or rolled BNC fleece, short and pre-moistened, are suitable for the application to allow for usability and easy re-swelling.

[0140] Loading of Lactobacillus strains into BNC The loading of Lactobacillus bacteria and mixtures thereof, as well as the reduction of pH, are described in Example 5. When Lactobacillus strains were loaded by spraying as described above, similar results were obtained regarding the distribution of bacterial cells on the BNC nonwoven fabric.

[0141] The L. delbrueckii subspecies bulgaricus strain DSM32749 was also shown to be suitable for women's health use, particularly in combination with L. plantarum strain DSM32758, or in combination with three strains including L. rhamnosus strain DSM32609. In this case, the protocol was designed to constitute its preferred anaerobic culture. Culture was performed in MRS medium under anaerobic conditions. All strains were also able to grow in artificial vaginal fluid (MSVF).

[0142] Furthermore, if potential is demonstrated, particularly regarding women's health (e.g., pH reduction, H2O2 production, or inhibition of pathogens (e.g., urinary tract pathogenic E. coli)), additional strains of Lactobacillus and / or Lactococcus species may be used in the product alone or in combination. In preferred embodiments, the strains are DSM33370 L. plantarum LN5, DSM33377 L. brevis LN32, DSM33368 L. plantarum S3, DSM33369 L. plantarum S11, DSM33376 L. paracasei S20, and DSM33375 L. paracasei S23. S23), DSM33374 L. reuteri F12, DSM33367 L. plantarum F8, DSM33366 L. plantarum S4, DSM33364 L. plantarum S28, DSM33363 L. plantarum S27, DSM33373 L. paracasei S18a The following strains are selected: S18a), DSM33365 Lactobacillus plantarum S18b, DSM33362 Lactobacillus plantarum S13, DSM32767 Lactococcus lactis sups. lactis, and Lactobacillus fermentum DSM32750.

[0143] Experimental Example 13: BNC mask / patch containing Propionibacterium acnes / Cutibacterium acnes for use as an anti-acne mask. Cutibacterium acnes was loaded onto glucose / NaCl-prepared nonwoven fabrics (as patches or masks) using vortex and spray loading techniques, then freeze-dried and packaged for storage as described in Experimental Example 9. Re-swelling and stability tests demonstrated that the described method is also suitable for this product application. This product example focuses on topical anti-acne application due to the beneficial effects of Cutibacterium acnes on pathogenic acne microflora after mask / patch application.

[0144] Experiment Example 14: Restoring the balance of the skin microbiome / BNC mask / patch containing Staphylococcus epidermidis that affects the skin microbiome Staphylococcus epidermidis was loaded onto glucose / NaCl-prepared nonwoven fabric (as a patch or mask) using vortex and spray loading techniques, followed by lyophilization and packaging for storage as described in Experimental Example 9. Re-swelling and stability tests demonstrated that the described method is also suitable for this product application. This product example focuses on the local rebalancing of the skin microbiome due to the beneficial effects of Staphylococcus epidermidis on the local microbiome composition after mask / patch application.

Claims

1. A method for loading microorganisms onto and / or into a pre-synthesized bacterial nanocellulose (BNC) fleece, comprising the steps of: synthesizing the bacterial nanocellulose (BNC) fleece; resuspending the microorganisms in a buffer or culture medium; and mixing the BNC fleece and the microorganisms at 3300 to 3500 rpm for 1 to 60 minutes at a temperature of 10 to 37°C, thereby loading the microorganisms onto and / or into the BNC fleece.

2. A method for loading microorganisms onto and / or into a pre-synthesized bacterial nanocellulose (BNC) fleece, comprising the steps of: synthesizing the bacterial nanocellulose (BNC) fleece; resuspending the microorganisms in a buffer or culture medium; injecting the microorganisms into the BNC fleece; and culturing at a temperature of 4 to 37°C for a maximum of 1 hour, thereby loading the microorganisms onto and / or into the BNC fleece.

3. A method for loading microorganisms onto and / or into a pre-synthesized bacterial nanocellulose (BNC) fleece, comprising the steps of synthesizing the bacterial nanocellulose (BNC) fleece, resuspending the microorganisms in a buffer or culture medium, and loading the microorganisms onto and / or into the BNC fleece by spraying.

4. The method according to any one of claims 1 to 3, wherein the microorganism is loaded as a cell in the growth phase or in a dormant state.

5. The method according to any one of claims 1 to 4, wherein the microorganism is wet or dry, and / or is pre-cultured or not pre-cultured.

6. The method according to any one of claims 1 to 5, wherein the BNC fleece is wet, dry, partially dry, or reswells in the buffer solution.

7. The method according to any one of claims 1 to 6, wherein the bacterial-synthesized nanocellulose (BNC) fleece is derived from Komagataeibacter.

8. The method according to any one of claims 1 to 7, wherein the bacterially synthesized nanocellulose (BNC) has a layered structure, and the layered structure is selected from a BNC comprising a network of cellulose fibers or nanowhiskers, a BNC comprising two or more different layers of cellulose fibrils, each layer comprising BNC derived from a different microorganism or a microorganism cultured under different conditions, a BNC comprising at least two different cellulose networks, or a BNC composite material further comprising a polymer.

9. The method according to any one of claims 1 to 8, wherein the bacterial-synthesized nanocellulose (BNC) is a BNC fleece having an average thickness of at least 0.5 mm.

10. The method according to any one of claims 1 to 9, wherein other substances that enable control of the resulting pore size / mesh size are added during the bacterial synthesis of the BNC.

11. The method according to any one of claims 1 to 10, wherein in a subsequent step, the loaded BNC fleece is cultured with a drying moisture binder, and the moisture binder is an osmotically and / or hygroscopically effective solution.

12. The aforementioned microorganisms include the genera Bifidobacterium, Carnobacterium, Corynebacterium, Cutibacterium, Lactobacillus, Lactococcus, Leuconostoc, and Microbacteria. Genera: Microbacterium, Oenococcus, Pasteuria, Pediococcus, Propionibacterium, Streptococcus, Bacillus, Geobacillus, Gluconobacter ter), Xanthomonas, Candida, Devariomyces, Hanseniaspora, Kluyveromyces, Komagataella, Lindnera, Ogataea, Saccharomyces, S The method according to any one of claims 1 to 11, wherein the probiotic bacterial strain or yeast strain is selected from the genera Schizosaccharomyces, Wickerhamomyces, Xanthophyllomyces, Yarrowia, Micrococcus, or a mixture thereof.

13. The method according to any one of claims 1 to 12, wherein the additional step is performed before, after, or in parallel with loading the microorganisms into the BNC fleece, and the BNC fleece is loaded with further components and / or nutrients selected from amino acids, fatty acid salts, anthocyanins, monosaccharides, and extracts.

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