Storage-stable microbial compositions

Abstract

The present invention relates to a method for producing a stable hydrogel-cell composition, comprising the steps of: a) providing a reaction mixture comprising at least one polymerizable substrate and at least one enzyme capable of polymerizing said at least one substrate, and cells; and b) incubating the mixture of step a) to form a stable hydrogel-cell composition.

Description

[Technical Field]

[0001] The present invention relates to the field of hydrogel-cell compositions. [Background technology]

[0002] Three-dimensional (3D) systems, including cell cultures, have found widespread applications in biomedical fields, such as cell spheroids for studying tumor behavior and cell encapsulation in cell-based transplantation therapy. Promising materials for cell encapsulation include hydrogels, i.e., soft, moist biomaterials with tunable properties, because they mimic the natural extracellular matrix (i.e., aqueous environment) by possessing excellent mass transport properties and exhibiting tissue-like elasticity. These properties allow cell growth, protect cells from environmental hazards, and preserve cell viability over extended periods of time.

[0003] Various categories of hydrogels for cell encapsulation have been reported in the art. Known hydrogels typically contain animal- or plant-derived proteins, such as gelatin or gluten. However, all of these materials exhibit relatively low mechanical flexibility and therefore cannot be used for processing. Known hydrogels may also contain substances that may be toxic to certain types of cells. Therefore, such hydrogels cannot be used for cell-based applications.

[0004] Hydrogels containing cellulose materials (e.g., cellulose nanofibers, cellulose nanocrystals, carboxymethylcellulose) have been developed for cell encapsulation. Known methods for producing such hydrogels generally require first dissolving cellulose to form cellulose microgels, which are then used for cell encapsulation.

[0005] For example, Serizawa et al. (ACS Macro Lett 2020, 9, 301-305) describe the neutralization-induced self-assembly of cellulose oligomers into crystalline nanoribbon network structures to fabricate physically crosslinked hydrogels for 3D cell culture.

[0006] A further example is disclosed in EP 4053141, which describes a cell culture medium composition for suspending cells containing cellulose oligomers. For example, this medium composition can be added to HeLa cells.

[0007] The cellulose oligomers in the composition do not solidify and keep the cells in suspension.

[0008] One further example is disclosed in JP 2017-201895, which describes a synthetic cellulose three-dimensional structure containing gelatin, to which animal cells can be added and grown on its surface.

[0009] Furthermore, JP 2018-145216 A describes an artificially synthesized three-dimensional cellulose structure used as a scaffold for animal cells.

[0010] EP 0928795 describes a process for producing natural organic polymers to be used as gelling agents for the preparation of hydrogels.

[0011] However, these methods are complex and highly inefficient. Furthermore, to achieve the desired physicochemical properties, cellulosic materials must be modified or combined with other materials. Chemical methods for cross-linking cellulose require toxic chemical cross-linking agents and catalysts, such as 2,2,6,6-tetramethylpiperidine-1-oxyl radical. These toxic chemicals adversely affect the viability of cells encapsulated by such cellulosic materials.

[0012] The shortcomings of hydrogels known in the art have heretofore hindered the development of stable hydrogel-cell compositions. It is therefore an object of the present invention to provide means and methods that overcome the shortcomings of the prior art for producing stable, biocompatible hydrogel-cell compositions that can be used in tissue engineering and biomedical applications, such as bioprinting. Summary of the Invention

[0013] Thus, the present invention provides a method for producing a stable hydrogel-cell composition, comprising: a) providing a reaction mixture comprising at least one polymerizable substrate and at least one enzyme that allows the at least one substrate to be polymerized, and cells; b) incubating the mixture of step a) to form a stable hydrogel-cell composition; The present invention relates to a method comprising:

[0014] Surprisingly, it has been found that cells can be stabilized in hydrogels when the hydrogels are enzymatically synthesized in the presence of cells. The method of the present invention is a bottom-up synthesis method from a polymerizable substrate that can form a polymer network upon mixing at least one polymerizing enzyme with cells. Thus, the polymer network of the present hydrogel-cell composition is constructed by enzymatic polymerization of at least one polymerizable substrate in the presence of cells. The bottom-up strategy of the present invention for synthesizing hydrogels allows for the stabilization of cells embedded in the hydrogel for a much longer period of time than methods that simply involve contacting the hydrogel with cells.

[0015] The method of the present invention further enables the formation of mechanically stable hydrogels that exhibit excellent biocompatibility during the entire process. Due to the enzymatic reaction that causes gelation in situ, cells can be embedded / encapsulated within the hydrogel, resulting in a more uniform and efficient formation. As mentioned above, this embedding / encapsulation method significantly extends the viability of cells contained within the hydrogel.

[0016] The use of polymerizable substrates and enzymes to form hydrogels is also advantageous because it allows for the creation of biocompatible hydrogel matrices that are beneficial for the cells embedded / encapsulated in the hydrogel and potential medical applications of the hydrogel-cell compositions of the present invention.

[0017] Thus, the method of the present invention for preparing hydrogels is fully applicable to cell embedding / encapsulation in terms of: a) obtaining a uniform distribution of cells within the hydrogel due to in situ gelation and encapsulation by premixing the cells in the aqueous reaction solution; b) the mild conditions used for gel synthesis, which preserve cell viability during encapsulation; and c) the biocompatible and eco-friendly materials used in the hydrogel composition, which make the system applicable to human and animal applications. Furthermore, the method of the present invention is a rapid, simple, and cost-effective process that does not require specialized equipment and makes the technology accessible to people without specialized knowledge or laboratory equipment (e.g., in pre-formulated kits).

[0018] The hydrogels obtainable by the method of the present invention can also be used in tissue engineering, where they take over the mechanical and other tasks of the extracellular matrix. The water-insoluble part can be enzymatically dissolved when growing cells excrete their own extracellular matrix. Since the degradation process must be controllable, cellulose fibers are particularly suitable for cells, preferably eukaryotic cells lacking cellulases, for example.

[0019] Another aspect of the present invention is a method for producing a stable hydrogel composition, comprising: a) providing a reaction mixture comprising at least one polymerizable substrate and at least one enzyme that allows said at least one substrate to be polymerized, and xanthan; b) incubating the mixture of step a) to form a stable hydrogel composition; The present invention relates to a method comprising:

[0020] The addition of xanthan to the reaction mixture used in the method of the present invention has been found to result in the synthesis of stable hydrogels with superior mechanical properties compared to hydrogel compositions containing other added polymers, e.g., polysaccharide-based ones. Furthermore, the hydrogels produced by the method of the present invention have been found to retain their mechanical properties for much longer periods of time compared to other hydrogels, particularly hydrogels containing other polymers (e.g., other polysaccharides). The addition of xanthan to the hydrogels produced by the method of the present invention confers additional mechanical stability to the hydrogel. This improved mechanical stability of the hydrogel combination significantly expands the range of use of this combination. An additional advantage of xanthan-containing hydrogels is their biocompatibility, which allows for much more efficient stabilization of biomolecules (e.g., enzymes) or cells embedded / encapsulated therein compared to other additives. This results in a product with a longer shelf life compared to hydrogels without xanthan, as bioactivity and enzymatic activity are retained within the hydrogel combination for extended periods of time.

[0021] Another aspect of the invention relates to a hydrogel-cell composition obtainable or obtainable by the method of the invention.

[0022] A further aspect of the present invention relates to Lactococcus lactis strain 10123, deposited at the DSMZ on February 4, 2022 under deposit number DSM 34156.

[0023] L. lactis strain 10123 exhibits surprising inhibitory properties against various bacterial species of the genus Staphylococcus, such as S. aureus, S. hominis, and E. hirae.

[0024] L. lactis is a well-known probiotic organism that has been widely used for thousands of years for various purposes, including cheese production. L. lactis is also known for producing bacteriocins (e.g., the lantibiotic nisin), which can act as inhibitors against certain types of other pathogenic bacteria, such as Staphylococcus bacteria. However, typical Lactococcus lactis bacteria only release limited amounts of antimicrobially active peptides that directly inhibit other bacteria. Therefore, it is surprising that L. lactis strain 10123 in particular exhibits superior antibiotic properties against many microorganisms compared to known L. lactis strains.

[0025] The strains of the present invention also demonstrate surprising effectiveness against antibiotic-resistant microorganisms. The rapid emergence of antibiotic-resistant microorganisms, particularly bacteria, is occurring worldwide, threatening the effectiveness of antibiotics that have transformed medicine and saved millions of lives. Decades after the first patients were treated with antibiotics, bacterial infections are once again a threat. Therefore, providing means and methods to combat these antibiotic-resistant microorganisms is of great importance. L. lactis strain 10123 has been found to be capable of preventing and / or inhibiting the growth of antibiotic-resistant microorganisms.

[0026] In particular, S. aureus and its antibiotic-resistant form, methicillin-resistant Staphylococcus aureus (MRSA), are concerning human pathogens, causing, for example, skin infections, respiratory infections, and food poisoning. For example, antibiotic elimination of S. aureus dysbiosis, a severe skin disease, has shown promising improvement in atopic skin conditions in various studies. The use of the strains of the present invention in systemic and topical medicine may be useful in combating the spread of multidrug-resistant bacteria.

[0027] Another preferred application of the bacterial strains of the present invention relates to cosmetics. Up until now, cosmetics have mainly used prebiotic agents (favorable skin flora) or postbiotic agents (lysates or bacterial metabolic products). Bacterial components or killed bacteria also have immunomodulatory effects. However, the use of live bacteria offers additional advantages. This is particularly important for the direct inhibition of pathogens such as C. albicans or S. aureus.

[0028] L. lactis strain 10123 has been found to produce sufficient amounts of antimicrobially active substances so that it can also be used as a food additive (e.g., as a preservative). Another aspect of the present invention relates to compositions comprising Lactococcus lactis 10123.

[0029] Another aspect of the present invention relates to a kit for producing a stable hydrogel-cell composition, comprising at least one polymerizable substrate and at least one enzyme that enables the at least one substrate to polymerize. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 shows a scheme for the beta-1,4-glycosylation of cellobiose using alpha-D-glucose-1-phosphate (αGlc1P) as the donor, catalyzed by cellodextrin phosphorylase (CdP). [Figure 2] Figure 1 shows reaction mixtures containing various xanthan concentrations (0-0.5%, m / v). The reactions were carried out in 10 mM phosphate buffer (pH 7.0) with 25 mM cellobiose, 200 mM Glc 1-P, and 0.5 mg / mL cellodextrin phosphorylase (CdP) at 37 °C for 3 h. [Figure 3] FIG. 1 shows the amplitude sweep analysis profile for a synthetic cellulose hydrogel containing 0.4% xanthan. [Figure 4]FIG. 1 shows confocal laser scanning micrographs of live cells (white) encapsulated within hydrogels. [Figure 5a] FIG. 1 shows CFU of bacteria (Lb. rhamnosus) in gels over storage time. [Figure 5b] FIG. 1 shows bacterial viability (Lb. rhamnosus) in standard hydrogel (xanthan only) over storage time. [Figure 6a] FIG. 1 shows CFU of bacteria (L. lactis) in gels over storage time. [Figure 6b] FIG. 1 shows a comparative analysis of cell survival between (Lb. rhamnosus) embedded in a standard hydrogel (xanthan) and L. lactis in a hydrogel containing xanthan according to the invention. [Figure 7] Figure 1 shows the zone of inhibition on Mueller-Hinton agar inoculated with S. aureus. [Figure 8] FIG. 1 shows that after 24 hours of incubation without L. lactis 10123, S. aureus was inhibited in co-culture with L. lactis 10123 compared to S. aureus monoculture and co-culture of S. aureus with Lb. rhamnosus. [Figure 9a] FIG. 1 shows a blood agar plate inoculated with 10 CFU of S. aureus with 10 CFU / ml of L. lactis on top. [Figure 9b] Figure 1 shows the zone of inhibition of blood agar plates to which hydrogel droplets containing L. lactis were directly applied. [Figure 10] FIG. 1 shows pig skin models inoculated with S. aureus (left spots) and S. aureus and L. lactis (right spots). DETAILED DESCRIPTION OF THE INVENTION

[0031] As used herein, a "stable hydrogel-cell composition" refers to a composition comprising at least one hydrogel and (biological) cells embedded and / or encapsulated in the at least one hydrogel. A stable hydrogel-cell composition can be formed within 1 to 8 hours, preferably 1 to 5 hours, starting from contacting at least one polymerizable substrate and at least one enzyme with cells. "Stable" with respect to the composition means that the physicochemical properties of the hydrogel remain substantially unchanged in the presence of cells embedded and / or encapsulated in the hydrogel for at least 30 days, preferably at least 60 days, and more preferably at least 180 days, at temperatures between 2°C and 30°C, preferably between 4°C and 25°C. Furthermore, "stable" also refers to the viability of cells encapsulated and / or embedded in the hydrogel, which retain their function.

[0032] Using the methods of the present invention, it is possible to produce hydrogel-cell compositions in which the cells embedded and / or encapsulated therein remain viable for at least 30 days, preferably at least 60 days, and more preferably at least 180 days, at temperatures between 2°C and 30°C, preferably between 4°C and 25°C. The number of viable cells embedded and / or encapsulated in the hydrogel obtainable by the methods of the present invention is minimally reduced under these conditions. Minimal reduction in viable cell counts (CFU, colony-forming units) was observed over a 6-month period at room temperature (less than 2 log CFU / ml). The stress resistance of the compositions was also tested by subjecting them to three freeze-thaw cycles: storing them at -18°C for 24 hours, followed by another 24 hours at room temperature, then at 37°C for 24 hours, and finally at room temperature for 24 hours. Cell viability can be determined by enumerating the encapsulated cells on agar plates according to Arepally D. et al. (Curr Res Food Sci. 2020 Oct 1;3:235-242). However, other methods for determining the number of CFUs are known in the art. Depending on the type of cell, different methods may be used.

[0033] As used herein, "polymerizable substrate" refers to at least one molecule that can form a polymer network when mixed with at least one polymerizing enzyme. Preferably, the polymerizable substrate is at least one monomer. The at least one monomer can be crosslinked into a polymer or oligomer chain by covalent, ionic, or physical bonds. A reaction mixture containing at least one polymerizable substrate, at least one enzyme that allows the at least one substrate to polymerize, and cells is incubated under conditions that allow enzymatic polymerization of the substrate and do not adversely affect the viability or function of the cells to be embedded and / or encapsulated. Therefore, these conditions depend on the enzyme and cells used in the method of the present invention. These conditions are well known to those skilled in the art.

[0034] Another aspect of the present invention relates to a method for producing a stable hydrogel composition by providing and incubating a reaction mixture comprising at least one polymerizable substrate, at least one enzyme, and xanthan.

[0035] Xanthan is known as a common food additive and plays an important role in industrial applications as a thickener and emulsion stabilizer. It is typically added to water-based drilling fluids due to its pseudoplastic behavior and thermal stability. In accordance with the present invention, the addition of xanthan to a reaction mixture significantly contributes to improved cell biocompatibility, making it more environmentally friendly and safe for human consumption compared to other chemical additives, such as PEG. Therefore, a stable hydrogel composition formed containing the reaction mixture and xanthan can be used as a carrier suitable for cell encapsulation. The addition of xanthan also surprisingly results in much better storage stability of the hydrogel-cell composition, since the viability of cells within such a composition can be better preserved in the presence of xanthan.

[0036] According to a preferred embodiment of the present invention, the reaction mixture for producing the stable hydrogel composition further comprises cells.

[0037] According to a preferred embodiment of the present invention, the at least one polymerizable substrate is a sugar phosphate, preferably a hexose phosphate, more preferably glucose phosphate, even more preferably alpha-D-glucose-1-phosphate.

[0038] The use of sugars as polymerizable substrates in the method of the present invention is particularly preferred. Sugars are known components of carbohydrate-based hydrogels. Sugar monomers can be linked to each other by using corresponding enzymes that allow the sugar monomers to be polymerized. Bottom-up synthesis from monomers to oligomers and polymers has proven advantageous because it allows cells to be embedded in the polymer matrix. Since most enzymes that allow the polymerization of sugar monomers require phosphorylated sugars, the use of sugar phosphates as monomers is particularly preferred.

[0039] When at least one polymerizable substrate is a sugar phosphate, such as alpha-D-glucose-1-phosphate, sucrose can be added to the reaction mixture in step a) or used in a separate reaction step to form alpha-D-glucose-1-phosphate, as described by Zhong and Nidetzky (Biotechnol. J. 2020, 15, 1900349). For example, sucrose phosphorylase catalyzes the formation of alpha-D-glucose-1-phosphate and fructose from sucrose and phosphate ions.

[0040] According to another preferred embodiment of the present invention, alpha-D-glucose-1-phosphate is formed by incubating sucrose with sucrose phosphorylase. The use of sucrose phosphorylase to produce alpha-D-glucose-1-phosphate is particularly advantageous, for example, because sucrose is much more economical than alpha-D-glucose-1-phosphate. The use of sucrose as a polymerizable substrate avoids the use of alpha-D-glucose-1-phosphate, making the production of stable hydrogel compositions much more cost-effective.

[0041] The stable hydrogel composition of the present invention can also be prepared in a one-pot reaction using sucrose phosphorylase, phosphate ions, and sucrose as substrates. Thus, the reaction mixture of the present method can include at least one polymerizable substrate, followed by at least one enzyme capable of polymerizing the at least one substrate sucrose phosphorylase, phosphate ions, and sucrose. It has been found that the components of such a reaction mixture do not adversely affect each other.

[0042] "One-pot reaction," as used herein, refers to a strategy for improving the efficiency of chemical reactions by subjecting the reactants to only one reaction mixture. This is highly desirable because one-pot reactions can avoid lengthy separation processes and can increase production yields while saving time and resources through purification of intermediate compounds.

[0043] According to another preferred embodiment, the reaction mixture comprises at least one further monosaccharide, oligosaccharide, and / or polysaccharide, preferably at least one further oligosaccharide and / or polysaccharide.

[0044] The polymerizable substrate to be used in the method of the present invention is preferably a sugar monomer, however, to increase the rate of polymerization, it is possible to add at least one further monosaccharide, oligosaccharide, and / or polysaccharide to which the sugar monomer can be attached by the enzyme used.

[0045] According to another preferred embodiment, at least one additional monosaccharide is glucose, and / or at least one additional oligosaccharide is a disaccharide, preferably cellobiose.Cellobiose can be obtained, for example, by enzymatic or acidic hydrolysis of cellulose and cellulose-rich materials, such as cotton, jute, or paper.It has been found that the construction of cellobiose chains by controlled polymerization provides excellent tailorability and control of the final chemical structure.

[0046] Depending on the reaction conditions, which result in different degrees of polymerization (DP), long chain soluble oligosaccharides or insoluble materials can be formed.

[0047] According to a preferred embodiment of the present invention, the reaction mixture comprises 10 to 500 mM, preferably 15 to 400 mM, more preferably 20 to 300 mM of said at least one polymerizable substrate and / or said at least one further monosaccharide, oligosaccharide, or polysaccharide.

[0048] According to a further preferred embodiment of the present invention, a mixture of α-D-glucose-1-phosphate (alpha-D-glucose-1-phosphate) and cellobiose is used as the polymerizable substrate. Cellobiose can be glycosylated by beta-1,4-glycosylation using α-D-glucose-1-phosphate as the donor substrate. The donor substrate, α-D-glucose-1-phosphate, can be efficiently made available for an additional phosphorolysis reaction by an enzyme using starch or sucrose as the substrate.

[0049] It has been found that the degree of polymerization can depend on the molar ratio of cellobiose acceptor to alpha-D-glucose-1-phosphate donor used in the reaction. Efficient synthesis of hydrogels requires good control over the DP of the resulting product. Therefore, the reaction mixture can contain alpha-D-glucose-1-phosphate and cellobiose as polymerizable substrates in a molar ratio of preferably 20:1 to 1:1, more preferably 10:1 to 2:1, and even more preferably 8:1 to 5:1. These ratios, in particular, have been found to be advantageous for forming hydrogels capable of stabilizing cells embedded therein.

[0050] At least one phosphorylase can be used to initiate polymerization of the reaction mixture. Phosphorylases are enzymes that catalyze the addition of a phosphate group from an inorganic phosphate donor to an acceptor. Phosphorylase reactions typically involve minimal hydrolysis, resulting in the exclusion of reactions with water from the enzyme active site. Consequently, phosphorylases can also contribute to the synthetic construction of oligosaccharide or polysaccharide chains, which subsequently undergo oligomerization-induced self-assembly into hierarchically organized materials with tunable properties.

[0051] For this reason, the at least one enzyme making it possible to polymerize the above-mentioned substrates is preferably a phosphorylase, more preferably an oligosaccharide phosphorylase or a polysaccharide phosphorylase.

[0052] In particular, the enzymatic reaction of the method of the present invention is catalyzed by phosphorolysis of beta-1,4-glycosidically linked oligosaccharide substrates, resulting in polysaccharide chains constructed from hundreds or more linked glucosyl groups. Phosphorylase can act on beta-1,4-glycosidic linkages in oligosaccharide and polysaccharide substrates.

[0053] According to a preferred embodiment of the present invention, the at least one enzyme is cellodextrin phosphorylase.

[0054] For example, cellodextrin phosphorylase (EC 2.4.1.49) can reversibly catalyze the synthesis of cellulose and free phosphate (product) from alpha-D-glucose-1-phosphate and short-chain cellodextrins (substrates) by repeated beta-1,4-glycosylation. It is particularly preferred to use bacterial cellodextrin phosphorylase, preferably from Clostridia, more preferably from Clostridium cellulosi (e.g., GenBank ID CDZ24361.1).

[0055] According to another preferred embodiment of the present invention, the reaction mixture comprises 0.5 to 50 U / ml, preferably 0.5 to 30 U / ml, more preferably 1 to 20 U / ml, more preferably 1 to 10 U / ml of said at least one enzyme.

[0056] According to another preferred embodiment of the present invention, the reaction mixture contains at least one additional carbohydrate polymer, preferably in an amount of 0.1 to 1% by weight of the reaction mixture. The carbohydrate polymer is composed of long chains of carbohydrate monomers attached via glycosidic bonds. A carbohydrate polymer can be added to the reaction mixture of the present invention. Depending on the added carbohydrate polymer, the properties of the resulting polymer composition can be adjusted without the use of UV light or temperature changes.

[0057] According to another preferred embodiment of the present invention, the at least one further carbohydrate polymer comprises carboxylic acid, acetyl and / or sulfate groups.

[0058] To introduce additional properties into the hydrogel, modified carbohydrate polymers can be added to the reaction mixture. Modification of carboxylic acid groups can, for example, alter the hydrolysis resistance of the polymer as well as its thermal stability. Sulfate groups attached to sugar residues can contribute to the stabilization of the polymer composition by introducing terminal sulfate groups onto each polymer chain formed.

[0059] According to another preferred embodiment of the present invention, the at least one further carbohydrate polymer is xanthan.

[0060] Xanthan is a heteropolysaccharide primarily composed of D-mannose and D-glucose as major hexose units, along with D-glucuronic acid and pyruvic acid. Xanthan can chemically increase the viscosity of the liquid with which it is mixed. This ability, for example, makes xanthan gum a frequent ingredient in sauces and salad dressings in the food industry, as well as in cosmetics. Surprisingly, the addition of xanthan to the reaction mixture of the present invention has been found to improve the mechanical stability of the hydrogel and extend the viability of embedded cells. Furthermore, xanthan is an environmentally friendly ingredient and a surprising alternative to other chemical ingredients and stabilizers used in in situ hydrogel gelation, such as PEG.

[0061] The stable hydrogel composition formed is constructed from a polymeric matrix as a base that interacts with and / or associates with the xanthan.

[0062] According to another preferred embodiment of the present invention, the reaction mixture is obtained by combining the components of the reaction mixture in a buffer solution, preferably a phosphate buffered saline (PBS) buffer solution, preferably a buffer solution having a pH of 6.5 to 7.5. For example, an isotonic buffer solution having a neutral pH can be used as an optimal composition that maintains the enzymatic activity of phosphorylase and is non-toxic to most cells.

[0063] One surprising effect achievable by the methods of the present invention is the excellent viability of prokaryotic and eukaryotic cells embedded in the hydrogel composition. Moreover, cell overgrowth within the hydrogel may be limited, and viability remains stable over time. According to preferred embodiments of the present invention, the cells are bacterial, fungal, animal, or human cells.

[0064] According to another preferred embodiment of the present invention, the bacterial cell belongs to the order Lactobacillales, preferably the family Lactobacillaceae and / or the family Streptococcaceae, more preferably the genus Lactococcus and / or Lactobacillus.

[0065] According to another further preferred embodiment of the present invention, the bacterial cell is selected from the group consisting of Lactococcus lactis, preferably Lactococcus lactis 10123 (DSM34156), Lactobacillus rhamnosus.

[0066] According to another preferred embodiment of the present invention, the reaction mixture contains cells at a concentration of 0.5 to 50 mg / ml, preferably 0.5 to 40 mg / ml, more preferably 0.5 to 30 mg / ml, more preferably 1 to 20 mg / ml, more preferably 1 to 10 mg / ml. CFU / mg is 1.0 x 10 3 ~1.0×10 12 between 1.0 x 10 and 1.0 x 10 4 ~1.0×10 10 The CFU / mg may also depend on the type of cell and the purpose for which the hydrogel-cell composition is being used.

[0067] According to another preferred embodiment of the present invention, freeze-dried cells are added to the reaction mixture.

[0068] Lyophilization, also known as freeze-drying, is a process used to preserve biological materials by removing water from the sample. This typically involves the optional addition of a cryoprotectant (e.g., a sugar alcohol such as mannitol, sorbitol, or xylitol), followed by freezing the sample and then drying it at ultra-low temperatures under vacuum. The addition of lyophilized cells to the reaction mixture can extend the culture time and viability of embedded cells. Lyophilization can stabilize embedded cell cultures for long-term storage while minimizing damage that can occur from rigorously drying the sample.

[0069] According to another preferred embodiment of the present invention, the mixture of step a) is shaken in step b) at 50 to 1000 rpm, preferably 100 to 800 rpm, more preferably 200 to 600 rpm, more preferably 200 to 500 rpm. Shaking the mixture can contribute to uniform distribution of the components of the mixture.

[0070] Another aspect of the present invention relates to hydrogel-cell compositions obtainable by the above-described method. Such compositions with encapsulated and / or embedded cells exhibited long-term stability with live cells for at least 30, 60, or even 174 days at room temperature while retaining gel properties in the presence of cells. Therefore, the resulting compositions are a facile solution for maintaining the viability of encapsulated and / or embedded cells by improving their shelf life compared to other in-situ hydrogels.

[0071] According to a preferred embodiment of the present invention, the hydrogel-cell composition is contacted with a patch or wrap for topical application to body tissue, such as the skin, mucous membranes, or organ surfaces. Adhering the composition to the body as part of a patch or wrap is advantageous because it allows for controlled and constant administration of the hydrogel-cell composition.

[0072] The hydrogel-cell composition remains at the site of action until the patch / wrap is removed, at which point the patch also performs adhesive tasks such as wound closure. Administration of the composition via a patch or wrap optimizes the amount and rate at which the molecules are absorbed by the body and made available at the site of action. Furthermore, the patch or wrap may prevent premature dehydration of the hydrogel, e.g., harmful infection of the wound, and allows for easy and convenient self-administration of the composition. The patch or wrap does not require a specialist for placement; for example, it can be applied to the skin over an area as needed by the user. The structure of the patch or wrap can be configured to allow it to be worn directly on the user's skin and can be adapted depending on the required site of action.

[0073] According to another preferred embodiment of the present invention, the hydrogel-cell composition can be used in food production. The composition can contain microbial cells, such as bacteria or fungi, which can contribute to the production of dairy products such as cheese and yogurt, meat products such as salami, or food supplements. The role of microbial cells in food production is known in the art.

[0074] The hydrogel-cell compositions of the present invention may further be used to preserve food through the formation of inhibitory metabolites, such as organic acids (e.g., lactic acid, acetic acid, formic acid, or propionic acid) or ethanol. The hydrogel-cell compositions may help improve food safety through the inhibition of pathogens and the removal of toxic compounds. Furthermore, the compositions of the present invention can improve the nutritional value and organoleptic properties of foods.

[0075] According to another preferred embodiment of the present invention, the hydrogel-cell composition can be used in agriculture and / or animal husbandry. Additional compositions regularly used in the cultivation of plant crops can be mixed with the hydrogel. The composition can be selected from the group consisting of pesticides, herbicides, insecticides, molluscicides, fungicides, pheromones, and preferably fertilizers.

[0076] Another aspect of the present invention relates to a hydrogel-cell composition for use in treating skin and / or eye disorders or damage.

[0077] Due to the unique properties of the hydrogel-cell compositions of the present invention, which result in significantly extended viability of cells embedded in the hydrogel, the hydrogel-cell compositions of the present invention, or obtainable by the methods of the present invention, can be used to treat disorders or diseases related to the skin and / or eye. The hydrogel-cell compositions may be placed in direct contact with the skin and / or eye to promote the healing and / or tissue regeneration process. The cells present in the hydrogel-cell compositions are selected to have a therapeutic effect on the skin and / or eye. The respective cells are well known in the art.

[0078] Due to the mechanical and cytoprotective properties of the compositions of the present invention, the hydrogel-cell compositions can flexibly conform to the shape of the defect in the skin and / or eye, for example, enhancing proper wound closure and the tissue regeneration process due to the presence of cells or their corresponding secretions. After administration, the compositions of the present invention adapt to the shape of the defect (e.g., wound) during in situ polymerization, which accelerates the healing process.

[0079] According to another preferred embodiment of the present invention, the cells in the hydrogel-cell composition are selected from the group consisting of stem cells, induced pluripotent stem cells, macrophages, keratinocytes, endothelial cells, T lymphocytes, monocytes, oval cells, sperm, and combinations thereof. Some of these cell types are involved in wound healing and tissue remodeling and can be used to treat skin and / or eye disorders / diseases. Furthermore, the secretome of some of these cells can significantly contribute to wound healing and tissue repair mechanisms. Suitable secretomes include various growth factors (e.g., PDGF, TGF-β, β-FGF) and cytokines (e.g., NF-α, interleukin-1 (IL-1), and IL-6) known to facilitate skin and / or eye healing.

[0080] According to another preferred embodiment of the present invention, the hydrogel-cell composition can be administered topically onto or into the skin or eye and / or by injection, preferably via subcutaneous or intravitreal injection.

[0081] The present invention can be used to treat skin and / or eye disorders or damage.

[0082] The term "injury," as used herein, refers to physical trauma suffered by multiple layers of skin, eye, or epithelial tissue, which may be in the form of a cut, impact, burn, scratch, and / or abrasion.

[0083] According to another preferred embodiment of the present invention, the skin and / or eye disorder or injury is selected from the group consisting of acne, alopecia areata, atopic dermatitis (eczema), psoriasis, Raynaud's phenomenon, rosacea, vitiligo, acute wounds, chronic wounds, corneal abrasions, glaucoma, cataracts, burns and / or irritations to the skin and / or eyes, actinic keratosis, melanoma, sun damage or aging of the skin, and combinations thereof.

[0084] Another aspect of the present invention relates to a method for use in treating skin and / or eye disorders or damage.

[0085] According to another preferred embodiment of the present invention, the composition may contain Lactococcus lactis 10123. The composition of the present invention has shown a surprising inhibitory effect against pathogens, particularly Staphylococcus pathogens such as S. aureus. Furthermore, the composition can be mixed with a reaction mixture and at least one enzyme. The strain Lactococcus lactis 10123 has been found to be suitable for embedding in the hydrogel composition obtained as described above.

[0086] Another aspect of the present invention relates to compositions obtainable by the method according to the present invention, which may be effective against pathogenic bacteria such as S. aureus and may be used as an alternative to antibiotics in the food industry, agricultural industry, medicine, and cosmetics.

[0087] The compositions of the present invention can be used to treat or prevent skin infections caused by various pathogens, particularly bacterial or fungal pathogens. Surprisingly, it has been found that compositions obtainable by the methods of the present invention and containing, for example, Lactococcus lactis 10123, can be effectively used to inhibit or prevent the growth of pathogens on skin and / or wounds. Furthermore, the compositions of the present invention can be used to strengthen the barrier function of the skin and its mucous membranes, and to induce anti-inflammatory behavior in human cells by altering gene expression and cytokine secretion. For example, the relatively low pH due to the presence of Lactobacillus not only promotes the skin's natural, protective acidic environment, but also inhibits the growth of harmful bacteria, viruses, and fungi. These properties make the compositions of the present invention ideal for use as hygiene products, preferably as thorough hygiene products. Many women experience problems with bladder infections, especially after being treated with broad-spectrum antibiotics. Because this treatment regimen kills many different bacteria, it also affects the natural intestinal and vaginal flora, resulting in bacterial decolonization of the vaginal, rectal, and perineal mucosa. These bacteria can cause changes in the pH value within the mucosa, thereby weakening the natural defenses against pathogens. The compositions of the present invention may also have antiviral and / or antitumor effects, thus preventing the formation of tumors caused by viruses such as human papillomavirus. The compositions of the present invention can prevent the formation of carcinomas in the skin, eyes, and / or mucosa. After topical application of the composition, the positive influence of Lactobacilli on the tumor microbiota can prevent the development of precancerous conditions. Furthermore, the compositions can be used to inhibit the proliferation of viruses that lead to abnormal cell growth that causes precancerous lesions, cancer, or genital warts. The compositions of the present invention can also contribute to the equilibration of the natural skin and mucosal flora by maintaining physiological pH and inhibiting the growth of harmful bacteria and fungi.

[0088] According to another preferred embodiment of the present invention, the composition may be in a form suitable for topical application, such as an ointment, cream, gel, lotion, powder, or emulsion. Emulsions may be prepared by a high shear mixing process from at least two phases, such as a hydrophilic (aqueous) and a lipophilic (oily) phase.

[0089] Surprisingly, it has been found that compositions of the invention in emulsion form can be prepared by using ingredients that have been experimentally shown to be compatible with bacteria such as Lactococcus lactis 10123. Compatibility is measured by stirring a lyophilized sample of L. lactis into each ingredient and measuring the CFU per ml or per gram on blood agar after 7 days.

[0090] According to a preferred embodiment of the present invention, the emulsion comprises: a) providing an oil phase and a water phase by separately mixing and heating each phase; b) mixing the phases, and c) It can be prepared by cooling the mixture.

[0091] The first step involves mixing and heating an oil phase containing, for example, shea butter and / or sunflower seed oil. In the second step, an aqueous phase containing purified water, salt, and a gelling agent such as xanthan gum, as well as an emulsifier such as lecithin, is prepared and heated. In the third step, the two phases are mixed under high shear and cooling. A stable formulation can be produced that can be carefully mixed with the stable hydrogel composition of the present invention. The hydrogel has been shown to be mechanically stable for at least three months when mixed with the emulsion of the present invention.

[0092] According to another preferred embodiment of the present invention, additional active ingredients can be added to the emulsion. Active ingredients can include ingredients that serve as a food source for bacteria, such as ceramides, glucose, or buffers and acids, such as lactic acid. The compositions of the present invention can be applied to the skin, as well as the mouth, buccal cavity, vagina, or rectum, or to areas of the body that are operated on. The compositions of the present invention can be used preventively or curatively for a variety of conditions.

[0093] According to a further preferred embodiment of the present invention, the composition in the form of an ointment, cream, gel, lotion, powder, or emulsion can be contacted with a patch or wrap for topical application to the skin. As part of a patch or wrap, the composition can be applied to the skin where delivery of the microorganisms to the target area of ​​the skin is intended. The use of a patch or wrap may further prolong the survival of the bacteria during application by preventing dehydration and / or infection.

[0094] Another aspect of the present invention relates to a kit for producing a stable hydrogel-cell composition. The kit can be a multi-component system including at least one polymerizable substrate and at least one enzyme capable of polymerizing the at least one substrate described herein. After mixing the above-described components, the hydrogel composition of the present invention can be obtained.

[0095] According to another preferred embodiment, the at least one polymerizable substrate is as defined above.

[0096] According to another preferred embodiment, the at least one enzyme is as defined above.

[0097] According to another further preferred embodiment, the kit further comprises cells, in particular the cells described above. The cells may be lyophilized and added to the other components of the kit.

[0098] example Example 1: Enzymatic synthesis of hydrogels The enzyme used for hydrogel synthesis was cellodextrin phosphorylase (CdP, EC 2.4.1.49). This enzyme catalyzes the iterative β-1,4-glycosylation of cellobiose using αGlc 1-P as the donor substrate (Figure 1). In this experiment, CdP from Clostridium cellulosi (GenBank ID CDZ24361.1) was used. It was recombinantly expressed in Escherichia coli BL21(DE3) containing a plasmid vector (pET-21b(+)) carrying a codon-optimized gene. The gene contained N-terminal NdeI and C-terminal XhoI restriction sites for subcloning into pET-21b(+). Enzyme expression in E. coli was induced with isopropyl β-D-1-thiogalactopyranoside (0.25 mM) overnight at 18 °C. The enzyme was then purified using a pre-packed (1.6 cm x 2.5 cm, 5 mL) HisTrap FF crude column (GE Healthcare Europe, Vienna, Austria) on an AKTA prime plus (GE Healthcare Europe). The N-terminal His-tagged protein was eluted with imidazole (0.01–0.3 M). The purified protein was desalted using phosphate buffer (50 mM, pH 7.0) with Vivaspin Turbo 50 kDa cutoff tubing (Sartorius Stedim, Vienna, Austria). The purified enzyme solution was sterilized using a 0.22 μm syringe filter before use.

[0099] The synthesis reaction (total volume 0.5 mL) was tested at 37 and 45 °C on a ThermoMixer C (Eppendorf, Vienna, Austria), with 37 °C being preferred. The reaction solution (RS) was prepared in phosphate buffer solution (PBS, 10 mM, pH 7.0) containing 200 mM αGlc 1-P, 25 mM cellobiose, and CdP at concentrations of 0.1–0.8 mg / mL (i.e., 1–10 U / mL). The reaction mixture was incubated for 1–5 hours to obtain stable hydrogels.

[0100] Example 2: Enzymatic synthesis of hydrogel with xanthan Using xanthan concentrations of 0.2–0.5% (m / v) as an additive in the RS described above, homogeneous whitish hydrogels were obtained after 2 h of reaction at 37°C (Figure 2). Under these conditions, the conversion yield of donor αGlc 1-P was approximately 35–55 mol.%. A control reaction performed under identical conditions but lacking xanthan for 4 h prepared a "cellulose-only" gel, and the overall mixture exhibited poor mechanical stability (control in Figure 2). Therefore, these results suggest a role for the polymer additive in gelation. The presence of xanthan induced a macromolecular crowding effect at high concentrations (above 0.25%), as shown in Figure 2. Furthermore, the gelation properties of the thus obtained hydrogel (containing 0.4% xanthan) were confirmed by rheological means using a strain-controlled rheometer (MCR 502, Anton Paar, Austria) at 25 °C with a cone-and-plate measurement geometry (CP 50-1) with a diameter of 50 mm and a cone face of 1°. The linear viscoelastic range was measured using a strain sweep (0.01–100%) at a fixed frequency of 10 rad / s. The results of the amplitude sweep were presented by plotting the strain on the x-axis and the storage modulus G' and loss modulus G" on the y-axis (Figure 3). The linear viscoelastic region (LVE region) was determined (approximately 0.1%), at which the curve of the G' function was constant, indicating that testing could be performed without destroying the sample structure. The prepared hydrogel showed a G'>G" relationship in the LVE region, confirming that a gel-like structure had been established in the hydrogel.

[0101] Example 3: Encapsulation of cells in hydrogels To encapsulate cells (Lactobacillus and Lactococcus strains were tested) in the hydrogel, lyophilized cells were presuspended in sterile RS (0.5 mL) at a cell concentration of 1–10 mg / mL. The reaction was then carried out on a ThermoMixer C (Eppendorf, Vienna, Austria) at 37 °C and 300 rpm for 3 h. Cellulose-xanthan hydrogels encapsulating bacteria were obtained. The overall gel properties were maintained in the presence of encapsulated cells. Confocal laser scanning microscopy was used to visualize the encapsulated cells (living L. rhamnosus cells with green fluorescence), revealing their distribution within the hydrogel (Figure 4). Furthermore, encapsulated L. rhamnosus cells in cellulose hydrogels without xanthan ("cellulose only") demonstrated long-term storage stability, with viable cell loss of less than 1 log(cfu / mL) within 90 days and greater than 3 log(cfu / mL) after 90 days of storage at room temperature. Encapsulated L. rhamnosus cells in cellulose-xanthan hydrogels demonstrated long-term storage stability, with viable cell loss of less than 1 log(cfu / mL) within 60 days and less than 2 log(cfu / mL) after 90 days of storage at room temperature (Figure 5a). In comparison, cells in xanthan solutions (containing xanthan as the sole polysaccharide at concentrations of 0.1–0.5%) demonstrated greater than 6 log(cfu / mL) cell loss, as shown in Figure 5b. Overall, significant cytoprotective effects were achieved by encapsulating cells in cellulose hydrogels as well as in cellulose-xanthan hydrogels.

[0102] Example 4: Hydrogel encapsulation to maintain cell (L. rhamnosus and L. lactis) viability To test the ability of enzymatically prepared hydrogels to maintain cell viability, two different bacterial strains (L. rhamnosus and L. lactis) were encapsulated. The number of viable bacteria was assessed after predetermined time points. To measure the number of viable bacteria, 100 mg of gel was diluted with PBS and plated on agar plates at each time point. The results show a gradual loss of viable cells in the gel over its shelf life. Within 174 days, only about 2 logs were lost (Figure 6a). Compared to a known hydrogel (containing xanthan as the only polysaccharide), the stability of the hydrogel of the present invention (cellulose-xanthan hydrogel) was significantly increased (Figure 6b).

[0103] Another parameter measured was optical stability. The gel remained as a white, opaque mass at room temperature for at least 6 months. The addition of xanthan also resulted in good mechanical stability. This was tested by rotating the tube. The gel did not leak for at least 6 months. For Lb. rhamnosus, the odor was pungent for the first few days, but after a few weeks, a sour, yogurt-like odor developed. The odor of L. lactis was more neutral. The pH was also measured using pH strips. A gradual (within 4-6 months) decrease in pH from 7.5 to 4.5 was observed.

[0104] Example 5: Strain of L. lactis 10123 inhibits S. aureus L. lactis strains were isolated from raw milk in Graz, Styria. 5 The plates were inoculated with 100 μL of an overnight culture (ONC) of L. lactis. 8ONC was poured onto agar plates inoculated with swabs soaked in PBS containing CFU / ml S. aureus and a central well. After 24 hours of incubation at 37°C, zones of inhibition were visible for S. aureus (Figure 7), as well as MRSA, S. hominis, and E. hirae. No zones of inhibition were visible when cell-free supernatant of L. lactis ONC was used.

[0105] In another series of experiments, the ability to inhibit S. aureus was also tested against ONC using 1:1 MRS and LB-medium at 37°C for 24 hours. 6 CFU of S. aureus and 10 6 The culture density was adjusted relative to a standard of 0.5 MFU (McFarland units). Lb. rhamnosus was used as a co-culture material with S. aureus in the ONC. The CFU at the start of the experiment was verified by counting colonies on the plates and was at least 10 5 As shown in Figure 8, after 24 hours, no S. aureus was found in the co-culture, but 10 CFU / ml was detected in the S. aureus monoculture. 8 It is noted that greater than CFU / ml of S. aureus was shown. As also shown in the literature, the other test strains resulted in less than a 2 log reduction (compared to over an 8 log reduction with L. lactis 10123).

[0106] Example 6: Functionality of strain 10123 after encapsulation To investigate the maintenance of functionality of L. lactis strain 10123 after storage for 1 month in xanthan-containing hydrogels at room temperature, 10 6 Inoculate 10 CFU of S. aureus onto one side of the plate.7 A 100µm drop of gel containing 100 CFU / ml of L. lactis was applied. The gel itself was also applied to S. aureus. The results show that S. aureus colonization was reduced to the right of the gel application (Figure 9a), and an inhibition zone was visible where the gel droplet was applied directly (Figure 9b).

[0107] The functionality of the 10123 strain was also confirmed in a pig skin model (dermis). A marked spot (left) was inoculated with S. aureus, and another marked spot (right) was inoculated with S. aureus and L. lactis. After 24 hours of incubation at 37°C, visible differences could be observed between the two spots (Figure 10).

[0108] Example 7: Comparison with neutralization-induced self-assembly of cellulose oligomers The method of the present invention was compared with the method published by Serizawa et al. (ACS Macro Lett. 2020, 9, 3, 301-3059). The synthesis of cellulose oligomers was carried out as described by Serizawa et al. (See "Enzymatic Synthesis of Cellulose Oligomers" in the supplementary information).

[0109] As described by Serizawa et al., 4.5 mg of lyophilized cellulose oligomer was dissolved in 27.5 μL of 1 M NaOH solution and neutralized with 423 μL of 65 mM HCl solution. Immediately after, 200 μL of the neutralized solution was mixed with 200 μL of a cell suspension in PBS (for L. rhamnosus), and the resulting solution was transferred to a 96-well plate. The final concentration of cellulose oligomer was set to 0.5% (w / v), and the cell density was adjusted to 1.50 × 10 cells. 10 cells / mL (Method 1).

[0110] In a comparative example, cellulose oligomer synthesis and cell encapsulation were carried out according to the present invention (Method 2). The reaction solution was prepared as defined in Example 1. Cells (L. rhamnosus) were mixed with the reaction solution and 1.50 × 10 10 The cell density in cells / mL was obtained. The reaction was then carried out on a ThermoMixer C (Eppendorf, Austria) at 37°C with agitation at 300 rpm for 3 hours.

[0111] [Table 1]

[0112] From the results in Table 1, it can be seen that the method according to the present invention (Method 2) significantly increases the long-term storage stability of encapsulated cells compared to the literature method (Method 1). These findings suggest that the bottom-up synthesis of hydrogels combined with cells has a positive effect on cell viability compared to the method described by Serizawa et al.

Claims

1. 1. A method for producing a stable hydrogel-cell composition, comprising: a) providing a reaction mixture comprising at least one polymerizable substrate and at least one enzyme that allows said at least one substrate to be polymerized, and cells; b) incubating the mixture of step a) to form a stable hydrogel-cell composition; A method comprising:

2. 1. A method for producing a stable hydrogel composition, comprising: a) providing a reaction mixture comprising at least one polymerizable substrate and at least one enzyme that allows said at least one substrate to be polymerized, and xanthan; b) incubating the mixture of step a) to form a stable hydrogel composition; A method comprising:

3. The method of claim 2 , wherein the reaction mixture further comprises cells.

4. The method according to any one of claims 1 to 3, wherein the at least one polymerizable substrate is a sugar phosphate, preferably a hexose phosphate, more preferably glucose phosphate, even more preferably alpha-D-glucose-1-phosphate.

5. 5. The method of claim 4, wherein the alpha-D-glucose-1-phosphate is formed by incubating sucrose with sucrose phosphorylase.

6. The method of any one of claims 1 to 5, wherein the reaction mixture comprises at least one further monosaccharide, oligosaccharide, and / or polysaccharide.

7. 7. The method of claim 6, wherein the at least one further monosaccharide is glucose and / or the at least one further oligosaccharide is a disaccharide, preferably cellobiose.

8. 8. The method according to any one of claims 1 to 7, wherein the reaction mixture comprises 10 to 500 mM, preferably 15 to 400 mM, more preferably 20 to 300 mM of the at least one polymerizable substrate and / or the at least one further oligo- or polysaccharide.

9. 9. The method according to any one of claims 4 to 8, wherein the reaction mixture comprises alpha-D-glucose-1-phosphate and cellobiose as polymerizable substrate, preferably in a molar ratio of 20:1 to 1:1, preferably 10:1 to 2:1, more preferably 8:1 to 5:

1.

10. The method according to any one of claims 1 to 9, wherein the at least one enzyme is a phosphorylase, more preferably an oligosaccharide phosphorylase or a polysaccharide phosphorylase.

11. The method according to any one of claims 1 to 10, wherein the at least one enzyme is cellodextrin phosphorylase.

12. 12. The method according to any one of claims 1 to 11, wherein the reaction mixture comprises 0.5 to 50 U / ml, preferably 0.5 to 30 U / ml, more preferably 1 to 20 U / ml, more preferably 1 to 10 U / ml of the at least one enzyme.

13. 13. The method of any one of claims 1 to 12, wherein the reaction mixture comprises at least one further carbohydrate polymer, preferably in an amount of 0.1 to 1% by weight of the reaction mixture, and wherein the at least one further carbohydrate polymer is preferably xanthan.

14. 14. The method of claim 13, wherein the at least one carbohydrate polymer comprises a carboxylic acid group, an acetyl group, and / or a sulfate group.

15. 15. The method according to any one of claims 1 to 14, wherein the reaction mixture is obtained by combining the components of the reaction mixture in a buffer solution, preferably in a phosphate buffered saline (PBS) buffer solution, preferably in a buffer solution having a pH of 6.5 to 7.

5.

16. The method of any one of claims 1 to 15, wherein the cell is a bacterial cell, a fungal cell, an animal cell, or a human cell.

17. 17. The method of claim 16, wherein the bacterial cell belongs to the order Lactobacillales, preferably the family Lactobacillaceae and / or the family Streptococcus, more preferably the genus Lactococcus and / or Lactobacillus, and the bacterial cell is preferably selected from the group consisting of Lactococcus lactis, preferably Lactococcus lactis 10123 (DSM 34156), and Lactobacillus rhamnosus.

18. 18. The method of any one of claims 1 to 17, wherein the reaction mixture comprises the cells at a concentration of 0.5 to 50 mg / ml, preferably 0.5 to 40 mg / ml, more preferably 0.5 to 30 mg / ml, more preferably 1 to 20 mg / ml, more preferably 1 to 10 mg / ml.

19. The method of any one of claims 1 to 18, wherein freeze-dried cells are added to the reaction mixture.

20. 20. The method according to any one of the preceding claims, wherein the mixture of step a) is shaken in step b) at 50 to 1000 rpm, preferably 100 to 800 rpm, more preferably 200 to 600 rpm, more preferably 200 to 500 rpm.

21. A hydrogel-cell composition obtainable by the method according to any one of claims 1 to 20.

22. Lactococcus lactis strain 10123 deposited at the DSMZ on February 4, 2022 under accession number DSM 34156.

23. 13. A kit for producing a stable hydrogel-cell composition, comprising at least one polymerizable substrate and at least one enzyme making it possible to polymerize said at least one substrate, wherein said at least one polymerizable substrate is preferably a substrate as defined in any one of claims 4 to 9, and said at least one enzyme is preferably an enzyme as defined in any one of claims 10 to 12.

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