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 the 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 Art

[0002] Three-dimensional (3D) systems containing cell cultures have found widespread applications in the biomedical field, such as cell spheroids for studying tumor behavior and cell encapsulation for cell-based transplantation therapies. Promising cell encapsulation materials include hydrogels, soft and wet biomaterials with tunable properties, because they mimic the native extracellular matrix (i.e., the aqueous environment), have excellent mass transfer properties, and display tissue-like elasticity. These properties allow cell growth and protect cells from environmental hazards, thereby maintaining cell activity over a longer period of time.

[0003] Various types of hydrogels for cell encapsulation have been reported in the art. Known hydrogels usually contain proteins of animal or plant origin, such as gelatin or gluten. However, all of these materials show relatively low mechanical flexibility and therefore cannot be used for processing. Known hydrogels may also contain substances that are 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, carboxymethyl cellulose) have been developed for cell encapsulation. Known methods for producing such hydrogels generally involve initial cellulose dissolution 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 neutralization-induced self-assembly of cellulose oligomers into crystalline nanoribbon networks to produce physically cross-linked hydrogels for 3D cell culture.

[0006] Another example is disclosed in EP4053141. It describes a cell culture medium composition for suspended cells, which comprises cellulose oligomers. For example, the culture medium composition can be added to HeLa cells.

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

[0008] Another example is disclosed in JP2017201895, which involves a synthetic cellulose three-dimensional structure with gelatin. Animal cells can be added to the structure and grown on its surface.

[0009] Furthermore, JP2018145216 describes an artificially synthesized three-dimensional cellulose structure used as a scaffold for animal cells.

[0010] EP0928795 describes a method for preparing a natural organic polymer, which is used as a gelling agent for preparing hydrogels.

[0011] However, these methods are complex and extremely inefficient. In addition, in order to obtain the desired physicochemical properties, the cellulose material must be modified or combined with other materials. The chemical methods used to cross-link cellulose involve toxic chemical cross-linkers and catalysts, such as 2,2,6,6-tetramethylpiperdine-1-oxylradicals. These toxic chemicals have a negative impact on the viability of cells encapsulated by such cellulose materials.

[0012] Disadvantages of hydrogels known in the art have so far hampered the development of stable hydrogel-cell compositions. It is therefore an object of the present invention to provide means and methods that overcome the disadvantages of the prior art in order to produce stable and biocompatible hydrogel-cell compositions that can be used, for example, in tissue engineering and biomedical applications such as bioprinting. Summary of the invention

[0013] Therefore, the present invention relates to a method for producing a stable hydrogel-cell composition, comprising the following steps:

[0014] a) providing a reaction mixture comprising at least one polymerizable substrate and at least one enzyme capable of polymerizing the at least one substrate and cells, and

[0015] b) incubating the mixture of step a) to form a stable hydrogel-cell composition.

[0016] Surprisingly, the results demonstrate that cells can be stabilized in the hydrogel if the hydrogel is enzymatically synthesized in the presence of cells. The method of the present invention is a bottom-up synthesis method from a polymerizable substrate that is capable of forming a polymer network after mixing with at least one polymerase and cells. Thus, the polymer network of the hydrogel-cell composition of the present invention is constructed by enzymatic polymerization of at least one polymerizable substrate in the presence of cells. The bottom-up strategy for synthesizing the hydrogel of the present invention allows cells embedded in the hydrogel to be stabilized for a longer period of time compared to methods involving a step of simply contacting the hydrogel with the cells.

[0017] The method of the present invention allows the further formation of a mechanically stable hydrogel with excellent biocompatibility during the entire process. Due to the in situ gelation caused by the enzyme reaction, the cells can be embedded / encapsulated in the hydrogel, forming more uniformly and more efficiently. As mentioned above, this embedding / encapsulation method significantly prolongs the viability of the cells contained in the hydrogel.

[0018] The use of polymerizable substrates and enzymes to form the hydrogel is also advantageous because it can produce a biocompatible hydrogel matrix that facilitates cells embedded / encapsulated in the hydrogel and potential medical uses of the hydrogel-cell compositions of the present invention.

[0019] Thus, the method of preparing hydrogels of the present invention is well suited for cell embedding / encapsulation in view of: a) obtaining uniform distribution of cells within the hydrogel due to in situ gelation and encapsulation by premixing the cells in the aqueous reaction solution, b) mild conditions for gel synthesis, which maintain cell viability during encapsulation, and c) biocompatible and eco-friendly materials used in the hydrogel composition, making this system suitable for both human and animal based applications. Furthermore, the method of the present invention is a rapid, simple and cost-effective method that does not require specialized equipment and enables people without specialized knowledge or laboratory equipment to use the technology (e.g., in pre-formulated kits).

[0020] The hydrogels obtainable by the method of the present invention can also be used for tissue engineering. Among them, the hydrogels take on the mechanical and other tasks of the extracellular matrix. When the growing cells secrete their own extracellular matrix, the water-insoluble parts can be enzymatically decomposed. Since the degradation process must be controllable, for example, cellulose fibers are particularly suitable for cells, preferably eukaryotic cells lacking their own cellulase.

[0021] Another aspect of the present invention relates to a method for producing a stable hydrogel composition, comprising the steps of:

[0022] a) providing a reaction mixture comprising at least one polymerizable substrate and at least one enzyme capable of polymerizing the at least one substrate and xanthan gum, and

[0023] b) incubating the mixture of step a) to form a stable hydrogel composition.

[0024] It has been shown that the addition of xanthan gum to the reaction mixture used in the inventive method results in the synthesis of a stable hydrogel, which has excellent mechanical properties compared to the hydrogel composition comprising other added polymers (e.g., based on a polysaccharide). In addition, it has been shown that the hydrogel produced by the method of the present invention maintains its mechanical properties over a longer period of time compared to other hydrogels, particularly compared to the hydrogel comprising other polymers (e.g., other polysaccharides). Xanthan gum is added to the hydrogel produced by the method of the present invention so that the hydrogel has further mechanical stability. The mechanical stability of this enhanced hydrogel combination significantly increases the range of use of the combination. Another advantage of the hydrogel comprising xanthan gum is its biocompatibility, which allows more effective stabilization of the biomolecules (e.g., enzymes) or cells embedded therein compared to other additives. Compared to the hydrogel without xanthan gum, this makes the product have a longer shelf life, because in the hydrogel combination, biological and enzymatic activity retains a longer period of time.

[0025] Another aspect of the invention relates to a hydrogel-cell composition obtainable or obtained by the method of the invention.

[0026] Another aspect of the present invention relates to Lactococcus lactis strain 10123, which was deposited in DSMZ on February 4, 2022 with the deposit number DSM 34156.

[0027] Lactococcus lactis strain 10123 exhibits surprising inhibitory properties against various bacterial species of the genus Staphylococcus, such as, for example, S. aureus, S. hominis and E. hirae.

[0028] Lactococcus lactis is a well-known probiotic organism that has been widely used for different purposes, such as cheese production, for thousands of years. Lactococcus lactis is also known for bacteriocin production (such as the lantibiotic nisin), which can be used as an inhibitor against certain types of other pathogenic bacteria (such as bacteria of the genus Staphylococcus). However, ordinary Lactococcus lactis bacteria only release a limited number of antimicrobial active peptides, which cause inhibition in direct confrontation with other bacteria. Therefore, surprisingly, Lactococcus lactis strain 10123 shows excellent antibiotic properties against many microorganisms compared to known Lactococcus lactis strains.

[0029] The strains of the present invention have been shown to be surprisingly effective against antibiotic-resistant microorganisms as well. Antibiotic-resistant microorganisms, particularly bacteria, are rapidly emerging worldwide, jeopardizing the efficacy of antibiotics, which 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, it is very important to provide means and methods to combat these antibiotic-resistant microorganisms. Lactococcus lactis strain 10123 has been shown to be able to prevent and / or inhibit the growth of antibiotic-resistant microorganisms.

[0030] In particular, Staphylococcus aureus and its antibiotic-resistant form, methicillin-resistant Staphylococcus aureus (MRSA), are a human pathogen of concern and are the cause of, for example, skin infections, respiratory infections and food poisoning. For example, in different studies, elimination of Staphylococcus aureus disorders (a serious skin disease) has a very good improvement by antibiotics. The use of the strains of the invention in systemic and topical medicines may be a useful application in combating the spread of multidrug-resistant bacteria.

[0031] Another preferred application of the strains of the present invention relates to cosmetics. So far, cosmetics mainly use prebiotics (beneficial skin flora) or postbiotics (lysates or bacterial metabolites) agents. In addition, bacterial components or dead bacteria have immunomodulatory effects. However, the use of live bacteria brings additional benefits. This is particularly important in the direct inhibition of pathogens (such as Candida albicans (C.albicans) or Staphylococcus aureus).

[0032] Lactococcus lactis strain 10123 produces sufficient amounts of antimicrobial active substances, so that the strain can also be used as a food additive (eg, as a preservative). Another aspect of the present invention relates to a composition comprising Lactococcus lactis 10123.

[0033] 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 capable of polymerizing the at least one substrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Shown is a scheme for the β-1,4-glycosylation of cellobiose catalyzed by cellodextrin phosphorylase (CdP) using α-D-glucose-1-phosphate (αGlC1P) as a donor.

[0035] Figure 2Reaction mixtures containing different xanthan gum concentrations (0-0.5%, m / v) are shown. 25 mM cellobiose, 200 mM Glc1-P, 0.5 mg / mL cellodextrin phosphorylase (CdP) were used in 10 mM phosphate buffer (pH 7.0) at 37°C for 3 hours.

[0036] Figure 3 Shown is a curve of amplitude sweep analysis of a synthetic cellulose-hydrogel containing 0.4% xanthan gum.

[0037] Figure 4 Confocal laser scanning microscopy images of living cells (white) encapsulated in the hydrogel are shown.

[0038] Figure 5a The CFU of bacteria (Lactobacillus rhamnosus, Lb. rhamnosus) in the gel during storage time are shown.

[0039] Figure 5b Bacterial survival (Lactobacillus rhamnosus) in a standard hydrogel (xanthan gum only) over storage time is shown.

[0040] Figure 6a Shown are the CFU of bacteria (Lactococcus lactis) in the gel over storage time.

[0041] Figure 6b A comparative analysis of cell survival between Lactobacillus rhamnosus embedded in a standard hydrogel (xanthan gum) and Lactococcus lactis (L. lactis) embedded in a xanthan gum-containing hydrogel according to the present invention is shown.

[0042] Figure 7 Shown are the inhibition zones on Müller-Hinton agar inoculated with S. aureus.

[0043] Figure 8 Shown is the inhibitory effect of a co-culture of S. aureus with L. lactis 10123 compared to a monoculture of S. aureus and a co-culture of S. aureus with L. rhamnosus after 24 hours of incubation in the absence of L. lactis 10123.

[0044] Figure 9a Shown is a blood agar plate inoculated with 10Λ6 CFU of Staphylococcus aureus with 10Λ7 CFU / ml of Lactococcus lactis inoculated on top.

[0045] Figure 9b Shown are the inhibition zones of blood agar plates where drops of hydrogel containing Lactococcus lactis were applied directly onto the blood agar plates.

[0046] Fig.10 Shown are porcine skin models inoculated with S. aureus (left spot) and S. aureus and L. lactis (right spot). DETAILED DESCRIPTION

[0047] As used herein, "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. Starting from contacting at least one polymerizable substrate and at least one enzyme with the cells, a stable hydrogel-cell composition can be formed within 1 to 8 hours, preferably within 1 to 5 hours. With respect to the composition, "stable" means that the physicochemical properties of the hydrogel remain substantially unchanged at a temperature of 2°C to 30°C, preferably 4°C to 25°C, for at least 30 days, preferably at least 60 days, and more preferably at least 180 days, even in the presence of cells embedded and / or encapsulated in the hydrogel. In addition, "stable" also refers to the viability of the cells encapsulated and / or embedded in the hydrogel and maintains their functions.

[0048] Using the method of the present invention, a hydrogel-cell composition can be produced, wherein the cells embedded and / or encapsulated therein survive at least 30 days, preferably at least 60 days, and more preferably at least 180 days at a temperature of 2°C to 30°C, preferably 4°C to 25°C. Under these conditions, the number of live cells embedded and / or encapsulated in the hydrogel obtainable by the method of the present invention is reduced the least. The results show that at room temperature, the number of live cells (CFU, colony forming units) is minimized (less than 2log CFU / ml) even if it is reduced over a period of 6 months. The stress tolerance of the composition was also tested by performing 3 freeze-thaw cycles, wherein the composition was stored at -18 degrees for 24 hours, then stored at room temperature for the next 24 hours, then stored at 37 degrees for 24 hours, and finally stored at room temperature for 24 hours. According to the research of Arepally D et al. (Contemporary Food Science (Curr Res FoodSci.), October 1, 2020; 3: 235-242), the viability of the cells can be determined by enumerating the encapsulated cells on an agar plate. However, other methods are known in the art to determine the number of CFU. Depending on the type of cell, different methods can be used.

[0049] As used herein, "polymerizable substrate" refers to at least one molecule that can form a polymer network when mixed with at least one polymerase. Preferably, the polymerizable substrate is at least one monomer. The at least one monomer can be cross-linked with a polymer or oligomer chain by a covalent bond, an ionic bond or a physical bond. The reaction mixture (which includes at least one polymerizable substrate and at least one enzyme that can polymerize the at least one substrate, and cells) is incubated under conditions that allow enzymatic polymerization of the substrate and under conditions that do not negatively affect the vigor or function of the cells to be embedded and / or encapsulated. Therefore, the conditions depend on the enzyme and cell used in the inventive method. These conditions are well known to those skilled in the art.

[0050] Another aspect of the present invention relates to a method of preparing a stable hydrogel composition by providing and incubating a reaction mixture comprising at least one polymerizable substrate, at least one enzyme, and xanthan gum.

[0051] Xanthan gum is a common food additive, plays an important role as thickener, emulsion stabilizer in industrial applications, and is usually added to water-based drilling fluid due to its pseudoplastic behavior and thermal stability. The present invention shows that, compared with other chemical additives (such as PEG), adding xanthan gum in reaction mixture helps to significantly enhance cell biocompatibility, more environmentally friendly and safe, and even can be edible for humans. Therefore, the stable hydrogel composition comprising reaction mixture and xanthan gum can be used as a suitable carrier for cell encapsulation. Surprisingly, adding xanthan gum also makes the hydrogel-cell composition have better storage stability, because the vigor of cells in this type of composition can be better kept in the presence of xanthan gum.

[0052] According to a preferred embodiment of the present invention, the reaction mixture for preparing the stable hydrogel composition further comprises cells.

[0053] According to a preferred embodiment of the present invention, the at least one polymerizable substrate is sugar phosphate, preferably hexose phosphate, more preferably glucose phosphate, and even more preferably α-D-glucose-1-phosphate.

[0054] Particularly preferably, sugar is used as polymerizable substrate in the method of the present invention. Sugar is a known component of carbohydrate-based hydrogels. Sugar monomers can be linked to each other by using respective enzymes capable of polymerizing the sugar monomers. Bottom-up synthesis from monomers to oligomers and polymers has been shown to be advantageous because it can embed cells into polymer matrices. Since most enzymes capable of polymerizing sugar monomers require phosphorylated sugars, it is particularly preferred to use sugar phosphates as monomers.

[0055] In the case where at least one polymerizable substrate is a sugar phosphate (such as α-D-glucose-1-phosphate), sucrose can be added to the reaction mixture of step a), or can be used in a separate reaction step to form α-D-glucose-1-phosphate, as described by Zhong and Nidetzky (Biotechnol. J., 2020, 15, 1900349). For example, sucrose phosphorylase catalyzes the formation of α-D-glucose-1-phosphate and fructose from sucrose and phosphate ions.

[0056] According to another preferred embodiment of the present invention, α-D-glucose-1-phosphate is formed by incubating sucrose with sucrose phosphorylase. It is particularly advantageous to use sucrose phosphorylase to produce α-D-glucose-1-phosphate because sucrose is more economical than, for example, α-D-glucose-1-phosphate. Using sucrose as a polymerizable substrate avoids the use of α-D-glucose-1-phosphate, thereby making the production of a stable hydrogel composition more cost-effective.

[0057] The stable hydrogel composition of the present invention can also be prepared in a one-pot reaction with sucrose phosphorylase, phosphate ions and sucrose as substrates. Therefore, in addition to at least one polymerizable substrate and at least one enzyme capable of polymerizing the at least one substrate, the reaction mixture of the present method can include sucrose phosphorylase, phosphate ions and sucrose. It has been shown that the components of such a reaction mixture do not negatively affect each other.

[0058] As used herein, "one-pot reaction" refers to a strategy to improve the efficiency of a chemical reaction in which the reactants are subjected to only one reaction mixture. This is highly desirable because one-pot reactions can avoid lengthy separation processes and purification of intermediate compounds, saving time and resources while increasing yields.

[0059] According to another preferred embodiment, the reaction mixture comprises at least one additional monosaccharide, oligosaccharide and / or polysaccharide, preferably at least one additional oligosaccharide and / or polysaccharide.

[0060] The polymerizable substrate used in the method of the present invention is preferably a sugar monomer. However, in order to increase the polymerization rate, at least one additional monosaccharide, oligosaccharide and / or polysaccharide may be added to which the sugar monomer can be linked by the enzyme used.

[0061] According to another further preferred embodiment, the at least one additional monosaccharide is glucose and / or the at least one additional oligosaccharide is a disaccharide, preferably cellobiose. For example, cellobiose can be obtained by enzymatic or acid hydrolysis of cellulose and cellulose-rich materials such as cotton, jute or paper. It has been shown that the final chemical structure can be excellently adjusted and controlled by precise polymerization to build up the cellobiose chain.

[0062] Depending on the reaction conditions leading to different degrees of polymerization (DP), long-chain soluble oligosaccharides or insoluble species can be formed.

[0063] According to a preferred embodiment of the present invention, the reaction mixture comprises 10-500 mM, preferably 15-400 mM, more preferably 20-300 mM of the at least one polymerizable substrate and / or the at least one additional mono-, oligo- or polysaccharide.

[0064] According to another preferred embodiment of the present invention, a mixture of alpha-D-glucose 1-phosphate (alpha-D-glucose 1-phosphate) and cellobiose is used as a polymerizable substrate. Cellobiose can be glycosylated by β-1,4-glycosylation using alpha-D-glucose 1-phosphate as a donor substrate. The alpha-D-glucose 1-phosphate donor substrate can be effectively obtained by an additional phosphorolysis reaction using an enzyme using starch or sucrose as a substrate.

[0065] The results show that the extent of polymerization may depend on the molar ratio of the cellobiose acceptor and the α-D-glucose 1-phosphate donor used in the reaction. The efficient synthesis of hydrogels requires good control of the DP of the formed product. Therefore, the reaction mixture may include α-D-glucose-1-phosphate and cellobiose as polymerizable substrates, preferably in a molar ratio of 20:1 to 1:1, preferably 10:1 to 2:1, and more preferably 8:1 to 5:1. In particular, these ratios have been shown to be advantageous in forming hydrogels that are able to stabilize cells embedded therein.

[0066] At least one phosphorylase may 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 little hydrolysis, as reactions with water are excluded from the enzyme active site. Thus, phosphorylases may aid in the synthetic construction of oligo- or polysaccharide chains that then undergo oligomerization-induced self-assembly to form hierarchically organized and property-tunable materials.

[0067] Therefore, the at least one enzyme capable of polymerizing the above substrate is preferably a phosphorylase, and more preferably an oligosaccharide phosphorylase or a polysaccharide phosphorylase.

[0068] In particular, the enzymatic reaction of the method of the present invention can be catalyzed by phosphorylation of the β-1,4 glycosidic bonds of the oligosaccharide substrate, thereby producing a polysaccharide chain constructed of hundreds or more linked glucosyl groups. The phosphorylase can act on the β-1,4 glycosidic bonds in oligosaccharide and polysaccharide substrates.

[0069] According to a preferred embodiment of the present invention, the at least one enzyme is cellodextrin phosphorylase.

[0070] For example, cellodextrin phosphorylase (EC 2.4.1.49) can reversibly catalyze the synthesis of cellulose and free phosphate (product) from α-D-glucose-1-phosphate and short-chain cellodextrins (as substrates) by repeated β-1,4-glycosylation. It is particularly preferred to use bacterial cellodextrin phosphorylases, preferably from Clostridia, more preferably Clostridium cellulosi (e.g., GenBank ID CDZ24361.1).

[0071] According to another preferred embodiment of the present invention, the reaction mixture comprises 0.5 U / ml to 50 U / ml, preferably 0.5 U / ml to 30 U / ml, more preferably 1 U / ml to 20 U / ml, more preferably 1 U / ml to 10 U / ml of the at least one enzyme.

[0072] According to another preferred embodiment of the present invention, the reaction mixture further comprises at least one additional carbohydrate polymer, preferably in an amount of 0.1-1wt% of the reaction mixture. The carbohydrate polymer is composed of long chains of carbohydrate monomers connected via glycosidic bonds. The 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 using UV light or temperature changes.

[0073] According to another preferred embodiment of the present invention, said at least one additional carbohydrate polymer comprises carboxyl groups, acetyl groups and / or sulphate groups.

[0074] Modified carbohydrate polymers can be added to the reaction mixture to impart additional properties to the hydrogel. For example, modification of the carboxyl groups can cause changes in the resistance to hydrolysis and thermal stability of the polymer. Sulfate groups bound to sugar residues can help stabilize the polymer composition by introducing terminal sulfate groups on each polymer chain formed.

[0075] According to another preferred embodiment of the present invention, the at least one additional carbohydrate polymer is xanthan gum.

[0076] Xanthan gum is a heteropolysaccharide, mainly composed of D-glucose and D-mannose as main hexose units and D-glucuronic acid and pyruvic acid. Xanthan gum can chemically increase the viscosity of the liquid it mixes. Due to this ability, for example xanthan gum is often used as a component in sauces and salad dressings and cosmetics in the food industry. Surprisingly, it is confirmed that adding xanthan gum to the reaction mixture of the present invention enhances the mechanical stability of the hydrogel and prolongs the vigor of the embedded cells. In addition, xanthan gum is a kind of environmentally friendly composition, and is an unexpected substitute for other chemical compositions and stabilizers (such as PEG) for the gelation of in-situ hydrogels.

[0077] The formed stable hydrogel composition is constructed from the polymerizable substrate as a basis for interaction and / or cross-linking with the xanthan gum.

[0078] According to another preferred embodiment of the present invention, the reaction mixture is obtained by mixing its components in a buffer solution, preferably in a phosphate buffered saline (PBS) having a pH value of 6.5 to 7.5. An isotonic buffer solution having a neutral pH value can be used as an optimal composition for maintaining enzyme activity, such as phosphorylase, and is non-toxic to most cells.

[0079] A surprising effect that can be achieved with the method of the present invention is the superior viability of prokaryotic and eukaryotic cells embedded in the hydrogel composition. In addition, the overgrowth of cells in the hydrogel may be limited and the viability remains stable over time. According to a preferred embodiment of the present invention, the cells are bacterial cells, fungal cells, animal cells or human cells.

[0080] According to another preferred embodiment of the present invention, the bacterial cell belongs to the order Lactobacillales, preferably to the family Lactobacillaceae and / or Streptococcaceae, more preferably to the genus Lactococcus and / or Lactobacillus.

[0081] According to another further preferred embodiment of the present invention, the bacterial cell is selected from Lactococcus lactis, preferably Lactococcus lactis 10123 (DSM 34156), Lactobacillus rhamnosus.

[0082] According to another preferred embodiment of the present invention, the reaction mixture comprises cells at a concentration of 0.5-50 mg / ml, preferably 0.5-40 mg / ml, more preferably 0.5-30 mg / ml, more preferably 1-20 mg / ml, more preferably 1-10 mg / ml. The CFU / mg may be between 1.0*10^3 and 1.0*10^12, preferably between 1.0*10^4 and 1.0*10^10. The CFU / mg may also depend on the type of cells and the intended use of the hydrogel-cell composition.

[0083] According to another preferred embodiment of the present invention, lyophilized cells are added to the reaction mixture.

[0084] Lyophilization, also known as freeze drying, is a method for preserving biological materials by removing water from a sample, which typically involves the selective addition of a lyoprotectant (such as a sugar alcohol such as mannitol, sorbitol, or xylitol), followed by freezing the sample and vacuum drying at very low temperatures. By adding lyophilized cells to the reaction mixture, the culture time and viability of the embedded cells can be extended. Lyophilization can stabilize the embedded cell culture for long-term storage while minimizing the damage that may be caused by rigorous drying of the sample.

[0085] According to another preferred embodiment of the present invention, the mixture of step a) is shaken at 50 rpm to 1000 rpm, preferably 100 rpm to 800 rpm, more preferably 200 rpm to 600 rpm, more preferably 200 rpm to 500 rpm during step b). Shaking the mixture can contribute to the uniform distribution of the ingredients of the mixture.

[0086] Another aspect of the present invention relates to a hydrogel-cell composition obtainable by the method as described above. Such a composition with encapsulated and / or embedded cells exhibits long-term stability at room temperature with living cells for at least 30 days, 60 days or even 174 days while maintaining gel properties in the presence of cells. Thus, the obtained composition is a simple solution for keeping encapsulated and / or embedded cells alive and extending shelf life compared to other in situ hydrogels.

[0087] According to a preferred embodiment of the present invention, the hydrogel-cell composition is contacted on a patch or wrap for topical application to the skin, mucous membranes or tissues inside the body, such as the surface of an organ. Adherence of the composition to the body as part of a patch or wrap is beneficial because it allows controlled and sustained administration of the hydrogel-cell composition.

[0088] The hydrogel-cell composition remains at the site of action until the patch / wrap is removed, and the patch also acts as an adhesive, such as closing a wound. Applying the composition via a patch or wrap optimizes the amount and rate of molecules absorbed by the body and available at the site of action. In addition, the patch or wrap prevents early dehydration of the hydrogel, harmful infection of wounds, etc., and allows for quick and easy use of the composition via self-administration. It does not require professional placement, and it can be applied to areas such as the skin as desired by the user. The patch or wrap structure can be configured to be worn directly on the user's skin and can be adjusted to the desired site of action.

[0089] According to another preferred embodiment of the present invention, the hydrogel-cell composition can be used for food production. The composition can include microbial cells, such as bacteria or fungi, which can contribute to the production of dairy products such as cheese yogurt, meat products such as salami, or food supplements. The role of microbial cells in food production is known in the art.

[0090] The hydrogel-cell compositions of the present invention can further be used to preserve food by forming inhibitory metabolites, such as organic acids (e.g., lactic acid, acetic acid, formic acid, or propionic acid) or ethanol. The hydrogel-cell compositions can help improve food safety by inhibiting pathogens and removing toxic compounds. In addition, the compositions of the present invention can improve the nutritional value and organoleptic quality of food.

[0091] According to another preferred embodiment of the present invention, the hydrogel-cell composition can be used in agriculture and / or breeding. Other compositions commonly used in the cultivation of crop plants can be mixed with the hydrogel. The composition can be selected from the group consisting of pesticides, herbicides, insecticides, molluscicides, fungicides, pheromones, preferably fertilizers.

[0092] Another aspect of the invention relates to hydrogel-cell compositions for treating conditions or injuries of the skin and / or eye.

[0093] Due to the unique properties of the hydrogel-cell composition of the present invention, the viability of cells embedded in the hydrogel is significantly prolonged. The hydrogel-cell composition of the present invention or the hydrogel-cell composition obtainable by the method of the present invention can be used to treat skin and / or eye-related conditions or diseases. The hydrogel-cell composition can be directly contacted with the skin and / or eyes to promote healing and / or tissue regeneration processes. The cells present in the hydrogel-cell composition are selected to have a therapeutic effect on the skin and / or eyes. The corresponding cells are well known in the art.

[0094] Due to the mechanical and cytoprotective properties of the composition of the present invention, the hydrogel-cell composition can flexibly adapt to the shape of skin and / or eye defects and promote, for example, normal closure of wounds and tissue regeneration processes through the presence of cells or their respective secretomes. After application, the composition of the present invention aligns with the form of the defect (e.g., wound) during the in situ polymerization process, promoting the healing process.

[0095] According to another preferred embodiment of the present invention, the cells in the hydrogel-cell composition are selected from the group consisting of the following cells: stem cells, induced pluripotent stem cells, macrophages, keratinocytes, endothelial cells, T lymphocytes, monocytes, oocytes, sperm and combinations thereof. Some of these cell types are associated with wound healing and tissue remodeling and can be used to treat skin and / or eye conditions / diseases. In addition, for example, the secretory protein groups of some of these cells may significantly contribute to wound healing and tissue repair mechanisms. Suitable secretory protein groups 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 promote skin and / or eye healing.

[0096] According to another preferred embodiment of the present invention, the hydrogel-cell composition can be administered topically and / or by injection onto or into the skin or eye, preferably subcutaneously or intravitreally.

[0097] The present invention can be used to treat conditions or injuries of the skin and / or eyes.

[0098] As used herein, the term "injury" refers to physical trauma experienced by the skin, eye or multi-layered epithelial tissue. This can be in the form of a cut, electric shock, burn, scratch and / or scrape.

[0099] According to another preferred embodiment of the present invention, the condition or injury of the skin and / or eyes 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 irritation of the skin and / or eyes, actinic keratosis, melanoma, sun damage or aged skin and combinations thereof.

[0100] Another aspect of the invention relates to methods for treating conditions or injuries of the skin and / or eyes.

[0101] According to another preferred embodiment of the present invention, the composition may include Lactococcus lactis 10123. The composition of the present invention has been shown to exhibit a surprising inhibitory effect on pathogens, in particular pathogens of the genus Staphylococcus, such as Staphylococcus aureus. In addition, the composition may be mixed with a reaction mixture and at least one enzyme. The results show that strain Lactococcus lactis 10123 is suitable for embedding in the hydrogel composition obtained as described above.

[0102] Another aspect of the present invention relates to compositions obtainable by the method according to the present invention. The compositions of the present invention are effective against pathogenic bacteria such as Staphylococcus aureus and can be used as substitutes for antibiotics in the food industry, agricultural industry, medicine and cosmetics. The compositions of the present invention can be used to treat or prevent skin infections caused by various pathogens, particularly skin infections caused by bacterial or fungal pathogens. Surprisingly, it has been confirmed that the compositions obtainable by the method of the present invention and including Lactococcus lactis 10123 can be effectively used, for example, to inhibit or prevent the growth of pathogens on the skin and / or wounds. In addition, the compositions of the present invention can be used to enhance the barrier function of the skin and mucocutaneous membranes, and to induce anti-inflammatory behavior in human cells by changing gene expression and cytokine separation. For example, due to the presence of Lactobacilli, the relatively low pH value not only promotes the natural protective acidic environment of the skin, but also inhibits the growth of harmful bacteria, viruses and fungi. These characteristics make the compositions of the present invention ideal products for use as sanitary products, preferably as intimate hygiene products. Many women have problems, especially after using broad-spectrum antibiotics to treat bladder infections. Since this treatment regimen kills many different bacteria, it also affects the natural intestinal and vaginal flora, leading to bacterial miscolonization of the vaginal, rectal and perineal mucosa. These bacteria can cause changes in the pH of the mucosa, thereby weakening the natural protection against pathogens. The composition of the present invention can also have antiviral and / or antitumor effects, thereby preventing the formation of tumors caused by viruses (such as human papillomavirus). The composition of the present invention can prevent the formation of malignant tumors of the skin, eyes and / or mucosa. After topical application of the composition, the occurrence of precancerous conditions can be prevented due to the positive effects of lactobacilli on the tumor microbiome. In addition, the composition can also be used to inhibit the proliferation of viruses that cause abnormal cell growth that causes precancerous lesions, cancer or genital warts. The composition of the present invention can also help balance the natural skin and mucosal flora by maintaining physiological pH and inhibiting the growth of harmful bacteria and fungi.

[0103] According to another preferred embodiment of the present invention, the composition can be in a form suitable for topical application, such as an ointment, cream, gel, lotion, powder or emulsion. An emulsion can be prepared by a high shear mixing process from at least two phases, such as a hydrophilic (water) phase and a lipophilic (oil) phase.

[0104] Surprisingly it was found that the composition of the invention in the form of an emulsion can be prepared by using ingredients that have been experimentally proven to be compatible with bacteria such as Lactococcus lactis 10123. Compatibility was measured by stirring a Lactococcus lactis lyophilisate into each ingredient and measuring the CFU per ml or per gram on blood agar after 7 days.

[0105] According to a preferred embodiment of the present invention, the emulsion can be prepared by:

[0106] a) providing an oil phase and an aqueous phase by mixing and heating the oil phase and the aqueous phase, respectively,

[0107] b) mixing the phases, and

[0108] c) Cooling the mixture.

[0109] The first step involves mixing and heating an oil phase, which includes, for example, shea butter and / or sunflower oil. In the second step, an aqueous phase is prepared and heated, which includes purified water, salt and a gelling agent (such as xanthan gum) and an emulsifier (such as lecithin). In the third step, the two phases are mixed and cooled under high shear. A stable formulation can be generated that can be carefully mixed with a stable hydrogel composition of the present invention. It has been shown that when the hydrogel is mixed with an emulsion of the present invention, its mechanical stability lasts at least three months.

[0110] According to a preferred embodiment of the present invention, additional active ingredients can be added to the emulsion. The active ingredients can include ceramides, ingredients used as bacterial food sources, such as glucose, or buffers and acids (such as lactic acid). The composition of the present invention can also be applied to the skin and orally, intraorally, vaginally or rectally or in the operating area of ​​the body. The composition of the present invention can be used preventively or therapeutically for a variety of symptoms (condition).

[0111] According to another preferred embodiment of the present invention, the composition in the form of ointment, cream, gel, lotion, powder or emulsion can be contacted on a patch or wrapper to be topically applied to the skin. The composition can be applied to the skin as a part of a patch or wrapper, and is intended to deliver microorganisms to the target area of ​​the skin. Using a patch or wrapper can further extend the survival time of bacteria during application by preventing dehydration and / or infection.

[0112] 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 comprising at least one polymerizable substrate and at least one enzyme capable of polymerizing at least one substrate described herein. After mixing the components as described above, the hydrogel composition of the present invention can be obtained.

[0113] According to another preferred embodiment, the at least one polymerizable substrate is as defined above.

[0114] According to another preferred embodiment, the at least one enzyme is as defined above.

[0115] According to another further preferred embodiment, the kit further comprises cells, in particular cells as described above. The cells may be lyophilized and may be added to the other components of the kit.

[0116] Example

[0117] Example 1: Enzymatic synthesis of hydrogel

[0118] The enzyme used for hydrogel synthesis is cellodextrin phosphorylase (CdP, EC 2.4.1.49). This enzyme catalyzes the iterative β-1,4 glycosylation of cellobiose using αGlc1-P as a donor substrate ( Figure 1 ). In this experiment, CdP from Clostridium cellulosi (GenBank ID CDZ24361.1) was selected. It was recombinantly expressed in Escherichia coli BL21 (DE3) strain using a plasmid vector (pET-21b(+)) containing a codon-optimized gene. The gene carries N-terminal Nde I and C-terminal Xho I restriction sites for subcloning into pET-21b(+). Enzyme expression in Escherichia coli was induced with isopropyl β-D-1-thiogalactopyranoside (0.25 mM) at 18°C ​​overnight. Then The enzyme was purified using a pre-filled (1.6 cm × 2.5 cm; 5 mL) HisTrap FF crude column (GE Healthcare Europe, Vienna, Austria) on prime plus (GE Healthcare Europe). The N-terminal His-tagged protein was eluted using imidazole (0.01-0.3 M). The purified protein was desalted using phosphate buffer (50 mM, pH 7.0) and Vivaspin Turbo 50 kDa cut-off tube (Sartorius Stedim, Vienna, Austria). The purified enzyme solution was sterilized through a 0.22 μm syringe filter before use.

[0119] The synthesis reaction (total volume 0.5 mL) was tested on a ThermoMixer C (Eppendorf, Vienna, Austria) at 37°C and 45°C, preferably at 37°C. The reaction solution (RS) was prepared in a phosphate buffer solution (PBS, 10 mM, pH 7.0) containing 200 mM αGlc1-P, 25 mM cellobiose and CdP (concentration 0.1-0.8 mg / mL (i.e. 1-10 U / mL)). The reaction was carried out for 1-5 hours to obtain a stable hydrogel.

[0120] Example 2: Enzymatic Synthesis of Xanthan Gum-Containing Hydrogel

[0121] In the above RS, xanthan gum with a concentration of 0.2-0.5% (m / v) was used as an additive, and a uniform white hydrogel was obtained after reacting at 37°C for 2 hours ( Figure 2 ). Under these conditions, the conversion of the donor αGlc1-P was approximately 35-55 mol%. Control reactions performed under the same conditions (without xanthan gum) for 4 h to prepare "cellulose only" gels were observed to show poor mechanical stability of the overall mixture ( Figure 2 ). Therefore, these results indicate the role of polymer additives in gelation. The presence of high concentrations (≥0.25%) of xanthan gum induces macromolecular crowding effects, such as Figure 2 As shown. In addition, the gelation properties of the hydrogels thus obtained (with 0.4% xanthan gum) were examined by rheological methods at 25°C using a strain-controlled rheometer (MCR 502, Anton Paar, Austria) with a cone-plate measurement geometry (CP 50-1) with a diameter of 50 mm and a cone of 1°. The linear viscoelastic range was measured with a strain sweep (0.01-100%) at a fixed frequency of 10 rad / s. The results of the amplitude sweep show that the strain is plotted on the x-axis and the storage modulus G' and the loss modulus G" are plotted on the y-axis ( Figure 3 ). The linear viscoelastic region (LVE region) (~0.1%) was determined, where the curve of the G' function was a constant value, which means that the test was performed without destroying the sample structure. The prepared hydrogel showed G'>G" in the LVE region, which indicated that a gel-like structure was established in the hydrogel.

[0122] Example 3: Encapsulation of cells in hydrogels

[0123] In order to encapsulate cells (tested strains of Lactobacillus and Lactococcus) in hydrogels, freeze-dried cells were pre-suspended in sterilized RS (0.5 mL) at a cell concentration of 1-10 mg / mL. Then the reaction was carried out on a ThermoMixer C (Eppendorf, Vienna, Austria) at 37 ° C and 300 rpm stirring for 3 hours. A cellulose-xanthan hydrogel encapsulated with bacteria was obtained. In the presence of encapsulated cells, the overall gel properties were retained. At the same time, the encapsulated cells (Lactobacillus rhamnosus, live cells have green fluorescence) were visualized using a confocal laser scanning microscope, and the results showed that the cells were distributed in the hydrogel ( Figure 4 ). In addition, cells (Lactobacillus rhamnosus) encapsulated in cellulose hydrogels without xanthan gum ("cellulose only") showed long-term storage stability, with a viable cell loss of ≤1 log (cfu / mL) within 90 days of storage at room temperature and a cell loss of >3 log (cfu / mL) after 90 days of storage. Encapsulated cells (Lactobacillus rhamnosus) in cellulose-xanthan hydrogels showed long-term storage stability, with a viable cell loss of ≤1 log (cfu / mL) within 60 days of storage at room temperature and a viable cell loss of ≤2 log (cfu / mL) after 90 days of storage ( Figure 5a In contrast, cells in xanthan gum solutions (containing xanthan gum as the sole polysaccharide at concentrations of 0.1-0.5%) showed cell losses exceeding 6 log(cfu / mL), as shown in Figure 2. Figure 5b Overall, excellent cell protection was achieved by encapsulating cells in cellulose hydrogels as well as cellulose-xanthan gum hydrogels.

[0124] Example 4: Hydrogel Encapsulation for Preserving Cell Viability (Lactobacillus rhamnosus and Lactococcus lactis)

[0125] To test the ability of the enzymatically produced hydrogels to maintain cell viability, two different bacterial strains (Lactobacillus rhamnosus and Lactococcus lactis) were encapsulated. The number of viable bacteria was assessed after defined time points. To measure the number of viable bacteria, 100 mg of the gel was diluted in PBS and plated on agar plates at various time points. The results showed that the viable cells in the gel slowly decreased over the shelf life. Over 174 days, only about 2 log ( Figure 6a Compared with known hydrogels (containing xanthan gum as the only polysaccharide), the stability of the hydrogel of the present invention (cellulose-xanthan gum hydrogel) is greatly increased ( Figure 6b ).

[0126] Other parameters measured were optical stability. The gel lasted at least 6 months at room temperature as an opaque white mass. Mechanical stability was also good after the addition of xanthan gum. This was tested by turning the tube. The gel did not run off for at least 6 months. Lactobacillus rhamnosus had a pungent smell in the first few days, but after a few weeks a yogurt-like sour smell surrounded it. Lactococcus lactis had a more neutral smell. The pH was also measured using pH test paper. It can be seen that the pH slowly dropped (over 4-6 months) from 7.5 to 4.5.

[0127] Example 5: Lactobacillus 10123 strain inhibiting Staphylococcus aureus

[0128] A strain of Lactococcus lactis was isolated from raw milk (Graz, Styria). 100 μL of 10^5 overnight culture (ONC) of Lactococcus lactis was inoculated on a Müller-Hinton agar plate. The plate was inoculated with a swab soaked with 10^8 CFU / ml of Staphylococcus aureus in PBS, and the ONC was poured into an agar plate with a central hole. After incubation at 37 degrees for 24 hours, Staphylococcus aureus ( Figure 7 ) and MRSA, Staphylococcus aureus, Enterococcus hilaris. No inhibition zone was seen when the cell-free supernatant of Lactobacillus lactis ONC was used.

[0129] In another set of experiments, the ability to inhibit S. aureus at 37°C for 24 hours in ONC with 1:1 MRS and LB-Medium was also tested. 10^6 CFU of S. aureus and 10^6 CFU of Lactococcus lactis 10123 were inoculated in 10 ml of culture medium. The culture density was adjusted compared to the standard of 0.5 MFU (McFarland unit). Lactobacillus rhamnosus was used as a co-culture agent with S. aureus in ONC. The CFU at the beginning of the experiment was verified by counting colonies on the plates, where at least 10^5 CFU / ml was detected. Figure 8 The results in show that after 24 hours, no S. aureus were found in the co-culture, while more than 10^8 CFU / ml of S. aureus appeared in the S. aureus-monoculture. As shown in the literature, the other tested strains caused a reduction of less than 2 log (in contrast, L. lactis 10123 caused a reduction of more than 8 log).

[0130] Example 6: Functionality of 10123 strain after encapsulation

[0131] To investigate the functional retention of Lactococcus lactis strain 10123 after being encapsulated in a hydrogel containing xanthan gum and stored at room temperature for 1 month, a blood agar plate was inoculated with 10^6 CFU of S. aureus and a 100 μm gel containing 10^7 CFU / ml of L. lactis was applied on one side of the plate. The gel itself was also applied to S. aureus. The results showed that the colonization of S. aureus was reduced where the gel was applied on the right side ( Figure 9a ), and inhibition zones were also seen where the gel droplets were directly applied ( Figure 9b ).

[0132] The functionality of strain 10123 was also demonstrated on a porcine skin model (dermis). One marked spot (left) was inoculated with Staphylococcus aureus and the second marked spot (right) was inoculated with Staphylococcus aureus and Lactococcus lactis. After 24 hours of incubation at 37°C, a visible difference between the two spots could be observed ( Fig.10 ).

[0133] Example 7: Comparison with Neutralization-Induced Cellulose Oligomer Self-Assembly

[0134] The method of the present invention was compared with the method disclosed 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", Supplementary Information).

[0135] As described by Serizawa et al., freeze-dried cellulose oligomers (4.5 mg) were dissolved in NaOH solution (1 M, 27.5 L) and neutralized with HCl solution (65 mM, 423 L). The neutralized solution (200 μL) was immediately mixed with a cell suspension in PBS (200 μL; tested Lactobacillus rhamnosus), and the resulting solution was then transferred to a 96-well plate. The final concentration of cellulose oligomers was set to 0.5% (w / v) and the cell density was set to 1.50*10 10 cells / mL (Method 1).

[0136] In contrast, the synthesis of cellulose oligomers and cell encapsulation were performed according to the present invention (Method 2). The reaction solution was prepared as defined in Example 1. Cells (Lactobacillus rhamnosus) were mixed into the reaction solution to obtain 1.50*10 10 The reaction was then carried out at 37°C with stirring at 300 rpm for 3 hours on a ThermoMixer C (Eppendorf, Austria).

[0137] Table 1: Results of comparative experiments

[0138] Days Method 1 [CFU / mL] Method 2 [CFU / mL] 0 1.30E+10 1.20E+10 19 4.25E+06 1.00E+09

[0139] The results in Table 1 show that the method according to the present invention (Method 2) leads to a significantly improved long-term storage stability of encapsulated cells compared to the method in the literature (Method 1). These findings indicate 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. A method for producing a stable hydrogel-cell composition, comprising the following steps: a) providing a reaction mixture comprising at least one polymerizable substrate and at least one enzyme capable of polymerizing the at least one substrate and cells, and b) incubating the mixture of step a) to form a stable hydrogel-cell composition.

2. A method for producing a stable hydrogel composition, comprising the steps of: a) providing a reaction mixture comprising at least one polymerizable substrate and at least one enzyme capable of polymerizing the at least one substrate and xanthan gum, and b) incubating the mixture of step a) to form a stable hydrogel composition.

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 a glucose phosphate, even more preferably a-D-glucose-1-phosphate.

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

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

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

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

9. The method according to any one of claims 4 to 8, wherein the reaction mixture comprises α-D-glucose-1-phosphate and cellobiose as polymerizable substrates, 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 of any one of claims 1 to 10, wherein the at least one enzyme is cellodextrin phosphorylase.

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

13. The process according to any one of claims 1 to 12, wherein the reaction mixture comprises at least one additional carbohydrate polymer, preferably in an amount of 0.1 wt% to 1 wt% of the reaction mixture, wherein the at least one carbohydrate polymer is preferably xanthan gum.

14. The method of claim 13, wherein the at least one carbohydrate polymer comprises carboxyl, acetyl and / or sulfate groups.

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

5.

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

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

19. The method according to any one of claims 1 to 18, wherein: Lyophilized cells were added to the reaction mixture.

20. The method according to any one of claims 1 to 19, wherein the mixture of step a) is shaken during 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, which was deposited at DSMZ on February 4, 2022 under the deposit number DSM 34156.

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

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