WO2025229134A1 - Method for producing a gelling collagen component - Google Patents

Abstract

The invention relates to a method for producing a gelling collagen component from at least one non-gelling, recombinantly produced component; to a gelling collagen component that can be produced using the method according to the invention; to the use thereof; and to products containing the gelling collagen component according to the invention.

Description

DESCRIPTION

Method for producing a gelling collagen component

The invention relates to a method for producing a gelling collagen component from at least one non-gelling, recombinantly produced component, as well as a gelling collagen component that can be produced using the method according to the invention, its use and products containing the gelling collagen component according to the invention.

Collagen is an extracellular structural protein found in animals, occurring in various tissues, particularly connective tissue, as a component of the extracellular matrix. Tendons, ligaments, cartilage, and bones are especially rich in collagen. Collagen is not found in plants or single-celled organisms. With over 30% of the total protein content, collagen is the most abundant protein in the human body.

Collagens occur in various structurally and functionally distinct types, differing in their structure, function, and origin, among other things. The polypeptide chains that make up collagen are synthesized individually within the cell at the ribosomes of the endoplasmic reticulum as larger precursor molecules and exhibit extensive repetitive (Gly-X-Y)n sequences, where X and Y can be any amino acid, but are most commonly proline and 4-hydroxyproline.

These precursor polypeptide chains are post-translationally hydroxylated in the endoplasmic reticulum at proline and lysine residues of the polypeptide chain, forming hydroxyproline and hydroxylysine residues. This hydroxylation serves to stabilize adjacent collagen polypeptide chains of the right-handed triple helix (procollagen), which forms in the cell and consists of three of these precursor polypeptide chains.

The procollagen thus formed is glycosylated intracellularly, secreted by the cell in its glycosylated triple-helical form (tropocollagen), and subsequently formed into collagen through peptidase-mediated cleavage of the terminal residues. This collagen then assembles into collagen fibrils via fibrillogenesis, which are subsequently cross-linked covalently to form collagen fibers. Collagen is also frequently used in denatured form, then referred to as gelatin, or in its hydrolysates.

When gelatin or collagen are subjected to hydrolytic processes, particularly enzymatic hydrolysis, to obtain collagen peptides, collagen hydrolysates with a wide variety of compositions and application profiles can be produced, depending on the type and origin of the collagen used and the enzymatic conditions. These collagen hydrolysates, however, represent a mixture of peptides whose molecular weights are distributed across specific size ranges. The use of such collagen hydrolysates, for example as dietary supplements or cosmetic aids, has been known for some time, including for the prevention and/or treatment of conditions related to bones, joints, or connective tissue.

Although the use of collagen hydrolysates derived from animal materials offers advantages for many applications and consumer groups, it may also be less desirable for certain consumer groups and application profiles. Some consumer groups are fundamentally critical of or opposed to raw materials derived from animal materials, whether due to concerns about contamination with harmful microorganisms or agents, such as processing aids, or undesirable immune reactions, or for religious or ethical reasons. Furthermore, the manufacturing processes used to obtain collagen hydrolysates from animal materials often involve complex and expensive extraction, purification, and further processing steps. Against this backdrop, it is not surprising that methods have been developed to produce gelatin, collagen, collagen hydrolysates, and individual collagen peptides biotechnologically using recombinant genetic engineering.

WO 2006/052451 A2 discloses the production of recombinant type III collagen in Pichia pastoris- X^mmQn, which also expresses human prolyl hydroxylases. WO 2005/012356 A2 discloses the production of gelatin from human type I collagen and individual 50 kDa, 65 kDa, and 100 kDa collagen peptide species, each in fully hydroxylated, partially hydroxylated, and non-hydroxylated forms. Olsen et al. (Protein Expression and Purification, 2005, 40, pp. 346–357) discloses the recombinant production of an 8.5 kDa collagen peptide species from the al chain of human collagen m'Pichia pastoris. WO 01/34646 Document A2 also discloses the production of individual recombinant gelatin species, each with a defined molecular weight of 0 to 350 kDa resulting from the recombinant manufacturing process, which can be in non-hydroxylated, partially, or fully hydroxylated form. The document discloses the general applicability of individual recombinant gelatin species or mixtures thereof in the food, beverage, cosmetic, or pharmaceutical industries. The document also discloses further uses of the disclosed recombinant products as photographic compositions or as technical aids, e.g., in the semiconductor industry, papermaking, or similar applications. It is therefore known to use recombinant gelatin or collagen peptides of any size, individually or in mixtures, hydroxylated or non-hydroxylated, in a wide variety of technical and non-technical fields, for example, in the food industry.

However, a particular problem with the recombinant production of collagen peptides is that the expression of larger heterologous polypeptides or proteins in numerous known expression systems is either not possible or fraught with major challenges, so that satisfactory yields cannot be achieved.

Particularly important application areas for collagen peptides include the food industry, especially the confectionery industry; the pharmaceutical industry, particularly as a key component of hard and soft capsules; the cosmetics industry; the film industry; and medical technology. For these applications, the exceptionally good gelling properties of collagen, unmatched by other materials, play a crucial role. The gelling properties of gelatin and collagen peptides, especially their gel strength, can be expressed using the so-called Bloom value. Collagen peptides with a Bloom value below 50 generally do not exhibit gelling properties. Conversely, gelatin and collagen peptide preparations with a high Bloom value, and thus properties that enable their use in a wide range of industrial applications, have a particularly high economic value.

A particular goal is therefore to provide methods that allow gelling collagen components to be produced from non-gelling, recombinantly produced collagen peptides in a simple and efficient manner, in order to expand the range of applications or even open up new areas of application and increase the economic value of the starting materials. The present invention solves the underlying technical problem through the subject matter of the independent claims.

The invention relates in particular to a method for producing a gelling collagen component, comprising the steps of: a) providing at least one non-gelling, recombinantly produced collagen component, b) incubating the at least one non-gelling, recombinantly produced collagen component provided in step a) under conditions that lead to an intermolecular bond between the amino acid chains of at least two molecules of the at least one collagen component, c) obtaining a gelling collagen component with a Bloom value of at least 50.

According to the invention, it may be provided that the at least one non-gelling, recombinantly produced collagen component is a recombinantly produced collagen peptide, a mixture of several individual, recombinantly produced collagen peptides, or a hydrolysate of at least one recombinantly produced collagen peptide.

In a preferred embodiment of the present invention, the at least one non-gelling, recombinantly produced collagen component is a recombinantly produced collagen peptide, preferably a recombinantly produced collagen peptide with a molecular weight of at most 45 kDa, preferably at most 44 kDa, preferably 43 kDa, preferably at most 42 kDa, preferably at most 41 kDa, preferably at most 40 kDa, preferably at most 39 kDa, preferably at most 38 kDa, preferably at most 37 kDa, preferably at most 36 kDa, preferably at most 35 kDa, preferably at most 34 kDa, preferably at most 33 kDa, preferably at most 32 kDa, preferably at most 31 kDa, preferably at most 30 kDa.

According to a further preferred embodiment of the invention, the at least one non-gelling, recombinantly produced collagen component is a collagen peptide with a molecular weight of at least 10 kDa, preferably at least 11 kDa, preferably at least 12 kDa, preferably at least 13 kDa, preferably at least 14 kDa, preferably at least 15 kDa. kDa, preferably at least 16 kDa, preferably at least 17 kDa, preferably at least 18 kDa, preferably at least 19 kDa, preferably at least 20 kDa.

In a particularly preferred embodiment of the invention, the at least one non-gelling, recombinantly produced collagen component is a collagen peptide with a molecular weight in the range of 10 to 45 kDa, preferably 12 to 44 kDa, preferably 14 to 42 kDa, preferably 15 to 40 kDa.

In a preferred embodiment of the present invention, the at least one non-gelling, recombinantly produced collagen component comprises at least two, preferably at least three, preferably at least four, preferably at least five, different recombinantly produced collagen peptides.

Preferably, the at least one non-gelling, recombinantly produced collagen component comprises at most 10, preferably at most 9, preferably at most 8, preferably at most 7, preferably at most 6, preferably at most 5, preferably at most 4, preferably at most 3, preferably at most 2, different recombinantly produced collagen peptides.

In a preferred embodiment of the invention, the at least one non-gelling, recombinantly produced collagen component comprises 2 to 10, preferably 2 to 8, preferably 2 to 6, preferably 2 to 4, different recombinantly produced collagen peptides.

According to this preferred embodiment of the present invention, the at least one non-gelling, recombinantly produced collagen component is a mixture of several individual, recombinantly produced collagen peptides.

Particularly preferably, all of the recombinantly produced collagen peptides of the non-gelling, recombinantly produced collagen component independently possess a molecular weight of at most 45 kDa, preferably at most 44 kDa, preferably at most 43 kDa, preferably at most 42 kDa, preferably at most 41 kDa, preferably at most 40 kDa, preferably at most 39 kDa, preferably at most 38 kDa, preferably at most 37 kDa, preferably at most 36 kDa, preferably at most 35 kDa, preferably at most 34 kDa, preferably at most 33 kDa, preferably at most 32 kDa, preferably at most 31 kDa, preferably at most 30 kDa.

According to a further preferred embodiment of the invention, all of the recombinantly produced collagen peptides of the non-gelling, recombinantly produced Each collagen component independently has a molecular weight of at least 10 kDa, preferably at least 11 kDa, preferably at least 12 kDa, preferably at least 13 kDa, preferably at least 14 kDa, preferably at least 15 kDa, preferably at least 16 kDa, preferably at least 17 kDa, preferably at least 18 kDa, preferably at least 19 kDa, preferably at least 20 kDa.

In a particularly preferred embodiment of the invention, all of the recombinantly produced collagen peptides of the non-gelling, recombinantly produced collagen components independently each have a molecular weight in the range of 10 to 45 kDa, preferably 12 to 44 kDa, preferably 14 to 42 kDa, preferably 15 to 40 kDa.

According to a further preferred embodiment of the present invention, the at least one non-gelling, recombinantly produced collagen component consists of at least two recombinantly produced collagen peptides, wherein one recombinantly produced collagen peptide has a molecular weight of at most 25 kDa, preferably at most 20 kDa, particularly preferably 10 to 20 kDa, and another recombinantly produced collagen peptide has a molecular weight of at least 25 kDa, preferably at least 30 kDa, particularly preferably 30 to 45 kDa.

According to the invention, it can also be provided that the at least one non-gelling, recombinantly produced collagen component is a hydrolysate of at least one recombinantly produced collagen peptide.

According to this embodiment of the present invention, the non-gelling, recombinantly produced collagen component preferably has a mean molecular weight in the range of 10 to 45 kDa, preferably 12 to 44 kDa, preferably 14 to 42 kDa, preferably 15 to 40 kDa.

Preferably, the hydrolysate comprises at most 15%, preferably at most 14%, preferably at most 13%, preferably at most 12%, preferably at most 11%, preferably at most 10%, preferably at most 9%, preferably at most 8%, preferably at most 6%, preferably at most 4% of collagen peptides with a molecular weight of less than 10 kDa. In a further preferred embodiment of the present invention, the hydrolysate has at most 10%, preferably at most 9%, preferably at most 8%, preferably at most 7%, preferably at most 6%, preferably at most 5%, preferably at most 4%, preferably at most 3%, preferably at most 2%, preferably at most 1% of collagen peptides with a molecular weight of less than 5 kDa.

Preferably, the hydrolysate contains at most 5%, preferably at most 4%, preferably at most 3%, preferably at most 2%, preferably at most 1%, preferably at most 0.5% of collagen peptides with a molecular weight of less than 500 Da.

According to a preferred embodiment of the present invention, the hydrolysate contains at most 25%, preferably at most 20%, preferably at most 15%, preferably at most 10%, preferably at most 8%, preferably at most 6%, preferably at most 5%, preferably at most 4%, preferably at most 3%, preferably at most 2%, preferably at most 1% of collagen peptides with a molecular weight of over 40 kDa.

The hydrolysate particularly preferably contains at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% of collagen peptides with a molecular weight in the range of 10 to 40 kDa, preferably 10 to 35 kDa, preferably 15 to 30 kDa.

In a further preferred embodiment of the present invention, the non-gelling, recombinantly produced collagen component has an average molecular weight in the range of 10 to 45 kDa and comprises at most 15% of collagen peptides with a molecular weight of less than 10 kDa, at most 10% of collagen peptides with a molecular weight of less than 5 kDa, at most 5% of collagen peptides with a molecular weight of less than 500 Da, at most 25% of collagen peptides with a molecular weight of more than 40 kDa, and at least 40% of collagen peptides with a molecular weight in the range of 10 to 40 kDa.

Particularly preferably, the non-gelling, recombinantly produced collagen component has a mean molecular weight in the range of 15 to 40 kDa and comprises at most 10% of Collagen peptides with a molecular weight of less than 10 kDa, at most 5% of

Collagen peptides with a molecular weight of less than 5 kDa, at most 2% of

Collagen peptides with a molecular weight of less than 500 Da, at most 15% of

Collagen peptides with a molecular weight of over 40 kDa, and at least 60% of

Collagen peptides with a molecular weight in the range of 10 to 40 kDa.

According to a preferred embodiment of the present invention, the at least one non-gelling collagen component has a Bloom value of at most 45, preferably at most 40, preferably at most 35, preferably at most 30.

According to the invention, the at least one non-gelling collagen component may be a recombinantly produced collagen peptide that has an amino acid sequence occurring in collagens of types I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, preferably type I, II or III, preferably type I or type III, and in particular consists of such an amino acid sequence.

In a further preferred embodiment, the at least one non-gelling collagen component is a recombinantly produced collagen peptide comprising an amino acid sequence occurring in collagen from vertebrates, in particular fish, amphibians, reptiles, birds and mammals, especially in human, bovine, porcine, equine, ovine, piscine or avian collagen of types I, II or III, preferably type I or type III, and in particular consisting of such an amino acid sequence.

Particularly preferred is the at least one non-gelling collagen component a recombinantly produced collagen peptide comprising an amino acid sequence occurring in human, bovine, porcine, equine, ovine, piscine or avian collagen, in particular in human, bovine, porcine, equine, ovine, piscine or avian type I collagen, preferably in the al chain of the human, bovine, porcine, equine, ovine, piscine or avian type I or type III collagen, and in particular in the al chain of the bovine or porcine type I or type III collagen.

According to the invention, it can be provided that the at least one non-gelling collagen component is a recombinantly produced collagen peptide that has the amino acid sequence of a naturally occurring collagen peptide. In a further preferred embodiment of the present invention, the at least one non-gelling collagen component does not have a naturally occurring amino acid sequence. According to the invention, it can therefore also be provided that the at least one non-gelling collagen component has the amino acid sequence of a genetically modified collagen peptide, and in particular consists of such an amino acid sequence.

According to a preferred embodiment of the present invention, the recombinantly produced collagen peptide is a genetically modified collagen peptide in which at least 10%, preferably at least 25%, preferably at least 50%, preferably at least 75% of the lysines in the amino acid sequence of a naturally occurring collagen peptide, preferably a bovine or porcine type I or type III collagen, are replaced by another naturally occurring amino acid. Preferably, the other naturally occurring amino acid is selected from the group consisting of arginine, threonine, glutamine, proline, glutamic acid, alanine, and leucine.

According to the invention, the at least one non-gelling collagen component can be hydroxylated or non-hydroxylated. In a preferred embodiment of the invention, the at least one non-gelling collagen component is non-hydroxylated. Preferably, the at least one non-gelling collagen component is hydroxylated, in particular partially or completely hydroxylated.

In a further embodiment of the present invention, the at least one non-gelling collagen component is non-glycosylated. According to the invention, it can also be provided that the at least one non-gelling collagen component is glycosylated.

Preferably, the at least one non-gelling collagen component is hydroxylated and glycosylated. In a particularly preferred embodiment of the present invention, the at least one non-gelling collagen component is neither hydroxylated nor glycosylated.

In a preferred embodiment of the present invention, the at least one non-gelling collagen component provided in step a) is hydroxylated, in particular partially or completely hydroxylated, if it has a molecular weight, in particular an average molecular weight, of < 45 kDa, preferably < 40 kDa, preferably < 35 kDa, preferably < 30 kDa, preferably < 25 kDa, preferably < 20 kDa, particularly preferably < 15 kDa. Particularly preferred is the at least one non-gelling collagen component provided in step a) being hydroxylated, in particular partially or completely hydroxylated, if it has a molecular weight, in particular a mean molecular weight, in the range of 10 to 45 kDa, preferably 10 to 40 kDa, preferably 10 to 35 kDa, preferably 10 to 30 kDa, preferably 10 to 25 kDa, preferably 10 to 20 kDa.

Preferably, the incubation according to step b) takes place in a reaction mixture, preferably in a solution or suspension.

According to a preferred embodiment of the present invention, the intermolecular bond between the amino acid chains of at least two molecules in step b) is formed by the presence of at least one chemical and/or one enzymatic crosslinker.

In a preferred embodiment of the present invention, the intermolecular bonding between the amino acid chains of at least two molecules in step b) is effected by the presence of at least one chemical crosslinker.

Preferably, the chemical crosslinker is a low-molecular-weight crosslinker, in particular a low-molecular-weight crosslinker selected from the group consisting of genipin, nordihydroguaiaretic acid, citric acid, procyanidin, EDC, NHS, butanediol diglycidyl ether (BDDGE), pullulan dialdehyde (PDA), NHSF, quinone methide, THPP, maleimide, glutaraldehyde, methacrylic anhydride (MAA), desaminotyrosine (DAT), desaminotyrosyltyrosine (DATT), starch dialdehyde (DAS), chitosan, riboflavin, quercetin, chlorogenic acid, rutin, fluorescamine, ferulic acid, caffeic acid, gallic acid, tannic acid, ellagic acid, thiolation, disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG), bis(sulfosuccinimidyl)suberate, bis(sulfosuccinimidyl)glutarate, native chemical ligation, and PdBT. H2O2, AMAS, ANB-NOS, BMB, BMDB, BMH, BMOE, BMPH, BMPS, BM(PEG)2, BM(PEG)3, BS3(Sulfo-DSS), BS2G-dO, BS2G-d4, BS3-d0, BS3-d4, BS(PEG)5, BS(PEG)9, BSOCOES, DCC, DFDNB, DMA, DMP, DMS, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP (Sulfo-DSP), EDC, EGS, EMCA, EMCH, EMCS, GMBS, KMUH, LC-SDA, LC-SMCC, LC-SPDP, L-Photo-Leucine, L-Photo-Methionine, MBS, MPBH, Mts-Atf-Biotin, Mts-Atf-LC-Biotin, NHS, NHS azides, NHS PEG4 azides, NHS PEG12 azides, NHS phosphines, PDPH, PEG4-SPDP, PEG12-SPDP, PMPI, SB AP, SDA, SD AD, SIA, SIAB, SMCC, SM[PEG]2, SM[PEG]4, SM(PEG)6, SM[PEG]8, SM[PEG]12, SM(PEG)24, SMPB, SMPH, SMPT, SPB, SPDP, Sulfo-EGS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-LC-SDA, Sulfo-LC-SPDP, Sulfo-MBS, Sulfo-NHS, Sulfo-NHS-Phosphine, Sulfo-SANPAH, Sulfo-SBED, Sulfo-SDA, Sulfo-SDAD, Sulfo-SIAB, Sulfo-SMCC, Sulfo-SMPB, TMEA (trifunctional), TSAT (trifunctional) or combinations thereof.

In a preferred embodiment, the intermolecular bonding between the amino acid chains of at least two molecules in step b) is effected by the presence of at least one enzymatic crosslinker.

The enzymatic crosslinker is preferably selected from the group consisting of transglutaminase (EC 2.3.2.13), lysyl oxidase (EC 1.4.3.13), peroxidase (EC 1.11.1), sulfhydryl oxidase/thiol oxidase (EC 1.8.3.2), peptiligase (EC 3.4.22.10), sortase A (EC 3.4.22.70), sortase B (EC 3.4.22.71), sortase C (EC 3.4.22.72), sortase D (EC 3.4.22.73), butelase 1 (EC 3.4.22.34), trypsiligase (EC 3.4.21.4), subtiligase, thymoligase (EC 3.4.21.62), Tyrosinase (EC 1.14.18.1), laccase (EC 1.10.3.2) and combinations thereof.

The enzymatic crosslinker is particularly favored to be a transglutaminase, especially a bacterial transglutaminase.

According to the invention, it can also be provided that the enzymatic crosslinker is a peptide ligase, in particular a bacterial peptide ligase, preferably from Bacillus subtilis or Bacillus amyloliquefaciens.

In a preferred embodiment of the present invention, the intermolecular bonding between the amino acid chains of at least two molecules in step b) is effected by the presence of at least two enzymatic crosslinkers. According to the invention, it can be provided that the intermolecular bonding between the amino acid chains of at least two molecules in step b) is effected by the presence of at least one transglutaminase and at least one peptide ligase.

According to a further preferred embodiment of the present invention, the intermolecular bonding between the amino acid chains of at least two molecules in step b) is carried out by dehydrothermal crosslinking, UV irradiation or by electrostatic interaction. According to the invention, the intermolecular bond between the amino acid chains of at least two molecules in step b) can be a covalent or a non-covalent bond. In a particular embodiment of the present invention, the intermolecular bond between the amino acid chains of at least two molecules in step b) is a non-covalent bond. Preferably, the intermolecular bond between the amino acid chains of at least two molecules in step b) is a covalent bond.

In a preferred embodiment of the present invention, the intermolecular bond between the amino acid chains of at least two molecules in step b) is a chain extension, in particular a peptide ligation. According to this preferred embodiment, in step b) at least two molecules of the at least one non-gelling collagen component are linked together by a covalent bond between the C- and N-termini of the at least two molecules.

According to a preferred embodiment of the present invention, the intermolecular bond between the amino acid chains of at least two molecules in step b) is a cross-linking between side chains.

According to the invention, it can also be provided that the intermolecular bond between the amino acid chains of at least two molecules in step b) is a chain elongation, in particular a peptide ligation, and a cross-linking between side chains. According to this embodiment of the present invention, the gelling collagen component obtained in step c) is both chain-elongated and cross-linked.

It can be provided that, first, in step bl), the non-gelling collagen components provided in step a) undergo chain elongation, and then, in a subsequent step b2), the collagen components obtained from step bl) undergo cross-linking. Likewise, it can be provided according to the invention that, first, in step bl), the non-gelling collagen components provided in step a) undergo cross-linking, and then, in a subsequent step b2), the collagen components obtained from step bl) undergo chain elongation. According to a further embodiment of the present invention, both cross-linking and chain elongation occur in parallel in a single step b). This can be achieved by the The presence of two different enzymes can be achieved, for example, by the presence of at least one transglutaminase and at least one peptide ligase.

In a preferred embodiment of the present invention, when using an enzymatic crosslinker in step b), the at least one enzymatic crosslinker is subsequently inactivated, preferably thermally inactivated.

According to a preferred embodiment of the present invention, the at least one non-gelling collagen component provided in step a) is present in step b) in a concentration of at least 5 wt.%, preferably at least 6 wt.%, preferably at least 7 wt.%, preferably at least 8 wt.%, preferably at least 9 wt.%, preferably at least 10 wt.% (in each case based on the total weight of the reaction mixture).

In a preferred embodiment of the present invention, the at least one non-gelling collagen component provided in step a) is present in step b) in a concentration of at most 30 wt.%, preferably at most 25 wt.%, preferably at most 20 wt.%, preferably at most 15 wt.%, preferably at most 10 wt.%, preferably at most 9 wt.%, preferably at most 8 wt.%, preferably at most 7 wt.% (in each case based on the total weight of the reaction mixture).

Particularly preferably, the at least one non-gelling collagen component provided in step a) is present in step b) at a concentration of 6.66 wt% (based on the total weight of the reaction mixture). In a further preferred embodiment, the at least one non-gelling collagen component provided in step a) is present in step b) at a concentration of 10 wt% (based on the total weight of the reaction mixture).

According to a preferred embodiment of the present invention, the incubation of the at least one non-gelling, recombinantly produced collagen component provided in step a) takes place in step b) in the presence of divalent ions, preferably in the presence of calcium and/or magnesium.

In a particularly preferred embodiment of the present invention, the incubation of the at least one non-gelling, recombinant product provided in step a) is carried out produced collagen component in step b) in the presence of divalent ions, preferably in the presence of calcium and/or magnesium, in a concentration of 30 to 600 ppm, preferably 40 to 500 ppm, preferably 50 to 400 ppm.

Preferably, the counterion of the divalent ions, in particular calcium and/or magnesium, is Cl, SO4 or PO4.

In the presence of divalent ions, especially calcium and/or magnesium, with the corresponding counterions, a stabilizing effect on the formation of intermolecular bonds between the amino acid chains of at least two molecules of the at least one non-gelling collagen component has been observed.

According to a preferred embodiment of the present invention, the incubation of the at least one non-gelling, recombinantly produced collagen component provided in step a) takes place in step b) at a pH of at least 4, preferably at least 4.2, preferably at least 4.4, preferably at least 4.6, preferably at least 4.8, preferably at least 5, preferably at least 5.2, preferably at least 5.4, preferably at least 5.6, preferably at least 5.8, preferably at least 6.

In a further preferred embodiment of the present invention, the incubation of the at least one non-gelling, recombinantly produced collagen component provided in step a) takes place in step b) at a pH of at most 9, preferably at most 8.8, preferably at most 8.6, preferably at most 8.4, preferably at most 8.2, preferably at most 8, preferably at most 7.8, preferably at most 7.6, preferably at most 7.4, preferably at most 7.2, preferably at most 7.

Particularly preferably, the incubation of the at least one non-gelling, recombinantly produced collagen component provided in step a) takes place in step b) at a pH value in the range of 4 to 9, preferably 4.5 to 8.5, preferably 5 to 8, preferably 5.5 to 7.5, particularly preferably 6 to 7.

In a particularly preferred embodiment of the present invention, the at least one non-gelling, recombinantly produced collagen component provided in step a) has an average molecular weight in the range of 10 to 45 kDa, and the incubation in step b) takes place at a concentration of the at least one non-gelling, recombinantly produced Collagen component of at least 5 wt% (based on the total weight of the reaction mixture), at a pH of 5.5 to 7.5 and at a transglutaminase concentration of at least 0.05 vol% (based on the total volume of the reaction mixture).

According to a further particularly preferred embodiment of the present invention, the at least one non-gelling, recombinantly produced collagen component provided in step a) is hydroxylated, in particular partially or completely hydroxylated, has an average molecular weight in the range of 10 to 20 kDa, and the incubation in step b) takes place at a concentration of the at least one non-gelling, recombinantly produced collagen component of at least 10 wt.% (based on the total weight of the reaction mixture), at a pH of 5.5 to 7.5, and at a transglutaminase concentration of at least 0.05 vol.%, preferably at least 0.5 vol.% (based on the total volume of the reaction mixture).

In a preferred embodiment of the present invention, the gelling collagen component obtained in step c) has a Bloom value of at least 55, preferably at least 60, preferably at least 65, preferably at least 70, preferably at least 75, preferably at least 80, preferably at least 90, preferably at least 95, preferably at least 100, preferably at least 105, preferably at least 110, preferably at least 115, preferably at least 120, preferably at least 125, preferably at least 130, preferably at least 135, preferably at least 140, preferably at least 145, preferably at least 150, preferably at least 155, preferably at least 160, preferably at least 165, preferably at least 170, preferably at least 175, preferably at least 180, preferably at least 185, preferably at least 190, preferably at least 195, preferably at least 200.

According to a further preferred embodiment of the invention, the gelling collagen component obtained in step c) has a Bloom value of at most 500, preferably at most 475, preferably at most 450, preferably at most 425, preferably at most 400, preferably at most 375, preferably at most 350, preferably at most 325, preferably at most 300, preferably at most 290, preferably at most 280, preferably at most 270, preferably at most 260, preferably at most 250, preferably at most 240, preferably at most 230, preferably at most 220, preferably at most 210, preferably at most 200, preferably at most 190, preferably at most 180, preferably at most 170, preferably at most 160, preferably at most 150, preferably at most 140, preferably at most 130, preferably at most 120, preferably at most 110, preferably at most 100.

Particularly preferably, the gelling collagen component obtained in step c) has a Bloom value of 50 to 500, preferably 60 to 400, preferably 70 to 350, preferably 80 to 300, preferably 90 to 250, preferably 100 to 200.

According to a further preferred embodiment of the present invention, the gelling collagen component obtained in step c) has a Bloom value of 100 to 500, preferably 150 to 450, preferably 200 to 400, preferably 250 to 350.

In a further embodiment of the present invention, the gelling collagen component obtained in step c) has a viscosity of at least 2.5 mPas, in particular at least 5.0 mPas, in particular at least 10 mPas, preferably at least 15 mPas, preferably at least 20 mPas, preferably at least 25 mPas, preferably at least 30 mPas, preferably at least 35 mPas, preferably at least 40 mPas, preferably at least 45 mPas, preferably at least 50 mPas.

In a further embodiment of the present invention, the gelling collagen component obtained in step c) has a viscosity of at least 10 mPas, preferably at least 15 mPas, preferably at least 20 mPas, preferably at least 25 mPas, preferably at least 30 mPas, preferably at least 35 mPas, preferably at least 40 mPas, preferably at least 45 mPas, preferably at least 50 mPas at 50 °C.

According to a preferred embodiment of the present invention, the gelling collagen component obtained in step c) has an average molecular weight of at least 60 kDa, preferably at least 80 kDa, preferably at least 100 kDa, preferably at least 120 kDa, preferably at least 140 kDa, preferably at least 160 kDa, preferably at least 180 kDa, preferably at least 200 kDa, preferably at least 220 kDa, preferably at least 240 kDa, preferably at least 260 kDa, preferably at least 280 kDa, preferably at least 300 kDa, preferably at least 320 kDa, preferably at least 340 kDa, preferably at least 360 kDa, preferably at least 380 kDa, preferably at least 400 kDa.

In a preferred embodiment of the present invention, the gelling collagen component obtained in step c) comprises at most 25 wt.%, preferably at most 20 wt.%. %, preferably at most 15 wt.%, preferably at most 10 wt.%, preferably at most 5

wt%, collagen peptides with a molecular weight of < 45 kDa.

According to a further preferred embodiment, the gelling collagen component obtained in step c) comprises at most 15 wt.%, preferably at most 10 wt.%, preferably at most 5 wt.%, preferably at most 4 wt.%, preferably at most 3 wt.%, preferably at most 2 wt.%, collagen peptides with a molecular weight of < 30 kDa.

Particularly preferably, the gelling collagen component obtained in step c) comprises at most 10 wt.%, preferably at most 8 wt.%, preferably at most 6 wt.%, preferably at most 4 wt.%, preferably at most 2 wt.%, preferably at most 1 wt.%, collagen peptides with a molecular weight of < 15 kDa.

In a further preferred embodiment of the present invention, the gelling collagen component obtained in step c) comprises at least 75 wt%, preferably at least 80 wt%, preferably at least 85 wt%, preferably at least 90 wt%, preferably at least 95 wt%, cross-linked collagen peptides with a molecular weight of > 45 kDa, preferably > 90 kDa, particularly preferably > 180 kDa.

Preferably, the collagen component obtained in step c) is thermoreversibly gelling. According to this preferred embodiment, the gelling collagen component obtained in step c) can be dissolved again by heating and re-gelled.

The present invention also relates to a gelling collagen component with a Bloom value of at least 50, consisting of at least two covalently linked non-gelling collagen components.

Particularly preferred is the gelling collagen component with a Bloom value of at least 50, consisting of at least two covalently linked non-gelling collagen components, which can be produced, in particular, using the inventive method for producing a gelling collagen component.

Another aspect of the present invention relates to a consumable product comprising at least one gelling collagen component according to the invention, in particular at least one gelling collagen component that can be produced, and in particular produced, by the method according to the invention. In a preferred embodiment, the edible product also contains at least one natural or synthetically produced compound with at least one reactive group. Particularly preferred is the at least one natural or synthetically produced compound selected from at least one compound from a plant extract, especially polyphenols, pharmaceutical active ingredients, and dietary supplements.

According to a further preferred embodiment, the edible product is selected from the group consisting of hard capsules, soft capsules, foodstuffs, in particular confectionery, preferably gum sweets, in particular fruit gums, wine gums, foam gums, marshmallows, nougat, caramel candies, yogurt, foamed milk desserts, milk desserts in jelly form, spreads, meat and sausage products, broths and canned meats.

Preferably, the edible product contains, in addition to the at least one gelling collagen component according to the invention, in particular in addition to the at least one gelling collagen component that can be produced, in particular produced, using the method according to the invention, no naturally occurring collagen component, in particular no naturally occurring collagen and no collagen peptides of a naturally occurring collagen.

According to a preferred embodiment, the edible product contains, in addition to the at least one gelling collagen component according to the invention, in particular in addition to the at least one gelling collagen component that can be produced, in particular produced, using the method according to the invention, no naturally occurring or recombinantly produced collagen component.

In a preferred embodiment of the present invention, the edible product, in particular the confectionery, preferably the gum confectionery, in particular fruit gum, wine gum, foam gum, marshmallow, contains at least 2 wt.%, preferably at least 3 wt.%, preferably at least 4 wt.%, preferably at least 5 wt.%, preferably at least 6 wt.%, preferably at least 7 wt.%, preferably at least 8 wt.%, preferably at least 9 wt.%, preferably at least 10 wt.% (in each case weight percent based on the total weight of the edible product) of the at least one gelling collagen component according to the invention, in particular the at least one gelling collagen component that can be produced, in particular produced, by the method according to the invention. Preferably, the edible product, in particular the confectionery, preferably the gum confectionery, in particular fruit gums, wine gums, foam gums, marshmallows, contains at most 50 wt.%, preferably at most 45 wt.%, preferably at most 40 wt.%, preferably at most 35 wt.%, preferably at most 30 wt.%, preferably at most 25 wt.%, preferably at most 20 wt.%, preferably at most 15 wt.%, preferably at most 10 wt.%, preferably at most 9 wt.%, preferably at most 8 wt.%, preferably at most 7 wt.%, preferably at most 6 wt.% (each a weight percent based on the total weight of the edible product) of the at least one gelling collagen component according to the invention, in particular the at least one gelling collagen component that can be produced, in particular produced, by the process according to the invention.

The edible product, in particular the confectionery, preferably the gum confectionery, in particular fruit gum, wine gum, foam gum, marshmallow, particularly preferably contains 2 to 50 wt.%, preferably 2 to 40 wt.%, preferably 2 to 30 wt.%, preferably 2 to 25 wt.%, preferably 2 to 20 wt.%, preferably 2 to 15 wt.%, preferably 2 to 10 wt.%, preferably 2 to 8 wt.% (each wt. percent based on the total weight of the edible product) of the at least one gelling collagen component according to the invention, in particular the at least one gelling collagen component that can be produced, in particular produced, by the process according to the invention.

According to a preferred embodiment of the present invention, the edible product, in particular the confectionery, preferably the gum confectionery, in particular fruit gums, wine gums, foam gums, marshmallows, comprises at least one carbohydrate in an amount of at least 10 wt.%, preferably at least 15 wt.%, preferably at least 20 wt.%, preferably at least 25 wt.%, preferably at least 30 wt.%, preferably at least 35 wt.%, preferably at least 40 wt.%, preferably at least 45 wt.%, preferably at least 50 wt.% (each a weight percent based on the total weight of the edible product).

Another aspect of the present invention relates to a product selected from the group consisting of coatings, films, implants, injectables and additive manufacturing products, in particular 3D printed products, comprising at least one gelling collagen component according to the invention, in particular at least one gelling collagen component that can be produced, in particular produced, by the method according to the invention. The present invention further relates to the use of non-gelling collagen components to obtain gelling collagen components with a Bloom value of at least 50 by intermolecular bonding between the amino acid chains of at least two molecules of the at least one non-gelling collagen component.

Another aspect of the present invention relates to the use of a gelling collagen component according to the invention in a product selected from the group consisting of hard capsules, soft capsules, foodstuffs, in particular confectionery, coatings, films, implants, injectables and additive manufacturing products, in particular 3D printed products.

The embodiments described and statements made in connection with the inventive method also relate, mutatis mutandis, to the inventive gelling collagen component, the inventive edible products, the inventive products and the inventive uses, and vice versa.

In a preferred embodiment, the term "collagen" in connection with the present invention is understood in the manner customary in the trade, in particular as defined, for example, in WO 01/34646. In a preferred embodiment, the term "collagen" refers to collagen types I to XXVII. In a further preferred embodiment, the term "collagen" is understood to mean a peptide having the sequence glycine-proline, glycine-4-hydroxyproline, or glycine-X-4-hydroxyproline, preferably the repetitive motif (Gly-XY) _n , wherein X and Y can be any amino acid, preferably proline and 4-hydroxyproline. Particularly preferably, the term "collagen" is understood to mean a peptide having the repetitive motif (Gly-Pro-Y) _n and/or (Gly-X-Hyp) _m , wherein X and Y can be any amino acid.

The term “gelatin” in connection with the present invention is preferably understood in the manner customary in the trade, in particular as defined, for example, in WO 01/34646.

In connection with the present invention, the term "collagen peptide" preferably refers to a peptide that has an amino acid sequence occurring in collagen according to the above definition. A "collagen peptide" preferably also refers to a genetically modified collagen peptide obtained by modifying the amino acid sequence of a naturally occurring collagen peptide. The term “collagen peptide” is used in connection with the present invention without any limitation to a specific length of the amino acid chain or without any limitation to a specific molecular weight to denote a peptide or protein that has an amino acid sequence occurring in collagen according to the foregoing definition.

According to the invention, a "collagen component" is understood to be a single collagen peptide or a plurality of collagen peptides, which may be covalently or non-covalently linked to one another. In a preferred embodiment, the "non-gelling collagen component" is a single recombinantly produced collagen peptide with a specific molecular weight, a mixture of several single recombinantly produced collagen peptides, each with a specific molecular weight, or a hydrolysate obtained from at least one recombinantly produced collagen peptide with a specific molecular weight, comprising a plurality of collagen peptides with different molecular weights. A "non-gelling collagen component" according to the present invention is characterized by the fact that it is unable to form a viscoelastic network after cooling. In particular, a "non-gelling collagen component" according to the present invention is characterized by a Bloom value of less than 50.

In the context of the present invention, a “recombinantly produced collagen peptide” is understood to be a collagen peptide encoded by recombinant DNA.

According to the invention, "conditions leading to an intermolecular bond between the amino acid chains of at least two molecules of the at least one collagen component" are understood to mean conditions such as, in particular, temperature, pressure, time, light, and the presence or absence of additives such as salts, buffers, or cofactors, which are required for the formation of a covalent or non-covalent, preferably a covalent, intermolecular bond between the amino acid chains of at least two molecules of the at least one collagen component. The specific "conditions leading to an intermolecular bond between the amino acid chains of at least two molecules of the at least one collagen component" depend on whether the intermolecular bond in step b) of the process according to the invention is formed, for example, by means of a chemical or enzymatic crosslinker, by dehydrothermal crosslinking, UV irradiation, or electrostatic interaction. The specific conditions required to The methods for enabling an intermolecular bond between the amino acid chains of at least two molecules are known to those skilled in the art and depend on the method used.

The "bloom value" is a well-established unit of measurement for the gelling strength, or gel consistency, of gelatin or collagen peptides. By definition, the bloom value is the mass in grams (g) required for a 0.5-inch diameter stamp to deform the surface of a 6.67% gelatin/water mixture to a depth of four millimeters (mm) without tearing it. The measurement is standardized and performed at 10°C after the gelatin has been aged for 17 hours.

According to the invention, a "bloom value equivalent" is a value determined using a regression curve that corresponds to the "bloom value." To determine the "bloom value equivalent," the force (normal force FN) required to deform a gel surface by 0.5 mm using a 12.5 mm diameter die is measured. The normal force FN is measured using a rheometer (MCR 302e, Anton Paar Germany GmbH) at 10 °C and a defined aging process of the samples for 17 hours at 4 °C. Using different gelatin samples, each with a known "bloom value," the function of a regression curve can be determined by measuring the normal force FN as described above. The "bloom value equivalent" can then be calculated from the measured normal force FN of samples with an unknown "bloom value" using this regression curve. The “bloom value equivalent” is used in particular to determine the gelling strength or gel consistency or gel strength in the case of particularly small sample volumes.

Unless otherwise specified, “viscosity” is defined as the dynamic viscosity of a 6.67% aqueous solution at 60°C and expressed in mPa (according to GME Gelatine Monograph for the testing of edible gelatine).

According to the invention, "semi-stable" is understood to mean the sensorily detectable property of a gel to lose a significant amount of strength/structure at room temperature compared to its behavior at 4 °C. A "semi-stable" gel at room temperature is also characterized by its high shear sensitivity and partial liquefaction. In the context of the present invention, the terms "comprising" and "comprising" are understood to mean that, in addition to the elements explicitly covered by these terms, further, unmentioned elements may be present. In the context of the present invention, these terms are also understood to mean that only the explicitly mentioned elements are covered and no further elements are present. In this particular embodiment, the meaning of the terms "comprising" and "comprising" is synonymous with the term "consisting of." Furthermore, the terms "comprising" and "comprising" also encompass compositions that, in addition to the explicitly mentioned elements, contain further unmentioned elements that are, however, of a functionally and qualitatively subordinate nature. In this embodiment, the terms "comprising" and "comprising" are synonymous with the term "essentially consisting of."

In the context of the present invention, the term "and/or" means that all members of a group connected by the term "and/or" are disclosed both alternatively to one another and cumulatively to one another in any combination. For the expression "A, B and/or C", this means that the following disclosure content is to be understood: a) A or B or C or b) (A and B) or c) (A and C) or d) (B and C) or e) (A and B and C).

Further preferred embodiments are set forth in the dependent claims.

The invention is illustrated below, without limiting the general concept of the invention, by means of an illustration and exemplary embodiments.

This shows

Figure 1 shows the column chromatographic separation of a non-gelling collagen component (2) and a gelling collagen component (1) obtained from it by the inventive method.

Figure 2 shows the column chromatographic separation of a non-gelling collagen component (2), a gelling collagen component (1) obtained from it by the inventive method and a natural gelatin of animal origin (3). Figure 3 shows the molecular weight distribution in a hydrolysate of a recombinantly produced, hydroxylated collagen peptide with a mean molecular weight of 38 kDa (non-gelling collagen component).

Figure 4 shows the molecular weight distribution in a cross-linked, gelling collagen component produced using the inventive method.

Figure 5 shows the molecular weight distribution in a natural gelatin of animal origin with a Bloom value of 90.

Figure 6 shows the regression curve for determining the “Bloom value equivalent” from the measured normal force FN.

Example 1:

1.1 Cross-linking of recombinantly produced, hydroxylated collagen peptides of a hydrolysate

10 g of a hydrolysate of a recombinantly produced, hydroxylated collagen peptide with a mean molecular weight of 38 kDa were dissolved in 90 g of water at 50 °C. Subsequently, 0.1% (0.1 g) of transglutaminase WM (Ajinomoto) (TG) based on the total weight of the solution was added. The solution was stirred on a hot plate for 3 minutes. In the next step, approximately 20 g of the solution was filled into the cylinder of the Brookfield UL adapter, and the viscosity was measured on a rotational viscometer at 50 °C (instrument setting: 50.5 °C).

The remaining solution was placed in a water bath at 50 °C for 2 hours. After 2 hours, the solution was removed from the water bath, cooled to room temperature (by standing at room temperature), and refrigerated overnight. The following morning, the solution or gel was assessed (gelled, liquid, etc.).

Table 1: Viscosity measurement

1.2 Cross-linking of a single recombinantly produced, non-hydroxylated

Collagen peptides

The recombinantly produced, non-hydroxylated collagen peptide with a

A molecular weight of 16 kDa was cross-linked with transglutaminase (0.1%) as described in Example 1.1, prepared for viscosity measurement and measured.

Table 2: Viscosity measurement

1.3 Cross-linking of a commercially available collagen peptide of animal origin

The collagen peptide with a mean molecular weight of 12 kDa (Peptiplus XB - UHV 895091) was cross-linked with transglutaminase (TG) (0.1%) as described in Example 1.1, prepared for viscosity measurement, and measured. The pH of the solution was adjusted from 5.82 to 6.44.

Table 3: Viscosity measurement

Example 2:

Cross-linking of recombinantly produced, hydroxylated collagen peptides of a hydrolysate and subsequent granulation

200 g of a hydrolysate of a recombinantly produced, hydroxylated collagen peptide with an average molecular weight of 38 kDa were placed in a 2 L beaker and made up to 1000 g with hot tap water. The 20% solution was then stirred with a magnetic stir bar at 50 ± 1 °C until no lumps were visible. The pH of the solution was then adjusted to 6.2 ± 0.1 with 25% NaOH solution. Cross-linking was initiated by adding 1 ml of Stabizym TGL (liquid transglutaminase, Stabizym GmbH, Roßdorf, Germany) to achieve a final concentration of 0.1% of the solution. After 3 min, 20 ml of the solution were withdrawn using a Finn pipette and pipetted into the cylinder of the Brookfield UL adapter. The crosslinking process was monitored over time by measuring the viscosity at 50 °C (50.5 °C device setting).

Table 4: Viscosity measurement

When stirring the solution with a spatula in the beaker, it felt more viscous than its viscosity would suggest. Therefore, the solution was removed from the stirring plate and placed in a preheated 90 °C water bath. This allowed the solution to be quickly heated to 75–80 °C and the enzyme to be inactivated. The solution was then transferred to a photographic dish. The gelatin was poured into a container and cooled to room temperature. The container was then placed in the refrigerator overnight for further cooling. The cooled gelatin was cut into strips and ground using the meat grinder attachment of a KitchenAid® stand mixer (5-6 mm grinding plate). The gelatin was then dried on a wooden frame covered with insect netting at 20°C and 20% relative humidity for three days. Finally, the gelatin was crushed by hand in a plastic bag. The yield was 184.5 g.

Table 5: Viscosity measurement and determination of the Bloom value

Example 3:

Maximum crosslinking of recombinantly produced, hydroxylated collagen peptides of a hydrolysate with 0.1% TG WM based on solution

30 g of a hydrolysate of a recombinantly produced, hydroxylated collagen peptide with an average molecular weight of 38 kDa were weighed into a 600 ml beaker, and 261 g of hot tap water were added. The powder was completely dissolved on a hot plate at 50 ± 2 °C (contact thermometer) while stirring with a magnetic stir bar. The pH of the solution was 6.17. 0.3 g of transglutaminase WM (Ajinomoto) were weighed into a 50 ml beaker and dissolved in 20 ml of demineralized water while stirring with a small magnetic stir bar. The solution appeared slightly opaque. The enzyme solution was then poured into the collagen peptide solution. The remaining 19 g of water were used to rinse the beaker and also added to the solution (total mass 300 g). The solution in the beaker therefore had a concentration of 10% (not including the moisture content of the powder). The crosslinking reaction and time measurement started with the addition of the enzyme. After 7 minutes, 20 ml of solution were taken with a Finn pipette and filled into the cylinder of the Brookfield UL adapter. Viscosity measurement on the rotational viscometer (50 °C, instrument setting 50.5 °C) was started immediately. After a total of 10 minutes, the first viscosity value was read.

Table 6: Viscosity measurement

After 122 minutes, the significant increase in viscosity indicated that the solution in the cylinder was beginning to cross-link completely. The solution on the hot plate could still be stirred easily with a magnetic stir bar. Heating to 80 °C was immediately initiated to inactivate the transglutaminase (maximum energy input, contact thermometer set to 85 °C). After 9 minutes, 82 °C was reached. The solution in the beaker could still be stirred easily.

At approximately 50 °C, the solution was diluted to a final concentration of 6.67%, and 105 g were weighed into each of two Bloom glasses. The Bloom glasses were placed in a 10 °C water bath. The Bloom measurement was taken the following morning (after approximately 17 hours). As a control sample, a 6.67% solution of the uncrosslinked starting hydrolysate was prepared, and 105 g were also weighed into a Bloom glass and placed in the 10 °C water bath to temper.

Table 7: Determination of the Bloom value

Example 4:

Comparison of the properties of a gelling collagen component produced according to the invention with those of natural gelatin

The properties of a gelling collagen component (sample) produced by the method according to Example 2 from a hydrolysate of a recombinantly produced, hydroxylated collagen peptide with a mean molecular weight of 38 kDa were compared with the properties of a gelatin of animal origin with a Bloom value of 90 (control). Results of the viscosity and Bloom value determinations depending on the concentration used are shown in Table 8 below.

Table 8: Viscosity measurement and determination of the Bloom value of a material prepared with the inventive device

Process-produced gelling collagen component (P) compared with animal gelatin (K)

It is shown that gelling collagen components (sample) can be produced using the inventive method, which in different concentrations have Bloom values comparable to natural gelatin (control), but tend to have a higher viscosity at 60 °C.

Example 5:

Column chromatographic separation of a non-gelling collagen component and a gelling collagen component derived therefrom

The separation of a non-gelling collagen component (hydrolysate of a recombinantly produced, hydroxylated collagen peptide with an average molecular weight of 38 kDa; Bloom: 20 g) and a gelling collagen component produced by the inventive process (cross-mixing product using 0.1% transglutaminase; Bloom: 80 g) was carried out using a column (guard column: TSKgel SWXL Guardcolumn, Tosoh Bioscience GmbH; separation column: TSK 4000 SWXL, Tosoh Bioscience GmbH, particle size 8 pm, pore size 450 Å). For this purpose, 1 g ± 0.1 g of the respective collagen component and 99 g ± 1 g of distilled water were first weighed into a 150 ml beaker. After swelling for 30 min at room temperature, the components were completely dissolved in a water bath at approximately 60 °C. The 1% sample solutions were filtered through a 0.2 pL disposable filter. Subsequently, 30 pL of sample solution + 600 pL of SDS buffer + 30 pL of benzoic acid were pipetted into a reaction vessel and mixed. The subsequent determination was carried out using a column (stationary phase: TSK Gel 4000 SWXL with guard column) with SDS buffer as the mobile phase and at a flow rate of 0.5 l/min. mL/min at a detector at a wavelength of 214 nm. The chromatogram of the non-gelling collagen component (2) and the gelling collagen component (2) is shown in Fig. 1.

Example 6:

Column chromatographic separation of a non-gelling collagen component and a gelling collagen component derived therefrom

The separation of a non-gelling collagen component (hydrolysate of a recombinantly produced, hydroxylated collagen peptide with an average molecular weight of 38 kDa), a gelling collagen component produced by the inventive process (cross-linking product using 0.1% transglutaminase (Stabizyme)), and a natural gelatin of animal origin was carried out as described in Example 5 using a column. The chromatogram of the non-gelling collagen component (2), the gelling collagen component (1), and the natural gelatin of animal origin (3) is shown in Figure 2.

Example 7:

Determination of the molecular weight distribution

Subsequently, the molecular weight distribution in the non-gelling collagen component, the gelling collagen component produced by the inventive method, and the natural gelatin of animal origin from Example 6 was determined. The results are shown in Table 9 below and Figures 1 to 3.

Table 9: Molecular weight distribution in various collagen peptide preparations

Example 8:

Gel properties of different gelling collagen components produced using the inventive method

The gel properties of different gelling collagen components produced using the inventive method were evaluated in the following experimental setup. Stock solutions were prepared to test various concentration levels of the collagen peptides. 5 g (= 20%), 2.5 g (= 10%), 1.563 g (= 6.25%), and 0.625 g (= 2.5%) of each collagen peptide were placed in 50 ml centrifuge tubes and filled to the required level with 25 ml of water. The stock solutions were then incubated on a heated shaker at 50 ± 1 °C and 1000 rpm until the collagen peptides were completely dissolved. After cooling to room temperature, the pH was adjusted to 6.2 ± 0.1 by adding N₂NaOH solution. Using a micropipette (Research Plus, Eppendorf SE), 1 ml of each stock solution was transferred into a 3 ml snap-top vial. The corresponding volume of transglutaminase (Stabizym TGL liquid transglutaminase, Stabizym GmbH, Roßdorf, Germany) was then added: 0.5 pL (= 0.05%), 1 pL (= 0.1%), 2 pL (= 0.2%), 5 pL (= 0.5%), 10 pL (= 1%), 50 pL (= 5%), and 100 pL (= 10%). The samples were then incubated for 70 minutes at 50 ± 1 °C. After 70 minutes, the samples were incubated at 75 ± 1 °C for 10 minutes to inactivate the transglutaminase. Following cross-linking, the experimental samples were stored in a refrigerator at 4 °C for 17 hours.

After storage, the gel properties were sensorially evaluated at 4 °C. A measurement method was established to assess the gel thickness on a 1 ml volume scale, providing a relative estimate of the known "Bloom value method" as a "Bloom value equivalent." For this purpose, the force (normal force FN) required to deform the gel surface by 0.5 mm using a 12.5 mm diameter stamp is determined. The measurement is performed using a rheometer (MCR 302e, Anton Paar Germany GmbH) at 10 °C, with a predefined aging of the samples for 17 h at 4 °C. To enable a relative correlation with the "Bloom value," the measurement method was calibrated using reference samples. For this purpose, a 6.67% gelatin/water mixture of various gelatins derived from pigskin (SSW) with different Bloom values (Bloom: 103 g, 146 g, 157 g, 202 g, 248 g, 250 g, 260 g, 272 g) was prepared. One ml of each mixture was transferred into 3 ml snap-top vials and stored in a refrigerator at 4 °C for 17 hours. The samples were measured using the rheometer as described previously, and the Bloom values were plotted against the respective measured normal force FN (see Figure 6 and Table 10). A calibration function was created using a regression model (third-degree polynomial), which forms the basis for calculating the "Bloom value equivalents."

Table 10: Data for determining a regression curve

The function of the regression curve was determined to be y = 2.43E-06x ³ -0.001412x ² +0.2749x-14.43.

The coefficient of determination of the regression curve was R ² = 0.97.

The gelling behavior at room temperature (RT) was evaluated sensorially after storing the samples at 21 °C for 1 h. The measurement data and results are listed in Table 11 below.

Table 11: Determination of the gel properties of different gelling collagen components produced using the inventive method

* “KP1” is the hydrolysate of the recombinantly produced, hydroxylated collagen peptide with an average molecular weight of 38 kDa from Examples 1.1 and 2 to 7. “KP2” is the recombinantly produced, non-hydroxylated collagen peptide with a molecular weight of 16 kDa from Example 1.2.

Example 9:

Production of a fruit gum composition comprising gelling collagen components produced by the inventive method

Fruit gum compositions containing gelling collagen components produced using the inventive method were prepared and analyzed analogously to the experimental setup described in Example 8. Unlike the formulations described previously, the stock solutions of a recombinantly produced, hydroxylated collagen peptide with a mean molecular weight of 38 kDa were prepared in 25 ml of water with additional additives. In variant A, a 46% glucose solution with 0.18% LPE Orange (food coloring safflower) was used as the additive, and in variant B, a 23% glucose solution with 0.18% LPE Orange (food coloring safflower) was used. The results of the analyses are summarized in Table 12 below. Table 12: Determination of the gel properties of fruit gum compositions comprising gelling collagen components produced by the inventive method

* Additive A consisted of 0.18% LPE Orange (food coloring safflower) and 46% glucose. Additive B consisted of 0.18% LPE Orange (food coloring safflower) and 23% glucose. Additive A was 0.18% LPE Orange (food coloring safflower) and 46% glucose.

Claims

REQUIREMENTS 1. Method for producing a gelling collagen component, comprising the steps of: a) providing at least one non-gelling, recombinantly produced collagen component, b) incubating the at least one non-gelling, recombinantly produced collagen component provided in step a) under conditions that result in an intermolecular bond between the amino acid chains of at least two molecules of the at least one collagen component, c) obtaining a gelling collagen component with a Bloom value of at least 50. 2. The method according to claim 1, wherein the at least one non-gelling collagen component is a recombinantly produced collagen peptide with a molecular weight in the range of 10 to 45 kDa, preferably 20 to 40 kDa. 3. Method according to claim 1 or 2, wherein the at least one non-gelling collagen component is hydroxylated, in particular partially or completely hydroxylated. 4. Method according to claim 2 or 3, wherein the recombinantly produced collagen peptide is a genetically modified collagen peptide in which at least 10%, preferably at least 25%, preferably at least 50%, preferably at least 75% of the lysines in the amino acid sequence of a naturally occurring collagen peptide, preferably a bovine or porcine type I or type III collagen, are exchanged by another naturally occurring amino acid. 5. The method of claim 4, wherein the other naturally occurring amino acid is an amino acid selected from the group consisting of arginine, threonine, glutamine, proline, glutamic acid, alanine and leucine. 6. Method according to any one of claims 1 to 5, wherein the intermolecular bond between the amino acid chains of at least two molecules in step b) is effected by the presence of at least one chemical or enzymatic crosslinker. 7. Method according to any one of claims 1 to 5, wherein the intermolecular bond between the amino acid chains of at least two molecules in step b) is formed by dehydrothermal crosslinking, UV irradiation or by electrostatic interaction. 8. Method according to any one of claims 1 to 7, wherein the intermolecular bond between the amino acid chains of at least two molecules in step b) is a covalent bond. 9. Method according to any one of claims 1 to 8, wherein in step b) the intermolecular bond between the amino acid chains of at least two molecules is a chain elongation, in particular a peptide ligation. 10. Method according to any one of claims 1 to 9, wherein in step b) the intermolecular bond between the amino acid chains of at least two molecules is a cross-linking between side chains. 11. Gelling collagen component with a Bloom value of at least 50, consisting of at least two covalently linked non-gelling, recombinantly produced collagen components. 12. Gelling collagen component according to claim 11, produced by a method according to any one of claims 1 to 10. 13. A consumable product comprising at least one gelling collagen component according to claim 11 or 12. 14. Consumable product according to claim 13, wherein the product further comprises at least one natural or synthetically produced compound with at least one reactive group, in particular at least one natural or synthetically produced compound selected from at least one compound from a plant extract, in particular polyphenols, pharmaceutical active ingredients and food supplements. 15. A consumable product according to claim 13 or 14, selected from the group consisting of hard capsules, soft capsules, foodstuffs, in particular confectionery, preferably sweet gums, in particular fruit gums, wine gums, foam gums, marshmallows, Nougat, caramel candies, yogurt, foamed milk desserts, jelly milk desserts, spreads, meat and sausage products, broths and canned meats. 16. Product selected from the group consisting of coatings, films, implants, injectables and additive manufacturing products, in particular 3D printed products, comprising at least one gelling collagen component according to claim 11 or 12. 17. Use of non-gelling, recombinantly produced collagen components to obtain gelling collagen components with a Bloom value of at least 50 by intermolecular bonding between the amino acid chains of at least two molecules of the at least one non-gelling collagen component. 18. Use of a gelling collagen component according to claim 11 or 12 in a product selected from the group consisting of hard capsules, soft capsules, food products, in particular confectionery, coatings, films, implants, injectables, and additively manufactured products, in particular 3D-printed products. ```