EP3847190A1

EP3847190A1 - Recombinant production of a collagen peptide preparation and use thereof

Info

Publication number: EP3847190A1
Application number: EP19804652.6A
Authority: EP
Inventors: Stephan Hausmanns; Hans-Ulrich Frech; Steffen Oesser; Martin Hahn
Original assignee: Gelita AG
Current assignee: Gelita AG
Priority date: 2018-11-06
Filing date: 2019-11-06
Publication date: 2021-07-14
Also published as: CN113272321A; WO2020094728A1; CA3118176A1; EA202190906A1; US11673940B2; DE102019202606A1; CN113272321B; AU2019375455A1; JP2022513591A; US20210309723A1; KR20210090628A; BR112021005037A2; MX2021005254A

Abstract

The present invention relates to a method for producing recombinant collagen peptide preparations comprising recombinant collagen peptides, to collagen peptide preparations produced by means of said methods, to products containing the collagen peptide preparations, and to uses of the aforementioned preparations and products.

Description

DESCRIPTION

Recombinant production of a collagen peptide preparation and its use

The present invention relates to methods for producing collagen peptide preparations containing recombinant collagen peptides, the collagen peptide preparations produced by means of these methods, products containing the collagen peptide preparations and uses of the aforementioned preparations and products.

Collagen is an extracellular structural protein that is found in animals, for example in mammals, birds and fish. It is usually found there in the connective tissue, particularly as part of the extracellular matrix. Tendons, ligaments, cartilage and bones are particularly rich in collagen. However, collagens are not found in plants and unicellular organisms.

Collagens occur in different, structurally and functionally different types and differ among other things in terms of their structure, function and origin. The collagen-forming polypeptide chains are synthesized in the cell individually on the ribosomes of the endoplasmic reticulum in the form of larger precursor molecules and have extensive repetitive (Gly-XY) _n sequences, where X and Y can be any amino acid, but mostly proline and 4- Are hydroxyproline.

These precursor polypeptide chains are post-translationally hydroxylated on proline and fysin residues of the polypeptide chain in the endoplasmic reticulum to form hydroxyproline and hydroxylysine residues. The hydroxylation serves to stabilize neighboring collagen polypeptide chains of the right-handed triple helix formed in the cell from three of the precursor polypeptide chains (procollagen).

The procollagen so formed is intracellularly glycosylated, in the glycosylated triple helical form (tropocollagen) secreted by the cell and then formed by peptidase-mediated cleavage of the terminal residues collagen. In the course of a fibrillogenesis process, this assembles into collagen fibrils, which are then covalently cross-linked and form collagen fibers. Collagen is often also used in denatured form, which is then referred to as gelatin, or in the form of hydrolyzates.

If gelatin and collagen are subjected to hydrolytic processes, in particular an enzymatic hydrolysis, depending on the type of collagen used and its origin as well as enzymatic conditions, collagen hydrolyzates with a wide variety of compositions and application profiles can be produced. These collagen hydrolyzates are a mixture of peptides, the molecular weights of which are distributed over certain size ranges. The use of such collagen hydrolyzates, for example as a dietary supplement or as a cosmetic aid, has long been known, inter alia for the prevention and / or treatment of complaints which are associated with the bones, the joints or the connective tissue.

For example, WO 2012/065782 describes collagen hydrolyzates obtained from porcine rind gelatin, which are used to stimulate the biosynthesis of extracellular matrix proteins by skin cells and are particularly suitable for cosmetic purposes.

WO 2012/117012 discloses enzymatically hydrolyzed collagen from bovine split with an average molecular weight of 1500 to 8000 Da, which can be used together with a prebiotic for the prevention and / or treatment of osteoporosis.

Although the use of collagen hydrolysates derived from animal materials has advantages for many applications and consumer groups, the use of collagen hydrolyzates obtained in this way can also be less desirable with regard to certain consumer groups and application profiles. Certain groups of consumers from raw materials derived from animal materials are fundamentally critical or opposed, be it that contamination with health-threatening microorganisms or agendas, for example process aids, or unwanted immune reactions, or fear of religious or ethical motives. In addition, the manufacturing processes used for the production of collagen hydrolyzates obtained from animal materials often include complex and expensive digestion, cleaning and further processing steps. Finally, it can make sense for certain applications to provide a collagen hydrolyzate that is standardized, exactly and reliably defined with regard to its origin and composition, which can advantageously also be produced economically on an industrial scale. Against this background, it is not surprising that processes have been developed to produce gelatin and collagen as well as hydrolyzates thereof using recombinant genetic engineering.

For example, WO 2006/052451 A2 discloses the production of recombinant type III collagen in Pichia pastoris strains which also express human prolyl hydroxylases.

WO 2005/012356 A2 discloses the production of gelatin from human collagen type I and individual 50 kDa, 65 kDa and 100 kDa collagen peptide species, in each case in fully hydroxylated, partial and non-hydroxylated form.

WO 01/34646 A2 also discloses the production of individual recombinant gelatin species with a defined molecular weight which results in each case from the recombinant production route and which can be present in non-hydroxylated, partially or fully hydroxylated form.

However, the production of recombinant collagen or hydrolyzates thereof, which are distinguished by structural and functional properties which are similar or at least similar to those of collagen or collagen hydrolysis obtained from natural sources, is problematic. This is partly because of the natural formation Collagen is a comparatively complex physiological process characterized by a number of intra- and extracellular influencing factors, which also includes post-translational synthetic steps such as glycosylation and hydroxylation. These post-translational synthetic steps, in particular the precise positioning and the extent of the hydroxylation of proline and lysine, ensure the provision of stable tropocollagen, which ultimately associates with fibrils and fibers. Wang et al. (Engineering Biology, 2017 (1), 18-23) known that the current recombinant production of collagen is characterized by low yields, high costs and in particular by missing or different post-translational synthetic steps as occur in the formation of native collagen. It is also known that precisely these post-translational modifications are essential for applications which utilize the natural structure and function of collagen as well as for collagen or collagen hydrolysis.

Accordingly, to date no recombinantly produced collagens or collagen hydrolyzates are known which have a structure, in particular quality and quantity post-translational modifications, in particular the degrees of hydroxylation and glycosylation as well as the positions of hydroxylation and glycosylation, which are identical to natural collagen or collagen hydrolysed therefrom.

The provision of recombinantly produced collagen and collagen hydrolyzates with a property potential specifically derived from conventionally produced collagen or collagen hydrolyzates is not readily possible, inter alia due to the circumstances described above, in particular the diversity of the starting materials and production processes. In prokaryotic organisms in particular, which are suitable per se for the industrial production of recombinant proteins, the production of recombinant collagen is problematic, among other things, in that the post-translational synthetic steps usually have to be introduced into the cell by recombinant technologies and this results in an additional metabolic load which makes the expression and recovery of the desired collagen peptides difficult or impossible. In addition, the expression of foreign proteins can be toxic to the host cell, the production of recombinantly produced proteins in or from host cells can prove to be technically or economically impossible, the stability of the expression product obtained can be too low or other effects such as growth and reproductive disorders of the host cell occur.

There is therefore still a great need for the provision of recombinantly produced collagen hydrolyzates for applications in a wide variety of areas, in particular also for therapeutic purposes, in particular for the prophylaxis or treatment of conditions or diseases, the muscles, joints, bones and skin of humans and animals affect.

The present invention is therefore based on the technical problem of providing methods for producing collagen peptide preparations and the recombinant collagen peptide preparations thus obtained which overcome the abovementioned disadvantages, in particular those which are recombinant in a standardized, reliable and precisely defined form, even on a larger industrial and inexpensive scale, can be produced and the collagen hydrolyzates obtained from animal materials have comparable, in particular improved properties, in particular effectiveness, in particular biological activity in relation to maintaining the health of muscles, Joints, bones and skin as well as for the prophylaxis or treatment of diseases affecting the muscles, joints, bones and skin of humans and animals.

The present invention solves the underlying technical problem by providing the teachings of the independent claims, particularly the teachings of the preferred embodiments in the description and the appended claims.

The present invention relates in particular to a method for producing a collagen peptide preparation comprising recombinant collagen peptides, comprising the method steps a) providing an expression system which has at least one expression cassette, the expression cassette having at least one nucleotide sequence which has a collagen peptide with a molecular weight in one region from 8 to 100 kDa, b) incubating the expression system under conditions that allow expression of the collagen peptide, c) recovering the collagen peptide, d) hydrolyzing the collagen peptide under conditions that lead to the production of a collagen peptide preparation, the collagen peptide having an average molecular weight of 1 to 7 kDa and with a molecular weight in a range from 0.1 to 13.5 kDa and e) recovering the collagen peptide preparation.

The method according to the invention for the production of collagen peptide preparations is characterized in particular by the fact that at least one, preferably precisely defined, recombinantly produced collagen peptide of a specific size from 8 to 100 kDa is provided by hydrolysis to produce a recombinantly produced collagen peptide preparation which has a molecular weight distribution and structure, which advantageously result from the recombinant specific collagen peptide species used for the hydrolysis and the subsequent process, in particular the hydrolysis step, in particular

Hydroxylation profile, and which result despite its recombinant production characterized immediately and without further processing steps by an advantageous biological activity.

Due to their recombinant production method, the collagen peptide preparations provided according to the invention have clear differences in their structure, in particular with regard to the modifications introduced by post-translational synthetic steps, such as hydroxylations and glycosylations, to collagen hydrolyzates obtained from natural sources. Surprisingly, they can be provided in a wide variety of expression systems even on an industrial scale without undesirable contamination, while at the same time they have advantageous biological activity, in particular with regard to applications for maintaining and improving the health of bones, cartilage, skin, hair and nails.

The biological activity found according to the invention of the recombinant collagen peptide preparations provided here already applies to the preparations obtained directly from the hydrolysis, without the need for further processing steps.

The biological activity found according to the invention and attributable to the recombinantly produced collagen peptide preparations of the present invention can be determined in particular using in vitro tests for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, preferably using in vitro tests for stimulating the synthesis of extracellular matrix proteins or mRNA encoding these proteins in osteoblasts, fibroblasts and chondrocytes, in particular using the in vitro tests shown in Examples 3 to 7, in particular Examples 3 to 5, for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes.

In a preferred embodiment, the recombinant collagen peptide preparations of the present invention prepared according to the invention have in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one, preferably at least two, preferably in all, of the Examples 3 to 7, in particular Examples 3 to 5, shown in vitro tests for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, have a biological effectiveness. The recombinant collagen peptide preparations of the present invention which are produced according to the invention preferably have in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one, preferably in at least two, preferably in all, of the Examples 3 to 7, in particular Examples 3 to 5, presented in vitro tests for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, the same biological activity as collagen peptide preparations isolated from natural sources, in particular non-recombinantly produced collagen peptide preparations.

The recombinant collagen peptide preparations of the present invention which are produced according to the invention particularly preferably have in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one, preferably in at least two, preferably in all, of the Examples 3 to 7, in particular Examples 3 to 5, shown in vitro tests for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, have a better biological effectiveness than collagen peptide preparations isolated from natural sources, in particular non-recombinantly produced collagen peptide preparations.

In a preferred embodiment of the present invention, the expression system provided in step a) is a cell-based or cell-free expression system.

The expression system provided in step a), in particular the cell-based expression system, is preferably a host cell, in particular a prokaryotic or eukaryotic cell.

The expression system, in particular the cell-based expression system, is preferably a host cell selected from the group consisting of bacterial cell, yeast cell, fungal cell, mammalian cell, insect cell and plant cell.

The expression system, in particular the cell-based expression system, is preferably a bacterial cell, in particular of the species Escherichia coli or Bacillus subtilis. In a further preferred embodiment, the expression system, in particular the cell-based expression system, is a yeast cell, in particular of the species Saccharomyces cerevisiae, Pichia pastoris or Ogataea angusta (Hansenula polymorpha).

The expression system, in particular the cell-based expression system, is preferably a fungal cell, in particular of the type Aspergillus niger.

In a further preferred embodiment of the present invention, the expression system, in particular the cell-based expression system, is a mammalian cell, in particular a CHO cell, a HeLa cell or a HEK293 cell.

The expression system, in particular the cell-based expression system, is preferably an insect cell, in particular an Sf-9, Sf-2l or Tn-5 cell.

The expression system, in particular the cell-based expression system, is preferably a plant cell, in particular a maize or tobacco cell.

In a further preferred embodiment of the present invention, the expression system provided in step a) is an expression system, in particular a cell-based expression system, which is capable of hydroxylating proline, lysine or proline and lysine residues of the expressed collagen peptide. The expression system provided in step a) is preferably a host cell which is capable of hydroxylating proline, lysine or proline and lysine residues of the expressed collagen peptide.

The expression system provided in step a) is preferably an expression system, in particular a cell-based expression system, which has prolyl hydroxylase and / or lysyl hydroxylase activity. The expression system provided in step a) is preferably a host cell which has prolyl hydroxylase and / or lysyl hydroxylase activity.

In a preferred embodiment, the expression system provided in step a) is a cell-based expression system which has at least one expression cassette which comprises a polynucleotide sequence which codes for prolyl-4-hydroxylase. The expression system provided in step a) is particularly preferably a cell-based expression system which has at least one expression cassette which encodes a prolyl-4-hydroxylase Includes polynucleotide sequence, so that an in vivo hydroxylated collagen peptide preparation is obtained in process step e).

In a preferred embodiment, the expression system provided in step a) is a cell-based expression system which has at least one expression cassette which comprises a polynucleotide sequence coding for lysyl hydroxylase. The expression system provided in step a) is particularly preferably a cell-based expression system which has at least one expression cassette which comprises a polynucleotide sequence encoding lysyl hydroxylase, so that an in vivo hydroxylated collagen peptide preparation is obtained in process step e).

In a further preferred embodiment of the present invention, the expression system provided in step a) is a cell-based expression system which has at least one expression cassette which comprises a polynucleotide sequence coding for prolyl 4-hydroxylase and at least one expression cassette which comprises a polynucleotide coding for lysyl hydroxylase. The expression system provided in step a) is particularly preferably a cell-based expression system which has at least one expression cassette which comprises a polynucleotide sequence encoding prolyl-4-hydroxylase and at least one expression cassette which comprises a polynucleotide sequence encoding lysyl hydroxylase, so that the method step e) an in vivo hydroxylated collagen peptide preparation is obtained.

Accordingly, the present invention also includes a method for producing a collagen peptide preparation comprising recombinant collagen peptides, in particular an in vivo hydroxylated collagen peptide preparation, comprising the method steps a) providing a cell-based expression system which has at least one expression cassette, the expression cassette having at least one nucleotide sequence which Encoded collagen peptide with a molecular weight in a range from 8 to 100 kDa and wherein the cell-based expression system is capable of hydroxylating proline, lysine or proline and lysine residues of the expressed collagen peptide, b) incubating the expression system, in particular culturing the cell-based

Expression system, under conditions which allow the expression and hydroxylation of the collagen peptide, c) recovering the collagen peptide, in particular the in vivo hydroxylated collagen peptide, d) hydrolyzing the collagen peptide, in particular the in vivo hydroxylated

Collagen peptide, under conditions which lead to the production of a collagen peptide preparation which has collagen peptides, in particular in vitro hydroxylated collagen peptides, with an average molecular weight of 1 to 7 kDa and with a molecular weight in a range of 0.1 to 13.5 kDa and e) winning of the collagen peptide preparation, in particular the one hydroxylated in vivo

Collagen peptide preparation, hereinafter also referred to as collagen peptide preparation A.

With the aid of the aforementioned method, it is therefore advantageously possible to use a

To obtain collagen preparation with in vivo hydroxylated recombinantly produced collagen peptides with a specific molecular weight and a specific average molecular weight which, depending on the cell-based expression system used, are distinguished by a specific pattern of post-translational modifications, in particular hydroxylations and glycosylations. In this way it is advantageously possible, in particular, to obtain a collagen peptide preparation with biological activity directly, that is to say without the need to subsequently modify the collagen peptides of the collagen peptide preparation.

In a preferred embodiment, the recombinant collagen peptides prepared in accordance with the invention have in vivo hydroxylated collagen peptide preparation, that is to say collagen peptide preparation A, in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one, preferably in at least one Two, preferably all, of the in vitro tests shown in Examples 3 to 7, in particular Examples 3 to 5, for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, have a biological activity. The recombinant collagen peptides prepared according to the invention preferably have in vivo hydroxylated collagen peptide preparation, that is to say collagen peptide preparation A, in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one, preferably in at least two, preferably in all of the in vitro tests shown in Examples 3 to 7, in particular Examples 3 to 5, for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, the same biological activity as collagen peptide preparations isolated from natural sources, in particular collagen peptide preparations not recombinantly produced.

The recombinant collagen peptides prepared in accordance with the invention particularly preferably have in vivo hydroxylated collagen peptide preparation, that is to say collagen peptide preparation A, in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one, preferably in at least two , preferably in all of the in vitro tests shown in Examples 3 to 7, in particular Examples 3 to 5, for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, have a better biological activity than collagen peptide preparations isolated from natural sources, especially collagen peptide preparations not produced recombinantly.

According to a further embodiment of the present invention, the expression system provided in step a) is an expression system which is not capable of causing hydroxylation of proline, lysine or proline and lysine residues of the expressed collagen peptide, in particular that in step a) provided expression system no prolyl hydroxylase and lysyl hydroxylase activity.

The present invention thus comprises a method for producing a collagen peptide preparation comprising recombinant collagen peptides, in particular a non-hydroxylated collagen peptide preparation, comprising the method steps a) providing an expression system which has at least one expression cassette, the expression cassette having at least one nucleotide sequence which comprises a collagen peptide encoded with a molecular weight in a range from 8 to 100 kDa and the expression system is not capable of hydroxylating proline, lysine or proline and lysine residues of the expressed collagen peptide, b) Incubation of the expression system under conditions that the expression of the

Enable collagen peptide, c) recovering the collagen peptide, especially the non-hydroxylated collagen peptide, d) hydrolyzing the collagen peptide, especially the non-hydroxylated

Collagen peptide, under conditions which lead to the production of a collagen peptide preparation which has collagen peptides with an average molecular weight of 1 to 7 kDa and with a molecular weight in a range of 0.1 to 13.5 kDa and e) obtaining the collagen peptide preparation, in particular the non- hydroxylated

Collagen peptide, hereinafter also called collagen peptide preparation B.

In a preferred embodiment, the recombinant collagen peptides produced according to the invention have non-hydroxylated collagen peptide preparation, ie collagen peptide preparation B, in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one, preferably in at least one Two, preferably all, of the in vitro tests shown in Examples 3 to 7, in particular Examples 3 to 5, for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, have a biological activity.

The recombinant collagen peptides prepared according to the invention preferably have non-hydroxylated collagen peptide preparation, that is to say collagen peptide preparation B, in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one, preferably in at least two, preferably in all of the in vitro tests shown in Examples 3 to 7, in particular Examples 3 to 5, for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, the same biological activity as collagen peptide preparations isolated from natural sources, in particular collagen peptide preparations not recombinantly produced.

The recombinant collagen peptides prepared according to the invention particularly preferably have non-hydroxylated collagen peptide preparation, that is to say collagen peptide preparation B, in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one, preferably in at least two, preferably in all, of the in vitro tests shown in Examples 3 to 7, in particular Examples 3 to 5, for stimulating the synthesis of extracellular matrix proteins in osteoblasts, Fibroblasts and chondrocytes, a better biological activity than collagen peptide preparations isolated from natural sources, in particular non-recombinantly produced collagen peptide preparations.

In a preferred embodiment of the present invention, the collagen peptide obtained in process step c) is hydroxylated before carrying out process step d) in a process step xl) and in process step e) a prelysally, that is to say ex vivo hydroxylated, collagen peptide preparation is obtained before the hydrolysis.

Accordingly, the present invention furthermore comprises a method for producing a collagen peptide preparation comprising recombinant collagen peptides, in particular a collagen peptide preparation hydroxylated exlysally ex vivo, comprising the method steps a) providing an expression system which has at least one expression cassette, the expression cassette having at least one nucleotide sequence encodes a collagen peptide with a molecular weight in a range of 8 to 100 kDa and wherein the expression system is not capable of hydroxylating proline, lysine or proline and lysine residues of the expressed collagen peptide, b) incubating the expression system under conditions that allow expression enable the collagen peptide, c) obtaining the collagen peptide, xl) ex vivo hydroxylating the collagen peptide obtained in step c), d) hydrolyzing the collagen peptide, in particular the ex vivo hydroxylated collagen peptide, with Be conditions which lead to the production of a collagen peptide preparation which has collagen peptides, in particular ex vivo hydroxylated collagen peptides, with an average molecular weight of 1 to 7 kDa and with a molecular weight in a range from 0.1 to 13.5 kDa and e) Obtaining the collagen peptide preparation, in particular the prlysal ex vivo hydroxylated collagen peptide preparation, hereinafter also referred to as collagen peptide preparation C.

In a preferred embodiment, the recombinant collagen peptides prepared according to the invention have prlysally ex vivo hydroxylated collagen peptide preparation, ie collagen peptide preparation C, in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one at least two, preferably all, of the in vitro tests shown in Examples 3 to 7, in particular Examples 3 to 5, for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, have a biological activity.

Preferably, the recombinant collagen peptides prepared according to the invention have prlysally ex vivo hydroxylated collagen peptide preparation, ie collagen peptide preparation C, in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one, preferably in at least two , preferably in all of the in vitro tests shown in Examples 3 to 7, in particular Examples 3 to 5, for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, the same biological activity as collagen peptide preparations isolated from natural sources, especially collagen peptide preparations not produced recombinantly.

Particularly preferably, the recombinant collagen peptides prepared according to the invention have prlysally ex vivo hydroxylated collagen peptide preparation, that is to say collagen peptide preparation C, in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one Two, preferably all, of the in vitro tests for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3 to 7, in particular Examples 3 to 5, have better biological activity than collagen peptide preparations isolated from natural sources , in particular non-recombinantly produced collagen peptide preparations.

In a further preferred embodiment of the present invention, the collagen peptide obtained in process step c) is carried out after carrying out process step d) a process step x2) hydroxylated and in process step e) a postlysal, that is to say ex vivo hydroxylated collagen peptide preparation, that is to say after hydrolysis.

Accordingly, the present invention further comprises a method for producing a collagen peptide preparation comprising recombinant collagen peptides, in particular a postlysal ex vivo hydroxylated collagen peptide preparation, comprising the method steps a) providing an expression system which has at least one expression cassette, the expression cassette having at least one nucleotide sequence which Encoded collagen peptide with a molecular weight in a range from 8 to 100 kDa and wherein the expression system is not capable of hydroxylating proline, lysine or proline and lysine residues of the expressed collagen peptide, b) incubating the expression system under conditions which allow the expression of the

Enable collagen peptide, c) recover the collagen peptide, d) hydrolyze the collagen peptide under conditions necessary to produce a

Lead collagen peptide preparation that has collagen peptides with an average molecular weight of 1 to 7 kDa and with a molecular weight in a range of 0.1 to 13.5 kDa and x2) ex vivo hydroxylation of the collagen peptides obtained in step d)

Collagen peptide preparation, e) obtaining the collagen peptide preparation, in particular the post-lysing ex vivo hydroxylated collagen peptide preparation, hereinafter also referred to as collagen peptide preparation D.

In a preferred embodiment, the recombinant collagen peptides prepared according to the invention have postlysal ex vivo hydroxylated collagen peptide preparation, that is to say collagen peptide preparation D, in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one two, preferably all, of the in vitro tests shown in Examples 3 to 7, in particular Examples 3 to 5, for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, a biological effectiveness.

The recombinant collagen peptides prepared according to the invention preferably have postlysal ex vivo hydroxylated collagen peptide preparation, ie collagen peptide preparation D, in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one, preferably in at least two , preferably in all of the in vitro tests shown in Examples 3 to 7, in particular Examples 3 to 5, for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, the same biological activity as collagen peptide preparations isolated from natural sources, especially collagen peptide preparations not produced recombinantly.

The recombinant collagen peptides prepared according to the invention particularly preferably have postlysal ex vivo hydroxylated collagen peptide preparation, ie collagen peptide preparation D, in at least one in vitro test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one Two, preferably all, of the in vitro tests for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3 to 7, in particular Examples 3 to 5, have better biological activity than collagen peptide preparations isolated from natural sources , in particular non-recombinantly produced collagen peptide preparations.

According to a preferred embodiment of the present invention, the at least one nucleotide sequence of the at least one expression cassette is codon-optimized, that is to say that those codons in the nucleotide sequence which are derived from the translation system of the expression system provided, in particular the cell-based expression system, in particular the host cell provided, not used or not used with preference, replaced by those which are preferably used by the translation system of the expression system provided, in particular the cell-based expression system provided, in particular the host cell provided, without thereby changing the amino acid sequence of the encoded peptide or protein.

In a preferred embodiment of the present invention, the collagen peptide encoded by the nucleotide sequence is a collagen peptide of a vertebrate, in particular a mammal, for example human or a non-human mammal, for example horse, donkey, kangaroo, sheep, rodent, pig or cattle, a bird, for example chicken, a fish, an amphibian, a reptile or an invertebrate, for example jellyfish.

The collagen peptide coded by the nucleotide sequence preferably has a collagen 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, preferably type II, preferably type III, occurring amino acid sequence.

The type I, II or III collagen peptide encoded by the nucleotide sequence is preferred, preferably type I or II, particularly preferably type I.

The collagen peptide encoded by the nucleotide sequence preferably has one in collagen from vertebrates, in particular fish, amphibians, reptiles, birds and mammals, in particular in human, bovine, porcine, equine or avian collagen of types I, II or III, preferably type I, preferably Type II, preferably Type III, occurring amino acid sequence.

The collagen peptide encoded by the nucleotide sequence particularly preferably has an amino acid sequence which occurs in human collagen, in particular in human type I collagen, preferably in the al chain of human type I collagen.

Particularly preferably, the collagen peptide encoded by the nucleotide sequence has an amino acid sequence which occurs in non-human collagen, in particular in non-human type I collagen, preferably in the al chain of the non-human type I collagen, in particular one in bovine, porcine, equine or avian collagen amino acid sequence.

The collagen peptide encoded by the nucleotide sequence is preferably a naturally occurring collagen peptide. In a further preferred embodiment of the present invention, the collagen peptide encoded by the nucleotide sequence is not a naturally occurring collagen peptide. The collagen peptide encoded by the nucleotide sequence is preferably a genetically modified collagen peptide. In a particularly preferred embodiment of the present invention, the collagen peptide encoded by the nucleotide sequence is a genetically modified collagen peptide in which at least one Amino acid of the amino acid sequence of a naturally occurring collagen peptide, preferably at least one non-essential amino acid, in particular Ala, Asn, Asp, Glu, Ser, of the amino acid sequence of a naturally occurring collagen peptide, by at least one very specific amino acid, in particular by at least one essential amino acid, in particular Ile , Leu, Lys, Met, Phe, Thr, Trp, Val, His, Cys, Tyr, particularly preferably Trp.

According to the invention, the collagen peptide encoded by the nucleotide sequence is preferably a genetically modified collagen peptide in which the amino acid sequence of a naturally occurring collagen peptide has at least one amino acid, preferably at least one essential amino acid, in particular Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, His , Cys, Tyr, particularly preferably Trp, was added. It can be provided according to the invention that the at least one amino acid, preferably the at least one essential amino acid, in particular Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, His, Cys, Tyr, particularly preferably Trp, N-terminal , C-terminal and / or within the amino acid sequence of a naturally occurring collagen peptide was added.

According to a preferred embodiment of the present invention, the at least one nucleotide sequence encodes a collagen peptide with a molecular weight in a range of preferably 8 to 95 kDa, preferably 8 to 90 kDa, preferably 8 to 85 kDa, preferably 8 to 80 kDa, preferably 9 to 95 kDa , preferably 9 to 90 kDa, preferably 9 to 85 kDa, preferably 9 to 80 kDa, preferably 10 to 95 kDa, preferably 10 to 90 kDa, preferably 10 to 85 kDa, preferably 10 to 80 kDa.

In a particularly preferred embodiment of the present invention, the hydrolysis is an enzymatic or acid-catalyzed hydrolysis, preferably an enzymatic hydrolysis, preferably an acid-catalyzed hydrolysis. The collagen peptide obtained in step c) is particularly preferably hydrolysed by adding at least one bacterial or microbial protease, in particular at least one bacterial and / or microbial serine, cysteine, aspartate and / or metalloprotease, preferably at least one bacterial and / or microbial endoprotease, preferably of at least one bacterial and / or microbial exoprotease.

In a further preferred embodiment of the present invention, the hydrolyzing of the collagen peptide in step d) is carried out under conditions which lead to the production of a Lead collagen peptide preparation, which has collagen peptides of an average molecular weight of 1 to 3 kDa and with a molecular weight in a range of 0.1 to 10 kDa, preferably 0.18 to 10 kDa, preferably 0.2 to 10 kDa.

In a preferred embodiment of the present invention, the hydrolyzing of the collagen peptide in step d) is carried out under conditions which lead to the production of a collagen peptide preparation, the collagen peptide with an average molecular weight of 1 to 5 kDa and with a molecular weight in a range from 0.1 to 12 kDa , preferably 0.18 to 12 kDa, preferably 0.2 to 12 kDa.

The present invention also relates to a collagen peptide preparation produced by one of the aforementioned methods according to the invention, in particular a collagen peptide preparation, the collagen peptides having an average molecular weight of 1 to 7 kDa and having a molecular weight in a range from 0.1 to 13.5 kDa, preferably 0.18 to 13.5, preferably 0.2 to 13.5.

In a preferred embodiment of the present invention, the collagen peptide preparation produced by one of the abovementioned methods according to the invention, in particular the collagen peptide preparation, which has collagen peptides with an average molecular weight of 1 to 7 kDa and with a molecular weight in a range from 0.1 to 13.5 kDa, a non-hydroxylated, partially hydroxylated or fully hydroxylated collagen peptide preparation, preferably a non-hydroxylated collagen peptide preparation, preferably a partially hydroxylated collagen peptide preparation, preferably a fully hydroxylated collagen peptide preparation.

The collagen peptide preparation produced by one of the abovementioned methods according to the invention, in particular the collagen peptide preparation which has collagen peptides with an average molecular weight of 1 to 7 kDa and with a molecular weight in a range from 0.1 to 13.5 kDa, is preferably a collagen peptide preparation, at least 1 %, preferably at least 2%, preferably at least 3%, preferably at least 4%, preferably at least 5%, preferably at least 10%, preferably at least 15%, preferably 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% of the prolyl residues, preferably the lysyl residues, particularly preferably the prolyl and lysyl residues, the collagen peptides are hydroxylated, are preferably hydroxylated in vivo, are preferably ex vivo hydroxylated, especially prysalsal are ex vivo hydroxylated or post lysally ex vivo hydroxylated. The collagen peptide preparation produced by one of the abovementioned methods according to the invention, particularly the collagen peptide preparation which has collagen peptides with an average molecular weight of 1 to 7 kDa and with a molecular weight in a range from 0.1 to 13.5 kDa, is particularly preferred, a collagen peptide preparation, at most 95%, preferably at most 90%, preferably at most 85%, preferably at most 80%, preferably at most 75%, preferably at most 70%, preferably at most 65%, preferably at most 60%, preferably at most 55%, preferably at most 50%, preferably at most 45%, preferably at most 40%, preferably at most 35%, preferably at most 30%, preferably at most 25%, preferably at most 20%, preferably at most 15%, preferably at most 10%, preferably at most 5%, of the prolyl residues, preferably the lysyl residues, particularly preferably the prolyl and lysyl residues, the collagen peptides are hydroxylated, preferably hydroxylated in vivo, be are preferably ex vivo hydroxylated, in particular are pre-lysed ex vivo hydroxylated or post-lysally ex vivo hydroxylated.

In a further preferred embodiment of the present invention, the collagen peptide preparation produced by one of the abovementioned methods according to the invention, in particular the collagen peptide preparation, which has collagen peptides with an average molecular weight of 1 to 7 kDa and with a molecular weight in a range from 0.1 to 13.5 kDa , a collagen peptide preparation, 0.5 to 80%, preferably 1 to 75%, preferably 5 to 70%, preferably 5 to 65%, preferably 10 to 60%, preferably 15 to 55%, preferably 20 to 50%, preferably 25 up to 50%, preferably 30 to 50%, preferably 35 to 50%, preferably 40 to 50% of the prolyl residues, preferably the lysyl residues, particularly preferably the prolyl and lysyl residues, the collagen peptides are hydroxylated, are preferably hydroxylated in vivo, preferably ex vivo are hydroxylated, in particular are pre-lysed ex vivo hydroxylated or post-lysally ex vivo hydroxylated.

In a preferred embodiment of the present invention, the collagen peptide preparation produced by one of the methods according to the invention, in particular the collagen peptide preparation, which has collagen peptides with an average molecular weight of 1 to 7 kDa and with a molecular weight in a range from 0.1 to 13.5 kDa Collagen peptide preparation, the collagen peptides of which are glycosylated. The collagen peptides are preferably glycosylated in vivo, preferably ex vivo glycosylated. At least 1%, preferably at least 2%, preferably at least 3%, preferably at least 4%, preferably at least 5%, preferably at least 6%, preferably at least 7%, preferably at least 8% are preferred, preferably at least 9%, preferably at least 10%, preferably at least 15%, preferably at least 20%, of the hydroxyl residues glycosylated, preferably glycosylated in vivo, preferably ex vivo glycosylated.

In a further preferred embodiment of the present invention, the collagen peptide preparation produced using one of the methods according to the invention is a collagen peptide preparation whose collagen peptides are not glycosylated.

In a preferred embodiment of the present invention

Collagen peptide preparation, in particular the collagen peptide preparation which has collagen peptides with an average molecular weight of 1 to 7 kDa and with a molecular weight in a range from 0.1 to 13.5 kDa, an in vivo hydroxylated collagen peptide preparation, that is to say collagen peptide preparation A.

In a preferred embodiment of the present invention

Collagen peptide preparation, in particular the collagen peptide preparation which has collagen peptides with an average molecular weight of 1 to 7 kDa and with a molecular weight in a range from 0.1 to 13.5 kDa, is a non-hydroxylated collagen peptide preparation, that is to say collagen peptide preparation B.

In a further preferred embodiment of the present invention, the collagen peptide preparation, in particular the collagen peptide preparation which has collagen peptides with an average molecular weight of 1 to 7 kDa and with a molecular weight in a range from 0.1 to 13.5 kDa, is an ex vivo hydroxylated collagen peptide preparation, that is, collagen peptide preparation C or D.

The collagen peptide preparation, in particular the collagen peptide preparation which has collagen peptides with an average molecular weight of 1 to 7 kDa and with a molecular weight in a range from 0.1 to 13.5 kDa, is preferred, a pre-lysed, that is to say ex vivo hydroxylated collagen peptide preparation before the hydrolysis , that is collagen peptide preparation C.

According to a further embodiment of the present invention, the collagen peptide preparation, in particular the collagen peptide preparation, is the collagen peptide with an average molecular weight of 1 to 7 kDa and with a molecular weight in one range from 0.1 to 13.5 kDa, a post lysal, that is to say ex vivo hydroxylated collagen peptide preparation, that is to say after the hydrolysis, that is to say collagen peptide preparation D.

In particular, the present invention also relates to a collagen peptide preparation, in particular a hydroxylated or non-hydroxylated collagen preparation, which is produced by hydrolysis of collagen peptide having a molecular weight in a range from 8 to 100 kDa produced recombinantly in a host cell, the collagen peptide preparation being collagen peptides with an average molecular weight from 1 to 7 kDa and a molecular weight in a range from 0.1 to 13.5 kDa.

The presence of the characteristic peptides of the collagen peptide preparation which contribute to its effectiveness can be determined in particular by means of mass spectroscopy, preferably by means of ESI (electron spray ionization) or MALDI mass spectroscopy, the characteristic peptides occurring as peaks in the mass spectrum. In a molecular weight distribution determined by means of MALDI mass spectroscopy, the characteristic peptides have at least twice the intensity, more preferably at least four times the intensity compared to their surroundings.

The collagen peptide preparations according to the invention, in particular the collagen peptide preparation A, the collagen peptide preparation B, the collagen peptide preparation C and the collagen peptide preparation D, can additionally also have characteristic peptides with a size of 1,500 to 3,500 Da.

In a further preferred embodiment of the invention, the collagen peptide preparations according to the invention, in particular the collagen peptide preparation A, the collagen peptide preparation B, the collagen peptide preparation C and the collagen peptide preparation D, have at most 5.5%, preferably at most 5%, preferably at most 4.5%, preferably at most 4%, preferably at most 3.5%, of collagen peptides with a size of <500 Da.

According to this embodiment, the particularly low proportion of peptides with a size of less than 500 Da advantageously results in an improved taste of the collagen peptide preparations compared to the preparations known in the prior art, in particular in a reduced bitterness of the collagen peptide preparations.

Preferably 35 to 60%, preferably 35 to 55%, preferably 35 to 50%, preferably 36 to 48%, preferably 36 to 46%, preferably 37 to 45%, preferably 38 to 44%, of the collagen peptides the collagen peptide preparations according to the invention, in particular the collagen peptide preparation A, the collagen peptide preparation B, the collagen peptide preparation C and the collagen peptide preparation D, have a size in the range from 1500 Da to 3500 Da.

The collagen peptide preparations according to the invention, in particular the collagen peptide preparation A, the collagen peptide preparation B, the collagen peptide preparation C and the collagen peptide preparation D, preferably have at most 2.8%, preferably at most 2.75%, preferably at most 2.7%, preferably at most 2.65%, preferably at most 2.6%, preferably at most 2.55%, preferably at most 2.5%, preferably at most 2.45%, preferably at most 2.4%, preferably at most 2.35%, preferably at most 2.3%, collagen peptides with a size in the range from 7500 Da to 13500 Da.

According to a particularly preferred embodiment of the present invention, at least 93%, preferably at least 93.5%, preferably at least 94%, preferably at least 94.5%, preferably at least 95% of the collagen peptides of the collagen peptide preparations according to the invention, in particular of the collagen peptide preparation A, of the collagen peptide preparation B, the collagen peptide preparation C and the collagen peptide preparation D, have a size in the range from 500 Da to 7500 Da.

Preferably at least 94.5%, preferably at least 95%, preferably at least 95.5%, preferably at least 95.6%, preferably at least 95.7%, preferably at least 95.6%, preferably at least 95.7%, preferably at least 95.8%, preferably at least 95.9%, preferably at least 96%, preferably at least 96.1%, preferably at least 96.2%, preferably at least 96.3%, preferably at least 96.4%, preferably at least 96.5 %, the collagen peptides of the collagen peptide preparations according to the invention, in particular the collagen peptide preparation A, the collagen peptide preparation B, the collagen peptide preparation C and the collagen peptide preparation D, have a size in the range from 500 Da to 13500 Da.

In a preferred embodiment, according to the present invention, the collagen peptide preparation is administered locally, in particular topically, or systemically, in particular enterally, preferably orally.

According to a preferred embodiment of the invention, the collagen peptide preparation is administered in the form of a food supplement. The nutritional supplement according to the invention is particularly advantageously in the form of a solution, suspension or gel, for example in an ampoule, as granules or powder. Due to its good solubility, the collagen peptide preparation can also be added to various beverages without causing clouding.

According to a preferred embodiment of the present invention, the food supplement provided according to the invention contains no further proteins or protein hydrolyzates in addition to the collagen peptide preparation.

According to one embodiment of the invention, the food supplement according to the invention contains, apart from the collagen peptide preparation, no further physiologically active constituents, in particular no proteins or protein hydrolysates.

The invention also relates to a product comprising a collagen peptide preparation according to the invention and at least one additive.

The invention further relates to a food supplement comprising a collagen preparation according to the invention and at least one further constituent, in particular at least one food-acceptable additive.

In one embodiment, the collagen peptide preparation can be added to a food or luxury food, for example a chocolate bar, protein bar, cereal bar, milk, milk products, for example yogurt, whey or curd cheese and milk substitute, for example soy milk, rice milk, almond milk and coconut milk (so-called functional food ).

The invention thus also relates to a food or luxury product, comprising a collagen preparation according to the invention.

It can further be provided according to the invention to administer the collagen peptide preparation in the form of a pharmaceutical composition. The pharmaceutical composition according to the invention is particularly advantageously administered, for example, in the form of tablets, lozenges, chewable tablets, capsules, bite capsules, dragées, pastilles, juices, gels or ointments.

The present invention also relates to a pharmaceutical composition comprising a collagen peptide preparation according to the invention and at least one pharmaceutically acceptable additive. In a further embodiment, the collagen peptide preparation can be administered in the form of a cosmetic composition. The cosmetic composition according to the invention is particularly advantageously administered, for example, in the form of lotions, ointments, creams, gels, powders, syringes or sprays.

The present invention also relates to a cosmetic composition comprising a collagen peptide preparation according to the invention and at least one skin-compatible additive.

If, according to a preferred embodiment of the invention, the collagen peptide preparation is not used as the only physiologically active constituent of a product, in particular a food supplement, a food or luxury food, a pharmaceutical composition or a cosmetic composition, it can be combined with one or more further components which have a positive effect on general health, especially endurance performance. Such components are preferably selected from the group consisting of vitamin C, vitamins of the B, D, E and K series, omega-3 fatty acids, omega-6 fatty acids, conjugated linolenic acids, caffeine and its derivatives, guarana extract, Green tea extract, epigallocatechin gallate, creatine, L-carnitine, a-lipoic acid, N-acetylcysteine, NADH, D-ribose, magnesium aspartate, antioxidants such as anthocyanins, carotenoids, flavonoids, resveratrol, glutathione and superoxide dismutase (SOD) cannabinoid, cannabino Adaptogens such as Rhodiola rosea, Panax ginseng, Withania somnifera, Shiitake, Ganoderma lucidum Lepidium meyenii, minerals such as iron, magnesium, calcium, zinc, selenium and phosphorus, as well as other proteins, hydrolyzates and peptides such as soy, wheat and whey protein.

In a preferred embodiment of the invention, the collagen peptide preparation is administered in an amount of 1 to 40 g per day, preferably from 1 to 30 g per day, preferably from 1 to 20 g per day, preferably from 1 to 15 g per day, preferably from 2.5 to 30 g per day, preferably 2.5 to 20 g per day, preferably 2.5 to 15 g per day, preferably 2.5 to 10 g per day, preferably 4 to 15 g per day, preferably 4 to 12 g per day, more preferably from 5 to 25 g per day, preferably 5 to 15 g per day, more preferably from 10 to 25 g per day, preferably 12 to 22 g per day, and in particular from 12.5 to 20 g per day, particularly preferably 6 to 15 g per day, in particular from 2.5 to 7.5 g per day, preferably 2.5 to 5 g per day.

The present invention also relates to a collagen peptide preparation according to the invention for use in a therapeutic method for maintaining and improving the Bone health, for the prevention and / or treatment of osteoporosis, for the prevention and / or treatment of sarcopenia, for the prevention and / or treatment of degenerative loss of muscle mass, for the improvement of muscle strength, for the stimulation of fat loss, for the reduction of body weight.

In a preferred embodiment, the present invention also relates to the collagen peptide preparation according to the invention for use in a method for the prevention and / or treatment of bone diseases, in particular osteoporosis.

In a preferred embodiment, the present invention relates to the collagen peptide preparation according to the invention for use in a method for prevention and / or

Treatment of sarcopenia.

Treatment of degenerative loss of muscle mass.

Treatment of cartilage diseases, especially arthrosis or arthritis.

In a preferred embodiment, the present invention relates to the collagen peptide preparation according to the invention for use in a method for improving muscle strength.

Treatment of a pathological condition characterized by a reduced mitochondrial activity, in particular for the prevention and / or treatment of a pathological condition characterized by a reduced endurance performance.

In a preferred embodiment, the present invention relates to the collagen peptide preparation according to the invention for use in a method for stimulating fat loss.

In a preferred embodiment, the present invention relates to the collagen peptide preparation according to the invention for use in a method for reducing body weight. In a preferred embodiment, the present invention relates to the collagen peptide preparation according to the invention for use in a method for the prevention and / or treatment of degenerative joint diseases, in particular arthrosis, rheumatoid arthritis, rheumatic diseases, spondylitis and / or fibromyalgia.

In a preferred embodiment, the present invention relates to the collagen peptide preparation according to the invention for use in a method for the prevention and / or treatment of diseases of the tendons or ligaments.

In a preferred embodiment, the present invention relates to the collagen peptide preparation according to the invention for use in a method for the prevention and / or treatment of skin diseases, in particular psoriasis vulgaris, acne, atopic dermatitis, chronic pruritus and / or rosacea.

In a preferred embodiment, the present invention relates to the collagen peptide preparation according to the invention for use in a method for treating wounds, in particular chronic wounds, acute wounds and / or burn wounds.

Treatment of degenerative nerve disorders.

Treatment of dementia.

Treatment of Alzheimer's Disease.

Treatment of a pathological condition characterized by a decrease in mental performance. In a preferred embodiment, the present invention relates to the collagen peptide preparation according to the invention for use in a method for prevention and / or

Treatment of diseases associated with malfunctions of the blood-brain barrier, in particular the structure and / or function of the meninges.

Treatment of bowel diseases, especially inflammatory bowel diseases.

Treatment of diseases of the cardiovascular system, in particular the structure and / or function of the blood vessels, in particular the vessel wall, in particular for the prevention and / or treatment of high blood pressure and / or circulatory disorders.

Treatment of diseases of the tooth structure.

The present invention also relates to a collagen peptide preparation according to the invention for use in a non-therapeutic method for maintaining and improving bone health, for preventing osteoporosis, for preventing and / or treating sarcopenia, for preventing degenerative loss of muscle mass, for improving muscle strength, to stimulate fat loss, reduce body weight and / or prevent degenerative joint diseases.

In a preferred embodiment, the present invention further relates to the non-therapeutic use of the collagen peptide preparation according to the invention for the optical and structural improvement of the skin, in particular for reducing wrinkling, improving skin elasticity, increasing the elasticity of the skin, increasing the moisture content of the skin, reducing cellulite and / or reduction of stretch marks, especially stretch marks. In a further preferred embodiment, the present invention relates to the non-therapeutic use of the collagen peptide preparation according to the invention for accelerating the growth of the nails and / or reducing the brittleness of nails.

In a preferred embodiment, the present invention also relates to the non-therapeutic use of the collagen peptide preparation according to the invention for the optical and structural improvement of the hair, in particular for improving the hair quality, reducing split ends and / or reducing / delaying hair loss.

In a further preferred embodiment, the present invention relates to the non-therapeutic use of the collagen peptide preparation according to the invention for increasing the mitochondrial number and / or mitochondrial activity.

In a further preferred embodiment, the present invention relates to the non-therapeutic use of the collagen peptide preparation according to the invention to improve endurance performance.

In a further preferred embodiment, the present invention relates to the non-therapeutic use of the collagen peptide preparation according to the invention for improving mental performance.

In a preferred embodiment of the present invention, the collagen peptide preparation according to the invention is used alone, that is to say without further substances, for use in one of the applications provided according to the invention.

In a further embodiment of the present invention, the collagen peptide preparation according to the invention is used as the only agent having biological activity in an application provided according to the invention.

In a further preferred embodiment, the collagen peptide preparation according to the invention is used together with at least one further agent, in particular a further biologically active agent, in an application provided according to the invention.

The present invention also relates to methods for preventing and / or treating the aforementioned indications, in particular the aforementioned therapeutic indications, according to to which the human or animal body is administered a sufficient amount of the collagen peptide preparation according to the invention, optionally with an additive, for the therapeutic purpose.

The present invention also relates to a non-therapeutic method for improving muscle strength, for increasing muscle mass, for stimulating fat loss, for reducing body weight, for maintaining and / or improving bone health, for maintaining and / or for improving skin health, to maintain and / or improve intestinal health, to maintain and / or improve the structure of the blood vessels, to maintain and / or improve the health of the cardiovascular system, to maintain and / or improve the dental system, to maintain and / or improve the health of the nails and Hair of a human or animal body, for maintaining and / or increasing the mitochondrial number and / or mitochondrial activity, for maintaining and / or improving endurance performance or for maintaining and / or improving mental performance, wherein the human or animal body according to the invention at least one SLI collagen peptide preparation is administered.

The present invention further relates to a collagen peptide preparation according to the invention for use in a method for producing films, foils and coatings. The coatings can be, for example, paints and varnishes, in particular paints and varnishes with special optical effects, or coatings for producing self-cleaning surfaces.

In a preferred embodiment, the term “collagen” is understood in the context of the present invention in a manner customary in the art, in particular as defined, for example, in WO 01/34646. In a preferred embodiment, the term “collagen” relates 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 , where X and Y can be any amino acid, preferably proline and 4-hydroxyproline. The term “collagen” is particularly preferably understood to mean a peptide having the repetitive motif (Gly-Pro-Y) _n and / or (Gly-X-Hyp) _m , where X and Y can be any amino acid. The term “gelatin” is preferably understood in the context of the present invention in a manner customary in the art, in particular as defined, for example, in WO 01/34646.

In connection with the present invention, the term “collagen peptide” is preferably understood to mean a peptide which has an amino acid sequence occurring in collagen as defined above. A “collagen peptide” is preferably also understood to mean a genetically modified collagen peptide that was obtained by modifying the amino acid sequence of a naturally occurring collagen peptide, the collagen peptide preparation obtained from the “collagen peptide” obtained in step e) of the method according to the invention preferably in at least one in vitro Test for stimulating the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, in particular in at least one, preferably in at least two, preferably in all, of the in vitro tests for stimulation shown in Examples 3 to 7, in particular Examples 3 to 5 the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, a biological activity, preferably the same biological activity as collagen peptide preparations isolated from natural sources, in particular non-recombinantly produced collagen peptide preparations ate, particularly preferably has a better biological activity, such as collagen peptide preparations isolated from natural sources, in particular non-recombinantly produced collagen peptide preparations.

In connection with the present invention, the term “recombinant DNA” denotes an artificially produced or manipulated DNA molecule that was produced in vitro using genetic engineering methods. In a preferred embodiment, the recombinant DNA is composed of components from different organisms of origin.

In connection with the present invention, a “recombinant or recombinantly produced collagen peptide” is understood to mean a collagen peptide encoded by recombinant DNA.

In connection with the present invention, the term “expression cassette” is understood to mean a DNA segment which is responsible for the transcription of the information coded in this segment into an RNA, in particular into an mRNA, and at least one promoter and one protein-coding nucleotide sequence the rule has at least one promoter, at least one protein-coding nucleotide sequence and optionally a terminator.

In connection with the present invention, a “nucleotide sequence” is understood to mean the sequence of the nucleotides of a nucleic acid, in particular a strand of nucleic acid, in particular a strand of DNA or RNA. A “nucleotide sequence” is therefore to be understood both as an informational unit and as the DNA or RNA strand that physically manifests this information.

In connection with the present invention, an “expression system” is understood to mean a system in which targeted and controlled protein biosynthesis can take place. According to the invention, the term “expression system” encompasses both cell-free expression systems in which the components necessary for protein biosynthesis are not present within a cell, that is to say protein biosynthesis takes place outside a cell, and cell-based expression systems in which protein biosynthesis takes place within a living cell he follows. A cell-free expression system in connection with the present invention is preferably a lysate or an extract from E. coli, insect cells, wheat germ, tobacco cells or mammalian cells, in particular CHO cells or reticulocytes from rabbits, which contains the components necessary for protein biosynthesis, in particular has a translation and a transcription system.

In connection with the present invention, a “host cell” is understood to mean a living cell which is capable of expressing peptides or proteins encoded in foreign DNA, in particular in recombinant DNA.

In connection with the present invention, the terms “prelysal” and “postlysal” refer to a point in time before or after the hydrolysis, in particular before or after enzymatic or acid-catalyzed hydrolysis.

In connection with the present invention, the term “incubation” means both the cultivation of a cell-based expression system, in particular a host cell, and the action of certain conditions on a cell-free expression system.

According to the invention, the terms “recovering the collagen peptide” according to method step c) and “recovering the collagen peptide preparation” according to method step e) are used Process known to the person skilled in the art for isolating the collagen peptide or the collagen peptide preparation from a composition containing several components by means of known isolation processes, such as, for example, centrifugation processes, in particular differential centrifugation and / or density gradient centrifugation, chromatographic processes, in particular gel filtration, ion exchange, affinity and / or high-performance liquid chromatography, electrophoresis processes ,

Filtration process and / or extraction process, understood, wherein an enrichment and purification of the relevant component from the composition containing several components can preferably be achieved by sequential application of several isolation processes.

“Conditions that enable the expression of the collagen peptide” are understood according to the invention to mean conditions, such as in particular temperature, pressure, time, light and the presence or absence of inducers and / or repressors, which activate or intensify expression of the collagen peptide. In a preferred embodiment, the collagen peptide is expressed in the context of a high cell density fermentation, in particular under high pressure, preferably high air pressure. The specific conditions which enable expression of the collagen peptide are known to the person skilled in the art and depend on the expression system used and the expression cassette used, in particular the promoter contained therein. Depending on the structure of the expression cassette, the expression of the collagen peptide can be constitutive or inducible expression.

In connection with the present invention, the term “hydrolyzing the collagen peptide under conditions which lead to the production of a collagen peptide preparation” describes those conditions, in particular the type of hydrolysis, if appropriate the type and amount of the at least one enzyme used for the hydrolysis, pH value, duration of hydrolysis, Hydrolysis temperature, understood, which lead to the obtaining of collagen peptides with an average molecular weight of 1 to 7 kDa and with a molecular weight in the range of 0.1 to 13.5 kDa from a recombinantly produced collagen peptide with a molecular weight in the range of 8 to 100 kDa. Suitable conditions for obtaining collagen peptides with an average molecular weight of 1 to 7 kDa and with a molecular weight in the range of 0.1 to 13.5 kDa from a recombinantly produced collagen peptide with a molecular weight in the range of 8 to 100 kDa are, for example, in Example 1 specified. The percentages given in connection with the molecular weight distribution of the collagen peptides in the collagen peptide preparations according to the invention relate in each case to% by weight in relation to all collagen peptides contained in the collagen peptide preparation in question.

In connection with the present invention, the terms “comprising” and “having” are understood to mean that, in addition to the elements explicitly covered by these terms, further elements not explicitly mentioned can be added. In connection with the present invention, these terms are also understood to mean that only the explicitly named elements are detected and no further elements are present. In this particular embodiment, the meaning of the terms “comprising” and “having” is synonymous with the term “consisting of”. In addition, the terms "comprehensive" and "exhibiting" also include compositions that contain, in addition to the explicitly named elements, other elements not mentioned, but which are of a functionally and qualitatively subordinate nature. In this embodiment, the terms “comprising” and “having” are synonymous with the term “essentially consisting of”.

In connection with the present invention, the term “and / or” is understood to mean that all members of a group which are connected by the term “and / or” are disclosed both as an alternative to one another and in each case cumulatively in any combination. For the expression "A, B and / or C", this means that the following disclosure content is to be understood as: 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 result from the subclaims.

The invention is described below without restricting the general inventive concept with reference to figures, tables and the associated exemplary embodiments.

It shows

Figure 1 shows the percentage of individual collagen peptides of the comparison products 1 and 2 and the collagen peptide preparations according to Examples 1.3, 1.4 and 1.5 in defined molecular weight ranges. FIG. 2A shows a chromatogram of a 1% solution of the comparative product 1. The molecular weight is plotted on the abscissa using a logarithmic scale.

FIG. 2B shows a chromatogram of a 1% solution of the comparison product 2. The molecular weight is plotted on the abscissa using a logarithmic scale.

3A shows a chromatogram of a 1% solution of the collagen peptide preparation according to Example 1.3. The molecular weight is plotted on the abscissa using a logarithmic scale.

3B shows a chromatogram of a 1% solution of the collagen peptide preparation according to Example 1.4. The molecular weight is plotted on the abscissa using a logarithmic scale.

4 shows a chromatogram of a 1% solution of the collagen peptide preparation according to Example 1.5. The molecular weight is plotted on the abscissa using a logarithmic scale.

FIG. 5 shows a comparison of the stimulation of the collagen synthesis of primary human fibroblasts in the absence of a collagen peptide (control 1), in the presence of 0.5 mg / ml of a 100 kDa collagen peptide (control 2), of a non-hydroxylated one according to the invention

Collagen peptide preparation with an average molecular weight of 1.8 kDa (sample 1), a non-hydroxylated collagen peptide preparation according to the invention with an average

Molecular weight of 2.4 kDa (sample 2) or a non-hydroxylated collagen peptide preparation according to the invention with an average molecular weight of 3.4 kDa (sample 3). The error bars show the standard deviation.

FIG. 6 shows a comparison of the stimulation of the elastin synthesis of primary human fibroblasts in the absence of a collagen peptide (control 1), in the presence of 0.5 mg / ml of a 100 kDa collagen peptide (control 2), of a non-hydroxylated one according to the invention

Molecular weight of 2.4 kDa (sample 2) or a non-hydroxylated collagen peptide preparation according to the invention with an average molecular weight of 3.4 kDa (sample 3). The error bars show the standard deviation. FIG. 7 shows a comparison of the stimulation of the proteoglycan synthesis of primary human fibroblasts in the absence of a collagen peptide (control 1), in the presence of 0.5 mg / ml of a 100 kDa collagen peptide (control 2), a non-hydroxylated collagen peptide preparation according to the invention with an average molecular weight of 1 , 8 kDa (sample 1), of a non-hydroxylated collagen peptide preparation according to the invention with an average molecular weight of 2.4 kDa (sample 2) or of a non-hydroxylated collagen peptide preparation according to the invention with an average molecular weight of 3.4 kDa (sample 3). The error bars show the standard deviation.

8 shows a comparison of the stimulation of the collagen synthesis of primary human fibroblasts in the absence of a collagen peptide (control 1), in the presence of 0.5 mg / ml of a hydroxylated collagen peptide preparation according to the invention with a medium one

Molecular weight of 7 kDa (sample 4), a hydroxylated collagen peptide preparation according to the invention with an average molecular weight of 5.6 kDa (sample 5) or a hydroxylated collagen peptide preparation according to the invention with an average molecular weight of 1.3 kDa (sample 6). The error bars show the standard deviation.

FIG. 9 shows a comparison of the stimulation of the elastin synthesis of primary human fibroblasts in the absence of a collagen peptide (control 1), in the presence of 0.5 mg / ml of a hydroxylated collagen peptide preparation according to the invention with a medium one

10 shows a comparison of the stimulation of the proteoglycan synthesis of primary human fibroblasts in the absence of a collagen peptide (control 1), in the presence of 0.5 mg / ml of a hydroxylated collagen peptide preparation according to the invention with an average molecular weight of 7 kDa (sample 4), of a hydroxylated collagen peptide preparation according to the invention with an average molecular weight of 5.6 kDa (sample 5) or a hydroxylated collagen peptide preparation according to the invention with an average molecular weight of 1.3 kDa (sample 6). The error bars show the standard deviation.

Examples: Example 1 - Preparation of collagen peptides:

1.1 Hydrolysis of a non-hydroxylated collagen peptide of bovine origin, recombinantly produced in Pichia pastoris, with a size of 45 kDa with neutral protease

A 0.789% collagen solution was first heated to 50 ° C. in a 50 ml bottle in the cryostat. Subsequently, the temperature-collagen solution 200 ppm CaCl ₂ X2h ₂ 0 (based on dry substance (TS) of the collagen) were added and the pH of the solution with a 10% NaOH solution was adjusted to 6.2. In a next step, 0.8% Sumizyme BNP-L (based on TS of the collagen) was added to the collagen solution.

The average molecular weight of the collagen peptides after a hydrolysis time of 180 min was determined to be 5.04 kDa.

1.2 Hydrolysis of a non-hydroxylated collagen peptide of bovine origin, recombinantly produced in Pichia pastoris, with a size of 45 kDa with alkaline protease

A 0.792% collagen solution was first heated to 63 ° C. in a 50 ml bottle in the cryostat. Thereupon 200 ppm CaCl ₂ x2H ₂ 0 (based on TS of the collagen) were added to the tempered collagen solution and the pH of the solution was adjusted to 7.75 with 10% NaOH solution. In a next step, 0.3% Alcalase 2.4L (based on TS of the collagen) was added to the collagen solution.

The average molecular weight of the collagen peptides after a hydrolysis time of 180 min was determined to be 3.01 kDa.

1.3 Hydrolysis of a non-hydroxylated collagen peptide of human origin, recombinantly produced in Pichia pastoris, with a size of 25 kDa with alkaline protease

A 2.85% collagen solution was first heated to 63 ° C. in a 50 ml bottle in a cryostat. Then 200 ppm CaCl ₂ x2H ₂ 0 (based on TS of the collagen) were added to the tempered collagen solution and the pH of the solution was adjusted to 7.6 with 10% NaOH solution. In a next step, 0.3% Alcalase 2.4L (based on TS of the collagen) was added to the collagen solution. The average molecular weight of the collagen peptides after a hydrolysis time of 45 min was 1.8 kDa.

1.4 Hydrolysis of a non-hydroxylated collagen peptide of human origin, recombinantly produced in Pichia pastoris, with a size of 100 kDa with alkaline protease

First, a 2.85% collagen solution in a 50 ml bottle in the cryostat was heated to 63 ° C. Subsequently, the temperature-collagen solution 200 ppm CaCl (relative to TS of the collagen) were added ₂ X2h ₂ 0 and the pH value of the solution with 10% NaOH solution to 7.6. Then 0.4% Alcalase 2.4L (based on TS of the collagen) was added to the collagen solution.

After a hydrolysis time of 45 min, the solution had collagen peptides with an average molecular weight of 2.4 kDa. The collagen peptide preparation obtained according to the invention was used as sample 2 in example 6.

1.5 Hydrolysis of a non-hydroxylated collagen peptide of human origin recombinantly produced in Pichia pastoris with a size of 100 kDa with alkaline protease

A 1.5% collagen solution was heated to 63 ° C. in a 50 ml bottle in a cryostat. Subsequently, the temperature-collagen solution 200 ppm CaCl (relative to TS of the collagen) were added ₂ X2h ₂ 0 and the pH value of the solution with 10% NaOH solution to 7.6. Finally, 0.3% Alcalase 2.4L (based on TS of the collagen) was added to the collagen solution.

The average molecular weight of the collagen peptides after a hydrolysis time of 60 min was 1.8 kDa. The collagen peptide preparation according to the invention obtained was used as sample 1 in example 6.

1.6 Hydrolysis of a non-hydroxylated collagen peptide of human origin recombinantly produced in Pichia pastoris with a size of 25 kDa with alkaline protease A 2.85% collagen solution was first heated to 63 ° C. in a 50 ml bottle in a cryostat. Then 200 ppm CaCl ₂ x2H ₂ 0 (based on TS of the collagen) were added to the tempered collagen solution and the pH of the solution was adjusted to 7.6 with 10% NaOH solution. In a next step, 0.1% Alcalase 2.4L (based on TS of the collagen) was added to the collagen solution.

The average molecular weight of the collagen peptides after a hydrolysis time of 60 min was 3.4 kDa. The collagen peptide preparation according to the invention obtained was used as sample 3 in example 6.

1.7 Hydrolysis of a hydroxylated recombinantly produced in Pichia pastoris

Collagen peptide of bovine origin with a size of 45 kDa with alkaline protease

A 5.53% collagen solution was first heated to 55 ° C. in a 250 ml glass bottle in the cryostat. Then 200 ppm CaCl ₂ x2H ₂ 0 (based on TS of the collagen) were added to the tempered collagen solution and the pH of the solution was adjusted to 7.59 with 2% NaOH solution. In a next step, 0.2% Alcalase 2.4L (based on TS of the collagen) was added to the collagen solution.

The average molecular weight of the collagen peptides after a hydrolysis time of 150 min was determined to be 7 kDa. The collagen peptide preparation according to the invention obtained was used as sample 4 in example 7.

1.8 Hydrolysis of a hydroxylated recombinantly produced in Pichia pastoris

Collagen peptide of bovine origin with a size of 45 kDa with alkaline protease

A 5.00% collagen solution was first heated to 55 ° C. in a 100 ml glass bottle in the cryostat. Thereupon 200 ppm CaCl ₂ x2H ₂ 0 (based on TS of the collagen) were added to the tempered collagen solution and the pH of the solution was adjusted to 7.60 with 2% NaOH solution. Then 0.25% NZ37071 (based on TS of the collagen) was added to the collagen solution.

The average molecular weight of the collagen peptides after a hydrolysis time of 240 min was 5.6 kDa. The collagen peptide preparation according to the invention obtained was used as sample 5 in example 7. 1.9 hydrolysis of a hydroxylated collagen peptide of bovine origin recombinantly produced in Pichia pastoris and having a size of 45 kDa with alkaline protease

Starting from the collagen peptide hydrolyzate obtained according to Example 1.7, the higher molecular components of the hydrolyzate were removed using a concentrator (e.g. Vivaspin 20) with a size exclusion membrane of 5000 Da.

An average molecular weight of 1.3 kDa was determined for the collagen peptides thus obtained. The collagen peptide preparation according to the invention was used as sample 6 in example 7.

1.10 Gel chromatographic analysis of the collagen peptide hydrolyzates

The molecular weight distribution of the collagen peptide hydrolyzates obtained in Examples 1.3 to 1.5 and of two commercially available comparison products with an average molecular weight of 2.3 kDa and 1.7 kDa were determined by means of gel chromatography (Knauer, Germany). The statistical analysis was carried out by the software WinGPC (PSS GmbH, Mainz, Germany). The following parameters were used for gel chromatography:

Stationary phase: TSK 2000 SW XL (TOSOH Bioscience GmbH)

Mobile phase: 0.4 mol / l sodium dihydrogen phosphate, pH 5.3

Flow rate: 0.5 ml / min

Calibration standard: defined collagen type 1 fragments (FILK)

Detection: UV detection at 214 nm (Knauer)

Sample concentration: 1%

For the different collagen peptide hydrolyzates, the percentages of individual collagen peptides shown in Table 1 and in FIG. 1 resulted in defined molecular weight ranges.

Table 1: Evaluation of the percentages of the individual collagen peptides in defined molecular weight ranges. The analysis was carried out by means of gel chromatography using defined type I collagen peptide standards.

Chromatograms of a 1% solution of the comparison products and the collagen peptide preparations according to Examples 1.3 to 1.5 are shown in FIGS. 2 to 4. Due to the narrower molecular weight distribution, the absence of higher molecular peptides and the inventive hydrolysis, the proportion of peptides <500 Da can be significantly reduced and a product with an average molecular weight of less than 2 kDa can nevertheless be achieved. At the same time, the number of peptides in the preferred size range between 1500 and 3500 Da is increased significantly. While peptides> 1500 Da are classified by the specialist as neutral in taste, the low-molecular peptides <500 Da in particular contribute significantly to the bitterness of collagen peptide products.

Avoiding the formation of such peptides is a further advantage for the production of collagen peptides which are sensory and organoleptically excellent and which the consumer classifies as "neutral". The same also applies to products with a higher average molecular weight in the range from 3 to 7 kDa.

It can be deduced from the exemplary embodiments mentioned that the process according to the invention and thus the use of uniform recombinant collagen fragments as the starting material for the hydrolysis enable the formation of preferred individual peptides within a narrow molecular weight distribution which, depending on the choice of the hydrolysis conditions, has characteristic peaks, generally between 4 and 6 (Figures 3A, 3B, 4).

The formation of these individual peptides can be controlled in a targeted manner both through the choice of the starting fragment and the hydrolysis conditions, which is almost impossible when using animal starting materials because of their inhomogeneity. Example 2 - ex vivo hydroxylation of collagen fragments

For the post-translational modification (hydroxylation of proline residues) of collagen fragments ex vivo, a specific 4-OH prolyl hydroxylase (P4H) was used in the presence of the cofactors a-ketoglutarate, iron (II) ions and 0 ₂ , whereby the use of non-specific hydroxylases is also conceivable. For this, 8 mM of the collagen fragment in the presence of 14 mM α-ketoglutarate, 0.5 mM iron (II) sulfate and 1.5 mM L-ascorbic acid in 50 mM MES buffer (pH 6.5) and an enzyme solution in a total volume of Incubate 1 mL with shaking (300 rpm) at 37 ° C on a thermomixer for 14 to 18 hours. Alternatively, the incubation can also take place in an incubator under the aforementioned conditions.

Example 3 - Osteoblast activity (especially bone health):

To analyze the biological effectiveness of the collagen peptide preparation according to the invention with regard to maintaining bone health and the prophylaxis and treatment of bone diseases, its stimulating effect on the synthesis of matrix proteins and enzymes, which play a role in the structure and mineralization of the matrix, by osteoblasts in vitro examined. This is done by determining the expression of the corresponding mRNA using real-time PCR and a semi-quantitative evaluation (based on a control without collagen hydrolyzate).

For this purpose, human osteoblasts are first isolated from knee joints by incubating bone material with vigorous agitation at 37 ° C for 1 h in Hanks solution, supplemented with 7 mg / ml hyaluronidase type I and III-S and 5 mg / ml pronase. The digestion is then continued at 37 ° C. for 3-5 h in Hanks solution, supplemented with 16 mg / ml collagenase type CLS IV. The primary osteoblasts obtained are after the enzymatic digestion in HanTs Fl2 medium, supplemented with 10% fetal calf serum, 20 U / ml penicillin streptomycin, 50 pg / ml partricin, 0.05 mg / ml ascorbic acid and 0.15 mg / ml Cultivated glutamine. Alternatively, primary osteoblasts (item no. C-12760; 2019) can also be obtained from PromoCell GmbH, Heidelberg, Germany, to investigate their biological effectiveness. The cells are then cultivated in HanT's Fl2 medium, supplemented with 10% fetal calf serum, 20 U / ml penicillin streptomycin, 50 pg / ml partricin and 0.15 mg / ml glutamine. For the investigation of the biological effectiveness, monolayer cell cultures of the isolated human osteoblasts are incubated for a period of 24 h in a medium which is supplemented with 0.5 mg / ml of the respective collagen peptide preparation. A control is incubated in medium without preparation. The respective mRNA expression is then determined.

Example 4 - Fibroblast Activity (Especially Skin Health):

Example 4.1 - Stimulation of mRNA synthesis:

The stimulation of the synthesis of collagen (type I) as well as the proteoglycans biglycan and versican is investigated in vitro on human dermal fibroblasts (skin cells). For this, the cells are incubated for 24 hours each with 0.5 mg / ml of a low molecular weight or collagen peptide preparation according to the invention and then the expression of collagen RNA, biglycan RNA and versican RNA is determined by means of real-time PCR and semi-quantitative (based on a control without preparation) evaluated.

Example 4.2 - Stimulation of the synthesis of connective tissue proteins:

To determine the stimulation of the synthesis of connective tissue proteins by the collagen peptide preparations according to the invention, primary human dermal fibroblasts are first digested after enzymatic digestion in HAM's F12 medium, comprising 10% FCS, 20 U / ml penicillin streptomycin, 50 pg / ml partricin, 0.05 mg / ml ascorbic acid and 0.15 mg / ml glutamine, cultured. After a confluence of 80%, the respective culture medium is replaced by a medium without collagen peptide (control) or with 0.5 mg / ml of a collagen peptide preparation to be tested and the primary human fibroblasts for a period of at least 14 days, preferably 14 to 21 days , in particular 14 days, incubated in the respective medium. The expression of different proteins of the connective tissue can then be determined and evaluated by means of suitable assays (see for example Examples 6 and 7).

Example 5 - Chondrocvten Activity (Especially Cartilage Health):

For cell cultures, porcine or human chondrocytes are isolated from cartilage tissue in a known manner and sown on culture plates at a density of approximately 350,000 cells / cm. Ham's Fl2 medium with 10% fetal is used as the culture medium Calf serum, 10 mg / ml gentamycin and 5 mg / ml amphotericin B were used. As an alternative to 10 mg / ml gentamycin, 10 mg / ml penicillin-streptomycin can also be used. The cultivation was carried out at 37 ° C in an oxygen-reduced atmosphere (5% 0 ₂ , 5% C0 ₂ and 90% N ₂ ).

Determination of collagen biosynthesis:

The collagen (essentially type II) synthesized by the chondrocytes is quantified by radioactive labeling with ¹⁴ C-proline, which is incorporated into the collagen.

Radioactive ¹⁴ C-proline is first added to the culture medium and the chondrocytes are cultivated under these conditions until the time of the determination. In order to be able to distinguish between the built-in and non-built-in ¹⁴ C-proline during the detection, the isotope-containing culture medium is then replaced by pure culture medium for a period of 3 days. The culture medium is then discarded and the adherent cell lawn is mixed with distilled water in order to destroy the cell membranes due to osmotic stress and to release cytosolic, unbound ¹⁴ C-proline. The cell debris is pelleted with the synthesized extracellular matrix by centrifugation. The pellet is resuspended in fresh distilled water and mixed with a xylene scintillation cocktail. The amount of collagen synthesized can then be quantified by detecting the ¹⁴ C-proline with a beta counter.

Alternatively, the quantification can be carried out using the Sircol Collagen Assay Kit (item no. 054S5000, 2019, tebu-bio, Offenbach, Germany or Biocolor Ltd., UK) according to the manufacturer's instructions (see Examples 6 and 7).

Determination of proteoglycan biosynthesis:

The proteoglycans synthesized by the chondrocytes are quantified by an Alcian blue staining and photometric determination of the glycosaminoglycans (GAG), which are components of the proteoglycans.

In order to determine the GAG content in the cell culture, the culture medium is first discarded and the adherent cell lawn is rinsed with PBS buffer (pH 7). The cells are then fixed at 4 ° C. for 2 hours in a 10% formaldehyde solution in PBS. After removing the formaldehyde, the Alcian blue staining reagent (5% Alcian blue in 3% - acetic acid) on the cell turf and incubated overnight at 4 ° C. Unbound Alcian blue is discarded and washed out by carefully rinsing three or four times with PBS. By adding acidic guanidine solution (8 mol / l) the GAG complexes are released from the cell lawn. The amount of glycosaminoglycans can then be quantified photometrically at a wavelength of 620 nm.

Alternatively, the quantification can be carried out using the Blyscan Glycosaminoglycan Assay Kit (Item No. 054B3000, 2019, tebu-bio, Offenbach, Germany or Biocolor Ltd., UK) according to the manufacturer's instructions (see Examples 6 and 7).

Example 6 - Stimulation of the synthesis of collagen, elastin and proteoglycan in primary human fibroblasts by non-hydroxylated collagen peptide preparations according to the invention:

To determine the stimulation of the synthesis of collagen, elastin and proteoglycan, primary human dermal fibroblasts according to Example 4.2 were used over a period of at least 14 days, preferably 14 to 21 days, in particular 14 days, in medium without collagen peptide (control 1) and in the presence of 0.5 mg / ml of a 100 kDa collagen peptide (control 2), a collagen peptide preparation with an average molecular weight of 1.8 kDa (sample 1), a collagen peptide preparation with an average molecular weight of 2.4 kDa (sample 2) and one of a collagen peptide preparation with an average molecular weight of 3.4 kDa (sample 3).

Example 6.1 - Determination of stimulation of collagen synthesis

The determination of collagen synthesis by primary human dermal fibroblasts was carried out using the Sircol Collagen Assay Kit (article no. 054S5000, 2019, tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK) according to the manufacturer's instructions. The results of the experiment are shown in Table 2 and Figure 5.

Table 2: Determination of the optical density (OD) at a wavelength of 450 nm to determine the collagen synthesis according to the Sircol Collagen Assay (tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK).

Example 6.2 - Determination of stimulation of the synthesis of elastin

The determination of elastin synthesis by primary human dermal fibroblasts was carried out using the Fastin Elastin Assay (item no. 054F2000, 2019, tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK) according to the manufacturer's instructions. The results of the experiment are shown in Table 3 and Figure 6.

Table 3: Determination of the optical density (OD) at a wavelength of 450 nm to determine the elastin synthesis according to the Fastin Elastin Assay (tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK).

Example 6.3 - Determination of the stimulation of the synthesis of glycosaminoglycan The determination of the synthesis of glycosaminoglycans by primary human dermal fibroblasts was carried out using the Blyscan glycosaminoglycan assay (article no. 054B3000, 2019, tebu-bio, Offenbach, or Biocolor Ltd., UK) the manufacturer's instructions. The results of the experiment are shown in Table 4 and Figure 7.

Table 4: Determination of the optical density (OD) at a wavelength of 450 nm to determine the synthesis of glycosaminoglycans according to the Blyscan glycosaminoglycan

Assay (tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK).

Example 7 - Stimulation of the synthesis of collagen, elastin and proteoglycan in primary human fibroblasts by hydroxylated collagen peptide preparations according to the invention:

To determine the stimulation of the synthesis of collagen, elastin and proteoglycan, primary human dermal fibroblasts according to Example 4.2 were used in a medium without collagen peptide over a period of at least 14 days, preferably 14 to 21 days, in particular 14 days

(Control 1) and in the presence of 0.5 mg / ml of a collagen peptide preparation with an average molecular weight of 7.0 kDa (sample 4), a collagen peptide preparation with an average molecular weight of 5.6 kDa (sample 5) and a collagen peptide preparation with one average molecular weight of 1.3 kDa (sample 6) incubated. Example 7.1 - Determination of stimulation of collagen synthesis

The determination of collagen synthesis by primary human dermal fibroblasts was carried out using the Sircol Collagen Assay Kit (item no. Item no. 054S5000, 2019, tebu-bio, Offenbach, Germany, or Biocolor Ftd., UK) according to the manufacturer's instructions. The results of the experiment are shown in Table 5 and Figure 8. The average value determined for the untreated control (control 1) was standardized to 1 by default.

Table 5: Determination of the optical density (OD) to determine the collagen synthesis according to the Sircol Collagen Assay (tebu-bio, Offenbach, Germany, or Biocolor Ftd., UK).

Example 7.2 - Determination of stimulation of the synthesis of elastin

The determination of elastin synthesis by primary human dermal fibroblasts was carried out using the Fastin Elastin Assay (Article No. 054F2000, 2019, tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK) according to the manufacturer's instructions. The results of the experiment are shown in Table 6 and Figure 9. The average value determined for the untreated control (control 1) was standardized to 1 by default.

Table 6: Determination of the optical density (OD) to determine the elastin synthesis according to the Fastin Elastin Assay (tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK).

Example 7.3 - Determination of stimulation of the synthesis of glycosaminoglycan

The determination of the synthesis of glycosaminoglycans by primary human dermal fibroblasts was carried out using the Blyscan glycosaminoglycan assay (item no. 054B3000, 2019, tebu-bio, Offenbach or Biocolor Ltd., UK) according to the manufacturer's instructions. The results of the experiment are shown in Table 7 and Figure 10. The average value determined for the untreated control (control 1) was standardized to 1 by default.

Table 7: Determination of the optical density (OD) for determining the synthesis of glycosaminoglycans according to the Blyscan glycosaminoglycan assay (tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK).

Claims

EXPECTATIONS

1. A method for producing a collagen peptide preparation comprising recombinant collagen peptides, comprising the method steps a) providing an expression system which has at least one expression cassette, the expression cassette having at least one nucleotide sequence which has a collagen peptide with a molecular weight in a range from 8 to 100 kDa encoded, b) incubation of the expression system under conditions which enable expression of the collagen peptide, c) recovery of the collagen peptide, d) hydrolysis of the collagen peptide under conditions which lead to the production of a collagen peptide preparation, the collagen peptide having an average molecular weight of 1 to 7 kDa and having a molecular weight in a range from 0.1 to 13.5 kDa and e) recovering the collagen peptide preparation.

2. The method according to claim 1, wherein the expression system is a host cell selected from the group consisting of bacterial cell, yeast cell, fungal cell, mammalian cell, insect cell and plant cell.

3. The method according to any one of the preceding claims, wherein the collagen peptide encoded by the nucleotide sequence is a collagen peptide of a vertebrate, in particular a mammal, a bird, a fish, an amphibian, a reptile or an invertebrate.

4. The method according to any one of the preceding claims, wherein the expression system is a host cell which is capable of hydroxylating proline, lysine or proline and lysine residues of the expressed collagen peptide.

5. The method according to claim 4, wherein the host cell has at least one expression cassette which has a prolyl 4-hydroxylase-coding polynucleotide sequence and in process step e) an in vivo hydroxylated collagen peptide preparation is obtained.

6. The method according to claim 4 or 5, wherein the host cell at least one

Has expression cassette, which has a lysyl hydroxylase-encoding polynucleotide sequence and in process step e) an in vivo hydroxylated collagen peptide preparation is obtained.

7. The method according to any one of claims 1 to 3, wherein the expression system is not capable of causing a hydroxylation of proline, lysine or proline and lysine residues of the expressed collagen peptide.

8. The method according to claim 7, wherein the collagen peptide obtained in process step c) is hydroxylated in a process step xl) before carrying out process step d) and a prlysal ex vivo hydroxylated collagen peptide preparation is obtained in process step e).

9. The method according to claim 7, wherein the produced in process step d)

Collagen peptide preparation after carrying out process step d) is hydroxylated in a process step x2) and a postlysal ex vivo hydroxylated collagen peptide preparation is obtained in process step e).

10. The method according to any one of the preceding claims, wherein the hydrolysis is an enzymatic or acid-catalyzed hydrolysis.

11. Collagen peptide preparation which can be prepared by one of the processes of claims 1 to 10.

12. Collagen peptide preparation which is produced by hydrolysis of collagen peptide having a molecular weight in a range from 8 to 100 kDa produced recombinantly in a host cell, the collagen peptide preparation being collagen peptides with an average molecular weight of 1 to 7 kDa and a molecular weight in a range of 0. 1 to 13.5 kDa.

13. Collagen peptide preparation according to one of claims 11 or 12, which is non-hydroxylated, partially hydroxylated or completely hydroxylated.

14. Collagen peptide preparation according to one of claims 11 to 13, wherein the collagen peptide preparation is a pre-lysed ex vivo hydroxylated collagen peptide preparation or a post-lysed ex vivo hydroxylated collagen peptide preparation.

15. Collagen peptide preparation according to one of claims 11 to 14 for use in a method for maintaining and improving bone health, for the prevention and / or treatment of osteoporosis, for the prevention and / or treatment of sarcopenia, for the prevention and / or treatment of degenerative loss of muscle mass, to improve muscle strength, for the prevention and / or treatment of a pathological condition characterized by a reduced mitochondrial activity, in particular for the prevention and / or treatment of a pathological condition characterized by a reduced endurance performance, for the stimulation of fat loss, for the reduction of body weight and / or for the prevention and / or treatment of degenerative joint diseases, for the prevention and / or treatment of cartilage diseases, for the prevention and / or treatment of diseases of the tendons and ligaments, for the prevention and / or treatment of skin diseases, for Treatment of wounds, for the prevention and / or treatment of degenerative nerve diseases, for the prevention and / or treatment of dementia, for the prevention and / or treatment of Alzheimer's disease, for the prevention and / or treatment of a pathological condition characterized by a reduction in mental performance , for the prevention and / or treatment of diseases related to malfunctions of the blood-Him barrier, for the prevention and / or treatment of intestinal diseases, for the prevention and / or treatment of diseases of the cardiovascular system and / or for the prevention or treatment of Diseases of the tooth structure.

16. Non-therapeutic use of a collagen peptide preparation according to one of claims 11 to 14 for the optical and structural improvement of the skin, for accelerating the growth of the nails and / or reducing the brittleness of the nails, for the optical and structural improvement of the hair, for increasing the number of mitochondria and / or mitochondrial activity, to improve endurance performance and / or to improve mental performance.

17. A product comprising a collagen peptide preparation according to any one of claims 11 to 14 and at least one additive.

18. Food supplement comprising a collagen peptide preparation according to one of claims 11 to 14 and at least one food-acceptable additive.

19. A pharmaceutical composition comprising a collagen peptide preparation according to any one of claims 11 to 14 and at least one pharmaceutically acceptable additive.

20. Cosmetic product comprising a collagen peptide preparation according to one of claims 11 to 14 and at least one skin-compatible additive.