Cosmetic composition comprising recombinant bacterial collagen-like protein (CLP) and use thereof

Abstract

The present invention relates to a cosmetic composition comprising a recombinant collagen-like protein (CLP).

Description

[0001] The present invention relates to a cosmetic composition comprising recombinant collagen-like protein (CLP) as both an active ingredient and a functional ingredient, and uses thereof.

[0002] Collagen is a long-standing player in anti-aging cosmetics and is primarily derived from animal sources. As such, it doesn't meet today's increasingly stringent conscious beauty standards, with the growing demand for vegan, clean, and green ingredients.

[0003] Collagen in cosmetics is used in a wide range of applications, from topically applied skin care actives to functional ingredients in hair styling products. Different applications require tailored material properties and take advantage of the various material characteristics of collagen from animal sources.

[0004] Collagen from animal sources forms a stable triple helix structure. However, when the respective mammalian collagen sequences are expressed in lower organisms such as bacteria or yeast, the materials do not exhibit the critical degree of post-translational hydroxylation required to stabilize the triple helix structure. Consequently, collagen-like proteins produced recombinantly by fermentation are generally only available as single chains and do not fold into a stable triple helix.

[0005] For example, WO 2020 / 205848 A1 discloses topical preparations of recombinant collagen, and US 11,514,609 B2 describes recombinant collagen and elastin molecules and their uses. However, these products are in monomeric form and do not form a stable triple helical structure similar to natural collagen.

[0006] The subject matter of the present invention is therefore to provide a vegan collagen substitute for cosmetic applications, which can also adopt a triple helical structure.

[0007] The present invention therefore relates to a cosmetic composition comprising a recombinant bacterial collagen-like protein.Preferably, the recombinant bacterial CLP is a bacterial collagen-like protein from Streptococcus pyogenes.

[0008] Bacterial collagen-like proteins mimic animal-derived materials in all respects, including their triple-helical structure. In this sense, they are ahead of current fermentation-derived collagen-like products. The inherent structural properties of bacterial collagen, which are determined by their specific sequence, allow the material to adopt a stable, triple-helical structure that mimics natural, human collagen, even when expressed in a host organism. Furthermore, the material is also available as a single-chain product, demonstrating excellent versatility.

[0009] Additionally, due to the structural uniqueness of bacterial collagen, the use of bacterial collagen obviates the need to introduce specific additional enzymes into the host to allow for critical post-translational hydroxylations during the fermentation process, as these modifications are not required to stabilize the triple helical structure.

[0010] The present invention of using bacterial collagen-like proteins in cosmetics overcomes the limitation that vegan collagen obtained through fermentation is generally only available as a single-chain molecule for such applications, rather than as a stable triple-helical form. Therefore, the application of bacterial collagen-like proteins in cosmetics allows for the widespread use of vegan collagen in cosmetics—either as a triple helix or a single chain.

[0011] Therefore, the material is suitable for a wide range of personal care applications as an active and functional ingredient in topical skin care through to hair and eyelash styling products.

[0012] Structure of recombinant bacterial CLP

[0013] Collagen-like proteins (CLPs) of bacterial origin (the most relevant industrially is the product of Streptococcus pyogenes) have quite favorable mechanical properties, similar to those of collagens of higher eukaryotes, without the need for the complex maturation steps required by their eukaryotic counterparts. CLPs present a common structure: two α-helices that stabilize each other constitute the "V domain," which is followed by a rod-shaped structural collagen domain (CL). Following the collagen domain, a membrane anchor (GPI-like) is usually present at the C-terminus of the protein.

[0014] As described in various publications (Lukomski et al. 2002 (J. Biol. Chem., 277:27312-27318, 2002), Brodsky et al. 2009 (Protein Sci., 18:1241-1251, 2009)), the current understanding of this process is that the V domain is required for folding three Scl2 protein monomers into a triple helical structure in vitro (Lukomski et al. demonstrated in vivo folding without the V domain).

[0015] Although the V domain may have a positive effect on this process, it was found that it is not the only factor that allows the protein to fold. It was possible to show that correct folding also occurs in the absence of the V domain. The main factors identified were the concentration of the Scl2 monomer, temperature, time, pH and salt concentration.

[0016] Because the V domain is considered critical for the production of triple-helical Scl2, removal of this sequence has never been considered, leading to the following challenges:

[0017] The -V domain constitutes approximately one-third of the entire sequence and hinders protein export from the yeast Pichia pastoris, requiring a complex downstream process involving cell lysis to remove the target protein from the cell.

[0018] The -V domain itself has pathogenic properties and needs to be removed during the purification process. This is done by protease digestion. The use of proteases is quite expensive and it also needs to be removed in downstream processes.

[0019] The following overview shows the process steps required for product purification using the Scl2 construct with the V domain attached:

[0020] Cell separation (centrifugation)

[0021] ●Cell lysis (pressure homogenizer)

[0022] V domain removal (protease digestion)

[0023] Cell debris removal (pH shift, centrifugation)

[0024] ●Purification (solvent precipitation)

[0025] Washing (TFF)

[0026] ●Further purification (IEX)

[0027] Such a process is disclosed, for example, in Peng et al. (Appl. Microbiol. Biotechnol., 98: 1807-1815, 2014).

[0028] Production of recombinant bacterial CLP

[0029] Collagen-like proteins (CLPs) can be produced in the methylotrophic yeast Pichia pastoris or other hosts. Key features of this process compared to current processes known in the art include: 1) protein secretion into the culture supernatant, allowing high titers (>5 g / L) to be achieved in low-cost culture media; and 2) ease of protein purification from the supernatant due to the absence of complex components in the culture medium.

[0030] Surprisingly, the purified product from the supernatant of a Pichia pastoris culture secreting Scl2p displayed an unexpected profile compatible with a mature collagen-like sequence. Further analysis revealed how an intracellular enzyme, most likely the processing protease Kex2p, was able to remove the V domain protein sequence without the need for an additional protease step. Additionally, to modify the cleavage sites present in the final product, which lead to a significant accumulation of degradation products, the protein sequence was mutated to modify such cleavage sites and eliminate degradation. Surprisingly, the most effective performance was achieved when the non-polar amino acid (valine in the wild-type sequence) was mutated to a polar amino acid (glutamine).

[0031] It was surprisingly found that after secretion in the culture supernatant, recombinant CLPs can be correctly folded in the absence of the V domain during storage / freezing of the cell pellet prior to lysis. The present invention discloses a novel downstream process that includes the production of Scl2 proteins without the V domain and the deliberate integration of a folding step for the CL domain.

[0032] The method for producing recombinant collagen-like protein (CLP) comprises the following steps:

[0033] a) fermentation in a culture medium of a host cell expressing a CLP, said CLP having an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 1, wherein said amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1,

[0034] b) accumulation of CLP in the culture medium, wherein the fermentation broth is obtained,

[0035] c) separating the host cells from the fermentation broth to obtain a supernatant,

[0036] d) incubating the fermentation supernatant of step c) at no more than 25° C. for at least 1 hour for folding of CLP,

[0037] e) optionally purifying the CLP by at least one of the following: solvent precipitation, tangential flow filtration (TFF), ion exchange chromatography, reverse phase chromatography.

[0038] The host cell is preferably selected from bacteria, yeast or plant cells. Preference is given to using bacteria or yeast cells.

[0039] In a preferred embodiment, the host cell is a microorganism of the species Pichia pastoris, E. coli, P. putida or C. glutamicum comprising any of the polypeptides according to the present invention.

[0040] In a preferred embodiment, the microorganism is a yeast or bacterial cell of the genus Pichia pastoris, preferably Escherichia coli, Corynebacterium or Brevibacterium.

[0041] The microorganism may be one in which the nucleotide sequence is present in an overexpressed form.

[0042] The microorganism may be characterized in that the microorganism has the ability to produce and secrete fine chemicals. The fine chemicals are preferably collagen-like proteins.

[0043] Overexpression generally means an increase in the intracellular concentration or activity of a ribonucleic acid, protein (polypeptide) or enzyme compared to the starting strain (parent strain) or wild-type strain (if this is the starting strain). The starting strain (parent strain) means the strain to which measures are taken to cause overexpression.

[0044] Among overexpression methods, recombinant overexpression methods are preferred. These include all methods in which a microorganism is produced using a DNA molecule provided in vitro. Such DNA molecules include, for example, promoters, expression cassettes, genes, alleles, coding regions, etc. These are transformed into the desired microorganism by transformation, conjugation, transduction, or similar methods.

[0045] The extent of expression or overexpression can be determined by measuring the amount of mRNA transcribed from the gene, by determining the amount of polypeptide and by determining enzyme activity.

[0046] Surprisingly, it was found that truncated variants of collagen-like proteins, including variants with truncated V domains or without any V domains, resulted in increased production of collagen-like proteins and secretion into the fermentation medium. Further surprising was that the truncated variants were able to fold correctly in the absence of the V domain.

[0047] In a preferred embodiment of the present invention, the optionally purified CLP is folded in a further process step (step d). The CLP folding in step d) is carried out at a temperature of -80°C to 25°C, preferably at a temperature of 0°C to 20°C. In a preferred configuration, the folding is carried out in the presence of glycerol or a salt.

[0048] In another preferred embodiment, the folding of the CLP in step d) is performed for a time of 1 hour to 48 hours, preferably 1 hour to 24 hours.

[0049] In another preferred embodiment, the folding of CLP in step d) is performed with a CLP concentration of at least 1 mg / ml, preferably at least 4 mg / ml.

[0050] Protein sequence SEQ ID NO: 1 Streptococcus pyogenes collagen-like protein (CLP), full-length protein SEQ ID NO: 2 Streptococcus pyogenes CLP, truncated 3

[0051] SEQ ID NO:3 Streptococcus pyogenes CLP, truncated 5

[0052] SEQ ID NO:4 Streptococcus pyogenes CLP, without V domain SEQ ID NO:5 Streptococcus pyogenes CLP, truncated 5 (AGPR mutant) SEQ ID NO:6 Streptococcus pyogenes CLP, truncated 5 (QGPR mutant) SEQ ID NO:7 Streptococcus pyogenes CLP, truncated 5 (VGPA mutant) SEQ ID NO:8 Streptococcus pyogenes CLP, truncated 5 (SGPR mutant) SEQ ID NO:9 Streptococcus pyogenes CLP, truncated 5 (VGPK mutant)

[0053] In a preferred embodiment, the CLP has an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 1 and comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1.

[0054] It is preferred when the amino acid sequence comprises a deletion of 38 to 90 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1. This includes a complete deletion of the N-terminal V domain (comprising 74 amino acids) and different truncations of the V domain of at least 38 amino acids.

[0055] The present invention accordingly also relates to polypeptide variants of SEQ ID NOs: 2 to 9, which contain one or more insertions or deletions. Preferably, the polypeptide contains an insertion or deletion of at most 5, at most 4, at most 3, or at most 2 amino acids.

[0056] In a preferred embodiment, the amino acid sequence is at least 60% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0057] In a preferred configuration, the amino acid sequence is at least 90%, 92%, 94%, 96%, 97%, 98%, 99% or 100%, preferably 97%, particularly preferably 98%, very particularly preferably 99%, and extremely preferably 100% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0058] Cosmetic compositions and preparations

[0059] The cosmetic compositions according to the present invention provide one or more benefits for hair care and skin care, including, but not limited to, improving various signs of skin aging, such as fine lines, wrinkles, folds, and sagging, especially in the face and on the neck, by one or more of: increasing firmness, increasing elasticity, improving hydration, and reducing the presence of superficial fat tissue.

[0060] The cosmetic composition according to the present invention preferably comprises 0.001% to 30% w / w, 0.01% to 15% w / w or 0.1% to 10% w / w of CLP.

[0061] The cosmetic composition preferably further comprises at least one additional ingredient selected from the group consisting of emollients, emulsifiers, thickeners / viscosity regulators / stabilizers, UV protection filters, antioxidants, hydrotropes, solids and fillers, film formers, anti-caking agents, pearlescent additives, deodorant and antiperspirant active ingredients, insect repellents, self-tanning agents, preservatives, conditioners, fragrances, dyes, odor absorbers, superfatting agents, carrier materials, further skin actives and solvents. Substances that can serve as exemplary representatives of the individual groups are known to those skilled in the art and can be found, for example, in German patent application DE 10 2008001788.

[0062] With regard to further optional components and the amounts in which these components are used, reference is expressly made to the relevant handbooks known to the person skilled in the art, for example K. Schrader, "Grundlagen und Rezepturen der Kosmetika [Cosmetics—fundamentals and formulations]", 2nd edition, pp. 329 to 341, Hüthig Buch Verlag Heidelberg.

[0063] In a further aspect, the present invention provides a topical cosmetic formulation comprising the recombinant bacterial collagen-like protein. The topical cosmetic formulation may comprise at least one cosmetically acceptable carrier.

[0064] The delivery of active substances, such as CLPs, to and through the skin involves a complex interplay between the active substance, the type of carrier system ('vehicle'), the choice of excipients, skin type and location, and skin condition. A cosmetically acceptable carrier is defined as a substance that carries the selected active substance into contact with and through the skin at appropriate levels to provide a cosmetic effect. At the same time, the carrier must not affect skin health.

[0065] The challenge of topical drug delivery lies in transport across the stratum corneum (SC). To overcome this obstacle, the carrier must: maintain the solubility and stability of the active substance; release the active substance for uniform deposition on the skin; allow penetration and permeation of the SC skin barrier; promote distribution from the SC into and diffusion through the viable epidermis; maintain the active substance at the target site for a sufficient duration to provide a cosmetic effect; and limit systemic absorption. Furthermore, the carrier should be soothing, comfortable, easy to spread, and aesthetically pleasing.

[0066] Topical carriers can be classified based on their physical state, including semisolids (e.g., ointments, creams, gels) and liquids (e.g., emulsions, solutions, foams, sprays). Emulsions, gels, and solutions are single-phase, ointments and creams are two-phase, and foams are three-phase. Cosmetically acceptable carriers are such that the composition can be prepared as a decorative cosmetic, skin care, body care, rinse-off, or leave-on hair care composition.

[0067] According to one aspect, the cosmetically acceptable carrier comprises water. According to another preferred aspect, the carrier additionally comprises a surfactant. The at least one cosmetically acceptable carrier may therefore be an aqueous carrier in a single phase, a two-phase phase or an emulsion.

[0068] According to the above, the present invention also relates to the use of recombinant bacterial collagen-like protein in cosmetic applications. The recombinant bacterial collagen-like protein preferably comprises one of the protein sequences in SEQ ID 1 to 9 as defined above.

[0069] As an alternative embodiment, the recombinant bacterial collagen-like protein can be formulated as a nutricosmetic supplement for oral consumption. As used herein, the term "nutricosmetic" should be understood as products and ingredients that act as nutritional supplements to care for skin, nails, and hair. In some embodiments, the recombinant bacterial collagen-like protein can be formulated as a food product or food ingredient for oral consumption. Accordingly, the present invention provides oral formulations containing the recombinant bacterial collagen-like protein.

[0070] The oral formulation may further include at least one component selected from a carrier, a preservative and / or another edible ingredient. Thus, for example, the composition may include vitamins (e.g., vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, etc.), minerals (e.g., calcium, zinc, copper, manganese, chromium, molybdenum (molundenum), boron, etc.), sugars (e.g., cellulose, dextrose, maltose, etc.), and / or natural extracts (e.g., herbs, ginseng, echinacea, green tea, glucosamine, ω-3, lutein, folic acid, liver oil, fish oil, coffee extract, etc.). Preparations suitable for consumption by an individual (e.g., person) include, but are not limited to, instant mix powders, instant drinkable beverages, functional injections (functional shots), supplemental tablets, and capsules.

[0071] According to the above, the present invention also relates to the use of recombinant bacterial collagen-like protein as a nutritional cosmetic. The recombinant bacterial collagen-like protein preferably comprises one of the protein sequences in SEQ ID 1 to 9 as defined above.

[0072] Finally, the present invention provides a non-therapeutic method of improving the appearance of a subject's skin, hair and / or nails, the method comprising administering to the subject a cosmetic composition according to the present invention. The cosmetic composition may be administered topically or orally. Example

[0073] A) Production of bacterial collagen-like protein (CLP)

[0074] Collagen-like proteins are produced in yeast host cells Pichia pastoris by fermentation. In order to produce Scl2 from Streptococcus pyogenes in Pichia pastoris, the sequences of collagen-like proteins (full-length protein as well as truncated variants and V-domain-free variants) have been codon-optimized using different algorithms and cloned into secretion vectors for Pichia pastoris. The sequences used are summarized in SEQ ID NO: 1 to SEQ ID NO: 9. For each specific sequence, the vector was transformed into Pichia pastoris following the standard protocol, and a standard expression protocol (Damasceno, LM, Huang, CJ. & Batt, CA Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol 93, 31–39 (2012)) in fed-batch mode was applied. The collagen domain of Scl2p protein was detected in the supernatant of the cell culture via HPLC analysis. After fermentation, the supernatant has been separated from the biomass by centrifugation (12000 g, 5 min at room temperature).

[0075] Collagen domains based on the Scl2p proteins of sequences SEQ ID NO: 1 to SEQ ID NO: 9 can be produced under similar conditions using Escherichia coli, Bacillus choshinensis or Corynebacterium glutamicum. In the case of production in yeast or Corynebacterium glutamicum, the collagen domains are secreted by the cells. This method does not require cell lysis as an initial purification step. In the case of production in Escherichia coli, cell lysis is necessary to remove the collagen domains from the cells.

[0076] Full-length collagen-like protein, truncated variants (truncation 3) and V-domain-free variants (based on the gene scl2 from Streptococcus pyogenes) are also expressed in Bacillus pumilus. Therefore, the corresponding DNA sequences are cloned into suitable secretion vectors for Bacillus pumilus. Transformation of Bacillus pumilus with the newly constructed plasmids is completed according to Mizukami et al. 2010 (Curr Pharm Biotechnol 2010, 13: 151-258).

[0077] The Eppendorf (Hamburg, Germany) A parallel bioreactor system was used to analyze Bacillus pumilus strains for their ability to produce different collagen-like proteins in batch culture at 33°C and pH 7. Fermentation was performed using a 1 L reactor. The production medium (TM medium, Biomed Res Int 2017, 2017: 5479762) contained 10 g / L glucose. After fermentation, the supernatant was separated from the biomass by centrifugation and analyzed by SDS PAGE. For all three variants, the collagen domain of the Scl2p protein was produced.

[0078] The present invention relates to the invention of the present invention.Full-length collagen-like protein and no V domain variant (based on the gene scl2 from Streptococcus pyogenes) are also expressed in Corynebacterium glutamicum.Therefore, corresponding DNA sequence dna is cloned into (Biotechnology Techniques 1999,13:437-441.) for the shuttle vector of Corynebacterium glutamicum together with the signal peptide for protein secretion of upstream positioning.As described by the people such as Ruan (Biotechnology Letters 2015,37:2445–2452), by means of electroporation with the newly constructed plasmid transformed Corynebacterium glutamicum bacterial strain ATCC 13032.

[0079] The Eppendorf (Hamburg, Germany) A parallel bioreactor system was used to analyze the ability of Corynebacterium glutamicum strains to produce different collagens in fed-batch cultures at 30°C and pH 7. Fermentation was performed using a 1L reactor. The production medium contained 20g / L glucose in the batch phase, and the fed-batch phase was run with a glucose feed rate of 4g / L*h. After fermentation, the supernatant was separated from the biomass by centrifugation and used for HPLC analysis. For both variants, the collagen domain of the Scl2p protein was produced. Titers were higher for the truncated variants of the collagen-like protein than for the full-length variant.

[0080] The process steps are summarized below:

[0081] 1. Production of collagen-like proteins in yeast, Escherichia coli, or Corynebacterium

[0082] 2. Cell lysis (only for E. coli)

[0083] 3. Cell separation (filtration or centrifugation)

[0084] 4. Folding of the CL single chain to form a triple helical structure

[0085] 5. Further Purification by Solvent Precipitation and Ultrafiltration

[0086] 6. Freeze-drying of Purified CL Protein

[0087] B) Regarding the cosmetic effects and preparations of bacterial collagen-like protein (CLP)

[0088] A series of in vitro experiments were performed to evaluate the effects of bacterial collagen-like protein (CLP) on normal human dermal fibroblasts.

[0089] Example 1: Bacterial collagen-like protein (CLP) stimulates fibroblast production of the HAS1 hyaluronan synthase 1 gene.

[0090] Normal human dermal fibroblasts were evaluated for hyaluronan synthase 1 (HAS1) expression. The expression of HAS1 was analyzed using RT-qPCR for total RNA extracted from the cells. Fibroblasts were incubated with various concentrations of the exemplary bacterial collagen-like protein (CLP) of SEQ ID NO: 3 for 24 hours. Figure 1 As shown in , these fibroblasts expressed higher levels of HAS1 than untreated controls or fibroblasts incubated with two different collagen benchmark materials. When tested at 1 mg / ml, CLP stimulated HAS1 expression up to 452% of the control.

[0091] Example 2: Bacterial collagen-like protein (CLP) stimulates type I procollagen release by fibroblasts.

[0092] Normal human dermal fibroblasts were evaluated for type I procollagen release. Fibroblasts were incubated with various concentrations of the exemplary bacterial collagen-like protein (CLP) of SEQ ID NO 3 for 72 hours. After the incubation period, the culture supernatant was collected and analyzed for type I procollagen C-peptide (PIP) by enzyme-linked immunosorbent assay (ELISA). Figure 2 As shown in , these fibroblasts released higher levels of type I procollagen than untreated controls or fibroblasts incubated with two different collagen benchmark materials. Relative to untreated controls, CLP at 10 mg / ml stimulated up to 219% (170% at 3 mg / ml and 154% at 1 mg / ml) type I procollagen release by fibroblasts.

[0093] Bacterial collagen-like protein (CLP) can be used in a variety of product forms and formulations. In its triple-helical form, CLP has a solubility of >50 g / L in water. In its single-stranded form, CLP exhibits increased water solubility. Exemplary formulations containing CLP are listed below.

[0094] Example 3: Collagen cream.

[0095] Preparation: Heat phases A and B separately to 70-75°C. Add phase A to phase B while stirring. (If phase A must be added to the vessel first, phase B must not be added without stirring.) Homogenize. Cool while stirring gently.

[0096] Table 1 Composition of collagen cream

[0097]

[0098] Example 4: Deep anti-wrinkle smoothing cream with collagen.

[0099] Preparation: Heat phases A and B separately to approximately 80°C. Add phase A to phase B while stirring. (If phase A must be added to the vessel first, phase B must not be added without stirring). Homogenize. Cool to approximately 60°C while stirring gently and add phase C. Homogenize briefly. Cool while stirring gently and add phase D below 40°C.

[0100] Table 2 Composition of deep anti-wrinkle smoothing cream with collagen

[0101]

[0102]

[0103] Example 5: Lifting effect facial care collagen lotion.

[0104] Preparation: Heat phase A to 70°C and phase B to approximately 30°C. Add phase B to phase A without stirring. Homogenize. Cool slowly while stirring.

[0105] Table 3 Composition of the lifting effect facial care collagen emulsion

[0106]

[0107]

[0108] Example 6: Anti-Gravity Facial Mousse.

[0109] Preparation: Heat phase A to 40°C. Add phase A to phase B while stirring. (If phase A must be added to the vessel first, phase B must be added without stirring). Homogenize. Add phase C and homogenize briefly. Add phase D and stir thoroughly. Add phase E and stir thoroughly. Mix with the propellant (propane, butane, isobutane) in a ratio of 90 emulsion:10 propellant.

[0110] Table 4 Composition of Anti-Gravity Facial Mousse

[0111]

[0112]

[0113] Example 7: Enhance hair and scalp health with gentle cleansing.

[0114] Preparation: Heat phases A and B to 75-80°C. While stirring, slowly add phase B in steps. Cool while stirring gently. Add phase C below 40°C.

[0115] Table 5 Ingredients for enhancing hair and scalp health with gentle cleansing

[0116] Description of the accompanying drawings:

[0118] Figure 1 Effects on gene expression of hyaluronan synthase 1 (HAS1) in fibroblasts treated with an exemplary bacterial collagen-like protein (CLP) (C) and benchmark materials A and B are shown.

[0119] Figure 2Shown are the effects on type I procollagen release by fibroblasts treated with an exemplary bacterial collagen-like protein (CLP) (C) and benchmark materials A and B; ns: >0.05 not significant, *: significant between 0.01 and 0.05, **: very significant between 0.001 and 0.01; ***: <0.001 extremely significant, nc: cannot be calculated.

Claims

1. A cosmetic composition comprising a recombinant bacterial collagen-like protein, wherein the recombinant bacterial collagen-like protein has an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:

1. 2 . The cosmetic composition according to claim 1 , wherein the recombinant bacterial collagen-like protein is a collagen-like protein from Streptococcus pyogenes.

3. The cosmetic composition according to claim 1 or 2, wherein the recombinant bacterial collagen-like protein is obtained by fermentation in a host cell, and the host cell is a yeast cell, preferably Pichia pastoris, or a bacterial cell, preferably Escherichia coli, Brevibacillus choshinensis or Corynebacterium glutamicum.

4. The cosmetic composition according to any one of the preceding claims, wherein the amino acid sequence of the recombinant bacterial collagen-like protein comprises a deletion of 38 to 90 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:

1.

5. The cosmetic composition according to any one of the preceding claims, wherein the amino acid sequence of the recombinant bacterial collagen-like protein is at least 60% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO:

9.

6. The cosmetic composition according to any one of the preceding claims, wherein the amino acid sequence of the recombinant bacterial collagen-like protein is at least 90%, 92%, 94%, 96%, 97%, 98%, 99% or 100%, preferably 97%, particularly preferably 98%, and very particularly preferably 99% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO:

9.

7. The cosmetic composition according to any one of the preceding claims, wherein the amino acid sequence of the recombinant bacterial collagen-like protein is 100% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO:

9.

8. The cosmetic composition according to any one of the preceding claims, further comprising at least one additional ingredient selected from the group consisting of emollients, emulsifiers, thickeners / viscosity regulators / stabilizers, UV protection filters, antioxidants, hydrotropes, solids and fillers, film formers, anti-caking agents, pearlescent additives, deodorant and antiperspirant active ingredients, insect repellents, self-tanning agents, preservatives, conditioning agents, fragrances, dyes, odor absorbers, superfatting agents, carrier materials, additional skin actives, and solvents.

9. A topical preparation comprising a recombinant bacterial collagen-like protein.

10. The topical formulation according to claim 9, further comprising at least one cosmetically acceptable carrier.

11. The topical formulation of claim 10, wherein the at least one cosmetically acceptable carrier is an aqueous carrier in a single phase, a dual phase, or an emulsion.

12. An oral preparation comprising recombinant bacterial collagen-like protein.

13. The oral formulation according to claim 12, further comprising at least one ingredient selected from a carrier, a preservative and / or another edible ingredient.

14. Use of recombinant bacterial collagen-like protein in cosmetic applications.

15. Use of recombinant bacterial collagen-like protein as nutritional cosmetics.

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