Composition comprising rapeseed protein hydrolysate and use thereof

Abstract

A composition, preferably a cosmetic composition, comprising - a rapeseed protein hydrolysate comprising protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa; and - optionally a cosmetically acceptable further ingredient.

Description

[0001] COMPOSITION COMPRISING RAPESEED PROTEIN HYDROLYSATE AND USE THEREOF

[0002] Field of the invention

[0003] The present invention relates a composition comprising rapeseed protein hydrolysate and the use thereof. Further, the present invention relates to a cosmetic skin and scalp care method for the reduction and / or removal of senescent cells.

[0004] Background of the invention

[0005] Proteins are a crucial part of human diet, and this is for a major part met by proteins from animal origin, such as dairy proteins or protein from meat or fish. World population growth in combination with increasingly limited resources has resulted in the need for alternative protein sources to meet global protein demand. The production of plant-based foods requires less land and water and is associated with lower greenhouse gas emissions compared with animal-based foods.

[0006] Generally, the consumed proteins in feed or food are intact proteins, however for specific reasons, hydrolysed proteins may have advantages. Nearly fully hydrolysed proteins lead to mostly free amino acids and some oligopeptides, for use in for instance flavours (savoury flavours from hydrolysed yeast proteins, rich in glutamate). Such hydrolysates can be made by proteolysis, using (combinations of) proteases, or in the case of several yeast extracts, autolysis. Alternatively, these can be made by chemical hydrolysis using highly concentrated acids.

[0007] Also in cosmetics protein hydrolysates are used. As for example mentioned by Colnaghi et al in their article titled “Characterization of protein hydrolysates of cosmetic use by CE- MS”published in the Journal of Separation Sciencevolume 34, Issue 8, 2011 , pages 947-956, protein hydrolysates are applied in cosmetic formulae for substantivity, penetration, film formation, moisture retention and / or gloss.

[0008] For example, W02005107697A1 describes the use of an effective amount of a hydrolyzate of plant proteins, belonging to the cruciferous family, as a whitening and I or depigmenting active principle, in or for the preparation of a cosmetic and I or dermatological and I or pharmaceutical, wherein the hydrolyzate is for example obtained from rapeseed seeds.

[0009] Hydrolysis of proteins may proceed to the individual free amino acids, but sometimes it is desired to generate peptides of various length, depending on the application, which can be tuned by the strategy of hydrolysis, choice of proteases, sequence of addition of enzymes, environmental conditions (pH, temperature etcetera) and pretreatment (for instance heating). In feed and / or food applications there are various reasons for such hydrolysates, mostly for nutritional or functional reasons. However, within the context of cosmetic applications little is known about the type of hydrolysis.

[0010] Functional properties of protein hydrolysate may also improve compared to their unhydrolyzed origins, as is described by for instance Wouters et al. (Comp Rev Food Sci Safety 2016 15 p786 Relevance of functional properties of enzymatic plant protein hydrolysates in food systems)-. Depending on the strategy of hydrolysis, changes can be obtained that can include better solubility, better foaming, better emulsification, and may also impact gelation and water holding properties. Protein hydrolysates may for instance also act as plasticizer for use in films and casings (Zhang, C., et al. Food Chem. 2019 272, 694-701 The preparation and physiochemical characterization of rapeseed protein hydrolysate-chitosan composite films), and in more solid foods such as protein bars cheese or cheese alternatives or meat alternatives. Protein hydrolysates may also display anti-oxidative properties, by for instance improved chelating properties of specific protein hydrolysates, as has been mentioned particular for a hydrolysate of rapeseed protein isolate using a fungal protease, described by Durand et al. (Eur. J. Lipid Sci. Technol. 2021 , 123, 2000380 High Metal Chelating Properties from Rapeseed Meal Proteins to Counteract Lipid Oxidation in Foods: Controlled Proteolysis and Characterization and Kaugarenia_et al. Foods 2022, 11 , p2618 Optimization of Selective Hydrolysis of Cruciferins for Production of Potent Mineral Chelating Peptides and Napins Purification to Valorize Total Rapeseed Meal Proteins). In the latter it is even clear that using a fungal protease leads to hydrolysis of particularly cruciferin, and leaves napin mostly intact.

[0011] Proteases are generally divided into two classes endoproteases (endopeptidase, endo proteinases) and exoproteases (exopeptidases). An endoprotease cleaves a peptide bond between amino acids within the protein chain, not at the termini. The exoproteases will cleave such peptide bond between amino acids at the termini of the protein molecule, either from the N-terminus or the C-terminus. Typical examples of endoproteases are those found in the digestive tract, such as pepsin, trypsin and chymotrypsin, as well as subtilisin.

[0012] Subtilisin is probably the most common protease used in food and non-food (such as laundry and dishwashing detergents), because of its aggressive character and its costeffectiveness. It originates from Bacillus subtilis, but mostly industrially is mostly produced in Bacillus amyloliquefaciens. It is a serine protease with molecular weight of typically around 27kDa. See for instance Azrin et al Biotech and App Biochem - 2022 p1 -18 Versatility of subtilisin: A review on structure characteristics and applications. Disadvantage of hydrolysates prepared by subtilisin is that the protein hydrolysates are perceived highly bitter.

[0013] Flavourzyme is also promoted as a good protease in food industry, it is a fungal protease complex produced by Aspergillus oryzae, and it contains both endoprotease and exopeptidase activities. At pH 7, when exo-protease activity prevails, a high degree of hydrolysis may be obtained, but that is mostly due to the generation of single amino acids, hydrolysed from the termini of the protein. At pH 5 the endoprotease activity is said to be more dominant. Often a combination of subtilisin (ALCALASE™) and Flavourzyme can be used to obtain functional hydrolysates, and it is also claimed to limit the bitterness level of the hydrolysates formed. It is claimed that the exo-activity of Flavourzyme leads to debittering of the peptides. US10619177 describes how common proteases such as ALCALASE™ (tradename for subtilisin) and Flavourzyme (tradename for a fungal protease / peptidase complex produced by Aspergillus oryzae) are capable of hydrolysing soy protein and wheat gluten to a substantial extent at standard conditions (0.25% ALCALASE™ on 12% soy bean meal, containing approximately 50% protein, reached a degree of hydrolysis of 12.4% in 2 hours at 70°; subsequent addition of 3% Flavourzyme at 50°C and 4 hours incubation led to a degree of hydrolysis of 22.4%).

[0014] Another strategy to reduce bitterness of protein hydrolysates in food industry is by treating a pre-hydrolysed protein further with a proline-specific protease such as Delvoplant PSP or DelvoPlant® PSP, as has been described by Edens et al. (J Agric Food Chem 2005 53 p7950- 7957, Extracellular prolyl endoprotease from Aspergillus niger and its use in the debittering of protein hydrolysates).

[0015] Rapeseed protein is abundantly available and is used more and more for the production of rapeseed protein products for the food and beverage industry. However, use within the cosmetic industry is limited. As mentioned above, W02005107697A1 describes the use of an effective amount of a hydrolyzate of plant proteins, belonging to the cruciferous family, as a whitening and I or depigmenting active.

[0016] Life expectancy is constantly increasing in many countries around the globe. Accordingly, the demand for treatments counteracting or even reversing aging processes is constantly growing, not only for age-related diseases but also for dealing with the physical signs of aging. Thus, it is not surprising that the need for cosmetic products that may be applied topically on skin to combat the signs of skin aging, such as wrinkles and age spots, has been growing significantly lately.

[0017] Aging of the skin and hair is a complex process. Intrinsic aging is an inevitable physiological process that results in thin, dry skin, fine wrinkles, decreased elasticity, aberrant pigmentation, hair greying, and hair loss. Extrinsic aging is caused by external environment factors notably solar radiation and air pollution and results in coarse wrinkles, loss of elasticity, laxity, and rough-textured appearance. Other factors that can further contribute to an aged appearance include general poor health, an unhealthy diet, cigarette smoking, and alcohol.

[0018] Today, it is well established that cell senescence plays an important role in aging processes. Cell senescence is a physiological process and a tumour-suppressive cell fate characterized by a permanent and irreversible cell cycle arrest as well as acquisition of a pro- inflammatory and proteolytic secretome. Cells that become senescent grow in size and cease to replicate. In addition, senescent cells generate a potent mix of molecules known as the senescence-associated secretory phenotype (SASP), which provokes the immune system into an inflammatory state, disrupts tissue structure and function, and in turn encourages nearby cells to also become senescent. The SASP is one of the key characteristics that distinguish senescent cells from quiescent, terminally differentiated, and other types of non-proliferating cells.

[0019] Intrinsic aging is typically correlated with an increase of senescent cells, including senescent skin cells. Senescence of dermal fibroblasts for example is considered a key driver of the aging-related phenotype in skin. Senescence of hair follicle dermal papilla cells (HFDPCs) on the other hand is considered a key driver of hair loss and baldness. Hence, there is an ongoing need to find agents which are able to selectively get rid of these errant cells.

[0020] Senolytics, which are typically understood as agents able to selectively kill senescent cells, were the first potential senotherapy to be successfully tested in preclinical in vivo models. In other words, an agent is considered as having senolytic activity when its activity includes specifically inducing or promoting cell death, in particular apoptotic cell death, of senescent cells in the respective tissue. Several senolytic agents have been identified by now such as navitoclax. Said senolytic agents are able to selectively kill senescent cells by induction of apoptosis process, but not on non-senescent cells or normal cells.

[0021] A drawback of currently known senolytic agents is that they are often of synthetic origin, have rather complex chemical structures that require significant efforts to be obtained, exhibit low solubility in conventional cosmetic oils, and / or may have severe undesired side effects.

[0022] Therefore, there is an ongoing need for senolytic agents of natural origin which are, for example, obtainable in an economic manner from plant-based sources.

[0023] Summary of the invention

[0024] Surprisingly, the present inventors found that the hydrolysis of a rapeseed protein isolate (RPI) by a combination of an endoproteases such as aspergillopepsin 1 with a proline-specific endoproteases leads to a hydrolysate where most of the napin has been hydrolysed and which has advantageous characteristics when applied in a cosmetic product.

[0025] Accordingly, in a first aspect, the invention provides a composition, preferably a cosmetic composition, comprising a rapeseed protein hydrolysate comprising protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa; and optionally a cosmetically acceptable further ingredient.

[0026] Surprisingly, it has now been found that such rapeseed protein hydrolysate can exhibit antioxidant activity, collagen boosting activity and / or senolytic activity. As illustrated in the examples, it has now been surprisingly found that such rapeseed protein hydrolysate can exhibit a senolytic activity, preferably on human dermal fibroblasts and / or hair follicle dermal papilla cells (HFDPCs). The rapeseed protein hydrolysate can thus advantageously be used to treat the signs of skin and / or hair aging.

[0027] Hence, in a second aspect, the invention provides a use, preferably a cosmetic and / or non- therapeutic use, of a rapeseed protein hydrolysate as described herein or a composition comprising such a rapeseed protein hydrolysate for the reduction of damage to DNA in the presence of oxygen; and / or for the boosting of Collagen 1 production by human dermal cell; and / or for the treatment of the signs of aging; and / or for reducing or eliminating senescent skin cells.

[0028] More generally the invention provides a use, preferably a cosmetic and / or non-therapeutic use, of a rapeseed protein hydrolysate described herein or a composition comprising such a rapeseed protein hydrolysate as an active ingredient for the treatment of the signs of skin and / or hair aging. More preferably the invention provides a use, preferably a cosmetic and / or non- therapeutic use, of a rapeseed protein hydrolysate as an active ingredient for the treatment of the signs of aging by selectively reducing or eliminating senescent skin cells. In a third aspect, the invention provides a cosmetic skin and scalp care method, said method encompassing the step of applying, onto the skin or the scalp in need, at least an effective amount of a rapeseed protein hydrolysate as described herein or a composition as described herein. Preferably such method is a method for the reduction and / or removal of senescent cells in the skin and / or hair follicle.

[0029] Details of the invention are provided below.

[0030] Description of the figures

[0031] Figure 1. SDS electrophoresis gel from samples of exp 1.1 a, proteolysis of 5% RPI with resp. 0.1 % ALCALASE™ ; lane 1 : marker, lane 2, RPI before addition of ALCALASE™, lane 6, after 4 hours incubation.

[0032] Figure 2. SDS electrophoresis gel from samples of exp 1.1 b / c, proteolysis of 5 or 20% RPI with 1.0% ALCALASE™ ; lane 3, sample 1.1 b 5% RPI after 4 hr incubation with 1.0% ALCALASE™; lane 5-7, 9 sample 1.1 C with 20% RPI after, 1.5, 3, 5 hours incubation with 1.0% ALCALASE™; Lane 10: marker.

[0033] Figure 3. SDS electrophoresis gel from samples of exp 1 .4, proteolysis of 20% RPI with 1 .0% Delvoplant PSP at pH 4; lane 1 marker, lane 2-5 after 0, 1 .5, 3, 4 hours incubation.

[0034] Figure 4. SDS Page gel of samples A and B taken during incubation, lane 1 and 2 Marker M12, lanes 3 - 5, sample 2A taken at t=0, 3hr and 4hr respectively, lanes 6 - 8, sample 2B taken at t=0, 3hr and 4hr respectively.

[0035] Figure 5. SDS Page gel of samples C taken during incubation; lanes 1 - 4, sample 2C taken at t=0, 3hr, 4hr of only MAXIPRO™ AFP, and 4 hours MAXIPRO™ AFP + 18 hours Delvoplant PSP respectively. Righter lane Marker M12.

[0036] Figure 6. Growth curves of various strains. Bs: B. subtilis, Ec: E. coli, Pp: P. pastoris, and Sc: S. cerevisiae. X axis time in hours, Y axis Optical Density in Arbitrary Units. Markers for various combinations of growth Verduyn based media with the indicated nitrogen source, RPI: rapeseed protein isolate; 2A, 2B and 2C: hydrolysates described in example 2; urea: only urea; blank: no nitrogen source added.

[0037] Figure 7. DNA damage assay with gH2AX staining. In the plot are represented mean + / - SD of 3 independent gH2AX staining experiment (n=4 each). Data are represented as gH2AX absolute signal intensity per cell.

[0038] Figure 8. Collagen I expression. In the plot are represented mean + / - SD (n=4). Data are represented as Collagen I level relative to control (%). Data are compared with TGFb (Transforming growth factor beta) stimulation (positive control). Unpaired student t-test, *p<0.05; ***p<0.0005; ****p<0.0001 .

[0039] Figure 9. Senolytic assay. In the plot are represented mean + / - SD (n=4). Data are represented as residual senescent cells (%). Data are compared with Navitoclax treatment (a compound with a good senolytic activity). Unpaired Student t-test, *p<0.05; **p<0.005; ****p<0.0001 . Detailed description of the invention

[0040] Definitions

[0041] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0042] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, “an element” may mean one element or more than one element. When referring to a noun (e.g. a compound, an additive, etc.) in the singular, the plural is meant to be included. Thus, when referring to a specific moiety, e.g. a "strain", this means "at least one" of that strain, e.g. "at least one strain", unless specified otherwise.

[0043] Throughout the present specification and the accompanying claims, all transitional phrases such as ‘comprising,’ ‘including,’ ‘carrying,’ ‘having,’ ‘containing,’ ‘involving,’ ‘holding,’ ‘composed of’, and variations such as "comprises", "comprise", "includes" and "include" are to be interpreted inclusively and to be understood to be open-ended, i.e., to mean including but not limited to. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows. Only the transitional phrases ‘consisting of’ and ‘consisting essentially of shall be closed or semi-closed transitional phrases, respectively.

[0044] The term 'consisting essentially of as used herein means that the total amount of the listed ingredients ideally sums up to 100 wt. % (weight percent). It is however not excluded that small amount of impurities derived from the ingredients and / or the production process may be present.

[0045] When referring to a compound of which several isomers exist (e.g. a D and an L enantiomer), the compound in principle includes all enantiomers, diastereomers and cis / trans isomers of that compound that may be used in the particular aspect of the invention; in particular when referring to such as compound, it includes the natural isomer(s).

[0046] Unless explicitly indicated otherwise, the various embodiments of the invention described herein can be cross-combined.

[0047] The term "cosmetic composition " or “cosmetic preparation” refers in the present document to a composition suitable for cosmetic purposes. Suitably a cosmetic composition is herein understood to be a composition for cosmetic, that is, non-therapeutic use. More preferably the term "cosmetic composition " or “cosmetic preparation” refers in the present document to a composition which is applied to the surface of a mammalian keratinous tissue. The terms "cosmetic composition " or “cosmetic preparation” can comprise or consist of those cosmetic compositions as defined underthe heading "Kosmetika" in Rbmpp Lexikon Chemie, 10th edition 1997, Georg Thieme Verlag Stuttgart, New York as well as to cosmetic compositions as disclosed in A. Domsch, "Cosmetic Compositions", Verlag fiir chemische Industrie (ed. H. Ziolkowsky), 4thedition, 1992. More preferably the "cosmetic composition " or “cosmetic preparation” is a cosmetic preparation, respectively a cosmetic composition, that can be topically applied to mammalian keratinous tissue such as e.g. human skin or hair (including eyelashes, the eyebrows, the nails orthe lips), particularly human skin. Hence, the cosmetic composition is preferably a topical composition.

[0048] By a topical composition is herein understood a composition for, preferably external, use on keratinous tissue such as the skin.

[0049] The term “keratinous tissue" as used in this document refers to tissue containing keratine, more preferably it means the skin (body, face, contour of the eyes, scalp), head hair, eyelashes, eyebrows, bodily hairs, nails and / or lips. Preferably, the keratinous tissue is the skin and hair.

[0050] The term “cosmetic ingredient composition” or “ingredient composition” is preferably understood to comprise or consist of one or more ingredient(s) suitable for use in the formulation of a cosmetic product. Preferably such ingredient composition comprises one or more active ingredients and optionally one or more cosmetically acceptable excipients and / or one or more cosmetically acceptable carriers. The ingredient composition may for example be a pre-mix blend which can be readily incorporated into cosmetic compositions. Preferably the ingredient composition comprises equal to or less than 9 ingredients, most preferably in the range from equal to or more than 1 to equal to or less than 6 ingredients. Preferably the ingredient composition is an active ingredient composition comprising one or two active ingredients.

[0051] The term cosmetically acceptable carrier herein refers to all carriers and / or diluents conventionally used in cosmetic preparations. Preferably the cosmetically acceptable carrier comprises or consists of water, oil or a combination of water and oil.

[0052] The term "senolytic effect" or “senolytic activity” is suitably used in this document to describe that a substance has the ability to selectively kill senescent cells and / or reduces the number of senescent cells, when said senescent cells are brought in contact with said substance.

[0053] The term "anti-oxidant effect" or “anti-oxidant activity” is suitably used in this document to describe that a substance reduces oxidative damage to DNA when said DNA is brought in contact with oxygen and said substance.

[0054] The term "collagen boosting effect" or “collagen boosting activity” is suitably used in this document to describe that a substance stimulates Collagen I production by human dermal cells when said human dermal cells are brought in contact with said substance.

[0055] By an “active ingredient”, “active compound”, “active component”, “active agent” or simply “active” is herein understood a substance that has a certain activity. So-called “primary substances” are substances designed to achieve a specific intended effect in a cosmetic product.

[0056] An “senolytic component”, “senolytic ingredient”, “senolytic agent”, “senolytic active” or simply “senolytic” is understood to be a substance which has an senolytic activity.

[0057] An “anti-oxidant component”, “anti-oxidant ingredient”, “anti-oxidant agent”, “anti-oxidant active” or simply “anti-oxidant” is understood to be a substance which has an anti-oxidant activity.

[0058] An “collagen-booster component”, “collagen-booster ingredient”, “collagen-booster agent”, “collagen-booster active” or simply “collagen-booster” is understood to be a substance which has an anti-oxidant activity. By an “excipient” is herein understood an inactive substance formulated alongside the active ingredient in a composition. Whilst the active ingredient provides the primary cosmetic effect or desired outcome, the excipient helps deliver, protect, and stabilize the active ingredient and / or improves the product's usability and acceptability.

[0059] Rapeseed protein hydrolysate

[0060] The rapeseed protein hydrolysate as described herein suitably comprises protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa.

[0061] Rapeseed seeds are rich in oil and contain considerable amounts of protein that accounts for 17 to 25% of seed dry weight. Processing rapeseed for oil for human consumption produces rapeseed meal as a by-product which contains about 30 to 40% protein. The rapeseed used for this purpose is usually of the varieties Brassica napus and Brassica juncea. These varieties contain only low levels of erucic acid and glucosinolate, and are also known as Canola. Canola is a contraction of Canada and ola, for "oil low acid", but is now a generic term defined as rapeseed oil comprising <2% erucic acid and <30 mmol / g glucosinolate. The resultant rapeseed meal is currently used as a high-protein animal feed.

[0062] The predominant storage proteins found in rapeseed are cruciferins and napins (S.P. Perera, T.C. McIntosh, J.P.D. Wanasundra, Plant 2016, 5, p36, “Structural Properties of Cruciferin and Napin of Brassica napus (Canola) Show Distinct Responses to Changes in pH and Temperature”). Cruciferins are globulins and are the major storage protein in the seed. It is composed of 6 subunits and has a total molecular weight of approximately 300 kDa. Napins are albumins and are a low molecular weight storage protein with a molecular weight of approximately 14 kDa. Napins are more easily solubilized and in for example EP 1715752B1 a process is disclosed to separate out the more soluble napin fraction, preferably to at least 85 wt.%. Napins are primarily proposed for use used in applications where solubility is key. DE 10 2014 005466 A1 also describes a process for obtaining purified cruciferin and napin fractions. During the process, also a protein mixture of the two with 55-60% napins and 40-45% cruciferins is obtained. The solubility of this protein mixture is approximately 75%.

[0063] Rapeseed proteins can be also divided into various fractions according to the corresponding sedimentation coefficient in Svedberg units (S). This coefficient indicates the speed of sedimentation of a macromolecule in a centrifugal field. For rapeseed proteins, the main reported fractions are: 12S, 7S and 2S. Cruciferin and napin are the two major families of storage proteins found in canola / rapeseed. Napin is a 2S albumin, and cruciferin is a 12S globulin.

[0064] The terms “rapeseed protein hydrolysate” and “hydrolyzed rapeseed proteins” are used interchangeably herein.

[0065] Preferably, the rapeseed protein hydrolysate comprises at least 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or at least 70% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa. In a preferred embodiment, the present rapeseed protein hydrolysate comprises: a. 1 to 10% (w / w) of protein fragments having a molecular weight of more than 17 kDa; b. 10 to 30% (w / w) of protein fragments having a molecular weight of 3, 5 to 17 kDa; and / or c. 60 to 89% (w / w) of protein fragments having a molecular weight of less than 3.5 kDa, preferably wherein the sum of the protein fragments does not exceed 100% (w / w).

[0066] More preferably, the present protein hydrolysate comprises: a. 1 to 6% (w / w) of protein fragments having a molecular weight of more than 17 kDa; b. 15 to 30% (w / w) of protein fragments having a molecular weight of 3, 5 to 17 kDa; and / or c. 65 to 84% (w / w) of protein fragments having a molecular weight of less than 3.5 kDa, preferably wherein the sum of the protein fragments does not exceed 100% (w / w).

[0067] Preferably, the molecular weight as used herein is defined by high performance sizeexclusion chromatography, HP-SEC. Preferably, the HP-SEC comprises:

[0068] - mixing 100 pl of protein hydrolysate 5% solution 900 pl of a 0.5% sodium dodecyl sulphate (SDS) solution in purified water, to obtain a 10 times (v / v) dilution of protein hydrolysate;

[0069] - denature the diluted protein hydrolysate;

[0070] - 1 pl was further used for further analysis.

[0071] Preferably, the further analysis comprises: 1 pl of the clear solution was injected in a Waters ACQUITY HCIass-Bio UPLC system, equipped with a pump (Waters 186015041), autosampler (Waters 186015040), column heater (Waters 186015011) and photodiode array detector (Waters 186015032). The running buffer was 0.5% SDS solution in purified water. Separation was performed on a Phenomenex BioZen SEC-3, 1.8 pm, 4.6 mm X 150 mm column (Phenomenex 00F-4772-E0) at 50°C and 0.35 ml / min. Size exclusion chromatography (SEC) profiles were recorded using ultraviolet (UV) detection at 214 nm. The following standards were used: bovine serum albumin (Sigma P0914), chicken ovalbumin (Bio-Rad 1511901), equine myoglobin (Bio-Rad 1511901), lysozyme (Sigma, L6876), insulin (Sigma, I5500), glucagon (Sigma, G2044), [Glu1 ] Fibrinopeptide B (Sigma, F3261), glutathione oxidized (Sigma G4376), and glutathione reduced (G4251). These were dissolved in 0.5% SDS solution, denatured according to sample protocol, and injected. Their corresponding monomeric molecular weights and elution times were used to assign molecular weight fractions of >66 kDa, 66-44 kDa, 44-17 kDa, 17-14.4 kDa, 14.4-5.7 kDa, 5.7-3.5 kDa, 3.5-1 .6 kDa, 1 .6-0.6 kDa, 0.6-0.3 kDa and <0.3 kDa.

[0072] In a preferred embodiment, the rapeseed protein hydrolysate comprises protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa, wherein 40 to 65% (w / w) of the protein fragments is derived from cruciferins and 35 to 60% (w / w) of the protein fragments is derived from napins.

[0073] In yet another preferred embodiment, the rapeseed protein hydrolysate comprising protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa, wherein 40 to 65% (w / w) of the protein fragments is derived from cruciferins and 35 to 60% (w / w) of the protein fragments is derived from napins, wherein the protein hydrolysate comprises a. 1 to 10% (w / w) of protein fragments having a molecular weight of more than 17 kDa; b. 10 to 30% (w / w) of protein fragments having a molecular weight of 3, 5 to 17 kDa; and / or c. 60 to 89% (w / w) of protein fragments having a molecular weight of less than 3.5 kDa, preferably wherein the sum of the protein fragments does not exceed 100% (w / w).

[0074] In a preferred embodiment, the rapeseed protein hydrolysate has a degree of hydrolysis of between 1 and 30%, preferably between 2 and 20%, more preferably between 5 and 10%. Preferably the degree of hydrolysis is determined using the method described herein.

[0075] Preferably, the rapeseed protein hydrolysate has a degree of hydrolysis of 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30%.

[0076] Alternatively, the rapeseed protein hydrolysate may have a degree of hydrolysis of 10 to 90%, 15 to 85%, 20 to 80%, 25 to 75%, 30 to 70%, 35 to 65%, 40 to 60% or 45 to 55%, or any combination of the lower and upper limits of these ranges.

[0077] In a preferred embodiment, the rapeseed protein hydrolysate has a protein content of 40 to 100% (w / w). Such as 50 to 99%, 55 to 95%, 60 to 90%, 65 to 85% or 70 to 80%. Preferably the protein content is or comprises the present protein fragments.

[0078] Preferably the composition according to the invention is a cosmetic composition. In such cosmetic composition the rapeseed protein hydrolysate is preferably present as an ingredient.

[0079] The rapeseed protein hydrolysate may be present in a dissolved form (that is, in the form of a solution) or in the form of a concentrated liquid, a paste, a powder or a granulate. More preferably the rapeseed protein hydrolysate is present in a dissolved form. Cosmetic compositions preferably comprise a cosmetically acceptable carrier. Preferably the cosmetic composition comprises water, oil and / or another suitable carrier, such as glycerol. If present, the cosmetically acceptable carrier more preferably comprises or consists of water, oil or a combination of water and oil. Preferably the cosmetic composition is a solution, emulsion or dispersion, wherein the rapeseed protein hydrolysate is present in the composition in a dissolved, emulsified or dispersed form.

[0080] Preferably the rapeseed protein hydrolysate is present in the claimed composition in an amount in the range from equal to or more than 0.001 wt%, more preferably from equal to or more than 0.005 wt%, even more preferably from equal to or more than 0.01 wt%, still more preferably from equal to or more than 0.05 wt%, yet more preferably from equal to or more than 0.1 wt%, still yet more preferably from equal to or more than 0.5 wt%, to equal to or less than 50 wt%, more preferably to equal to or less than 20 wt%, even more preferably to equal to or less than 10 wt%, still more preferably to equal to or less than 8 wt%, yet more preferably to equal to or less than 5 wt%, still yet more preferably to equal to or less than 2 wt%., based on the total weight of the composition. Further suitable ranges for the amount of rapeseed protein hydrolysate are from 0.05 to 2 wt.% and from 0.1 to 1 wt.%, based on the total weight of the composition. Further preferred ranges for the amount of rapeseed protein hydrolysate are from 0.001 to 0.5 wt.%, 0.001 to 1 wt.- %, 0.01 to 1 wt-% as well as from 0.01 to 0.5 wt.%, in each case based on the total weight of the composition.

[0081] As described in the experimental part herein, the hydrolysate of a rapeseed protein isolate (RPI) is preferably a hydrolysate where most of the napin has been hydrolysed. More preferably the rapeseed protein hydrolysate is a rapeseed protein hydrolysate wherein equal to or more than 10 wt.%, even more preferably equal to or more than 20 wt.%, still more preferably equal to or more than 30 wt. %, yet more preferably equal to or more than 40 wt. %, yet still more preferably equal to or more than 50 wt.% and equal to or less than 100 wt% of the napin, based on the total weight of the rapeseed protein, has been hydrolyzed. Moreover, the applied and described rapeseed protein hydrolysate is a vegan hydrolysate.

[0082] Preferably the rapeseed protein hydrolysate is a rapeseed protein hydrolysate obtained by a process for preparing a hydrolyzed rapeseed proteins comprising the steps of: a) preparing an aqueous mixture comprising water and rapeseed proteins; b) contacting the aqueous mixture, comprising the water and the rapeseed proteins, with an aspergillopepsin 1 and / or a proline-specific endoprotease, at a pH in the range from 2 to 6, preferably 3 to 5, preferably at a temperature in the range from 30°C to 65°C, more preferably 40°C to 60°C, most preferably 45°C - 55°C, preferably for a time period in the range from 10 minutes to 40 hours, more preferably 30 minutes to 30 hours, most preferably 1 hour to 20 hours, to hydrolyze the rapeseed proteins; and / or c) retrieving the hydrolyzed rapeseed proteins from the aqueous mixture.

[0083] This combination of proteases can be a sequential addition (such as first aspergillopepsin 1 , and then proline-specific endoprotease, or another protease under its optimum condition and then adapting the pH and temperature for the proline-specific endoprotease), or a combination of both enzymes, preferably in an environment (temperature, pH) where both enzymes show high activity.

[0084] In a preferred embodiment, the process comprises first incubation of the rapeseed protein with aspergillopepsin 1 and thereafter incubation of the rapeseed protein with the proline-specific endoprotease.

[0085] The inventors found that this process hydrolyses both the cruciferins and napins. The potential advantage of hydrolysis of napin is that this leads to peptides of a reduced size rich in cysteine, leading to other material properties.

[0086] In another preferred embodiment, the above process comprises combined incubation of the rapeseed protein with aspergillopepsin 1 and proline-specific endoprotease.

[0087] The present aspergillopepsin 1 (herein also referred to as fungal endoprotease aspergillopepsin 1) is preferably an enzyme from the enzyme class EC 3.4.23.18.

[0088] A "proline-specific endoprotease" is herein preferably understood to be a protease that hydrolyses a protein or peptide at a position where the protein or peptide contains a proline-residue. A proline-specific endoprotease may have proline-specific endoprotease and / or proline-specific oligopeptidase activity (EC3.4.21 .26). A proline-specific endoprotease is preferably an enzyme that hydrolyses a peptide bond at the carboxy-terminal end of proline residues, resulting in a peptide and / or polypeptide fragment with a C-terminal proline.

[0089] A proline-specific endoprotease may for instance be derived from Aspergillus niger or Penicillium chrysogenum, such as disclosed in W02002 / 046381 and W02009 / 144269 respectively.

[0090] Other proline-specific endoproteases are known from WO2012 / 174127. WO2012 / 174127 discloses proline-specific protease from Botryotinia, fuckeliana, Aspergillus clavatus, Sclerotinia sclerotiotum, Mycosphaerelly graminicola, Neuropspora crasse, Talaromyces stipitatus and Gibberella zeae.

[0091] The skilled person is very well capable of determining suitable conditions for step (b) of the above-described process. A preferred temperature is a temperature of at least 50 degrees Celsius, preferably a temperature in the range of 50 to 65 degrees Celsius. A preferred starting pH is pH 3. Further an incubation time of at least 6 hours is preferred. Suitable enzyme concentrations may include a range of 1 % to 11 % of both enzymes (where the dosage of the formulated enzymes may be based on the protein powder), and / or may include enzymes with an activity (MAXIPRO™ AFP) of 1000-1100 SAPU / g, this is 10-11 SAPU / g protein up to 100-110 SAPU / g protein, or for MAXIPRO™ PSP with 5-9 PPU / g this is 0.05-0.09 PPU / g protein, up to 0.5 - 1 PPU / g protein.

[0092] Within this document Aspergillopepsin 1 of different batches with different names were used (MAXIPRO™ AFP and Maxamyl AFP), as well proline-specific endoprotease (DelvoPlant PSP and MAXIPRO™ PSP)

[0093] Suitably rapeseed protein isolate can be obtained by aqueous extraction of rapeseed meal or rapeseed ‘cake’ obtained after the oil was removed (partially) by pressing and or optionally extraction by an organic solvent, which results in the rapeseed protein isolate and a (wet) cake residue. While rapeseed protein isolate may have a protein content of higher than 80%, findings of this invention may also apply to a rapeseed protein concentrate with less than 80% protein, or even a rapeseed preparation with even lower protein concentration, such as rapeseed meal, or pressed rapeseed meal (pressed filter cake) or from the depleted wet cake residue that may contain substantial amount of protein.

[0094] In an alternative embodiment, the proteolysis from such rapeseed meal, press cake or depleted wet cake (residue) may occur in the presence of other enzymes, such as enzymes that further breakdown the cellular matrix and hence improve the accessibility for enzymes and release of the protein (hydrolysate) from the matrix, such as carbohydrases (cellulases, hemicellulases, pectinase) or phytase.

[0095] Preferably, the present rapeseed protein is rapeseed protein isolate.

[0096] Preferably, the present rapeseed protein comprises napins and cruciferins. Preferably the amount of cruciferins and napins is determined by Blue Native Page, HP-SEC or by sedimentation velocity (SV-AUC). Preferably, the amounts of cruciferins and napins are calculated based on the total amount of rapeseed protein. Or alternatively, the amounts of cruciferins and napins are calcuated based on the sum of cruciferins and napins present in the rapeseed protein. Preferably, the amounts of cruciderins and napins are determined by size exclusion chromatography (SEC). Preferably, the amounts of cruciderins and napins are determined by size exclusion chromatography (SEC) using the following test: samples of protein isolate are dissolved in a 500 mM NaCI saline solution and analyzed by High Performance SEC using the same solution as the mobile phase, followed by detection using measuring UV absorbance at 280 nm, wherein the relative contribution of cruciferin and napin (wt. %) was calculated as the ratio of the peak area of each protein with respect to the sum of both peak areas.

[0097] The rapeseed protein may suitably be in the form of an isolate or a concentrate. Rapeseed protein isolate may be prepared from cold-pressed rapeseed oil seed meal as described in WO 2018 / 007492 resulting in a product with a protein content of from 50-98% (w / w), or from 7095% (w / w) or of 90±5% (w / w). The rapeseed protein isolate may comprise of from 40-65% (w / w) cruciferins and of from 25-60% (w / w) napins as verified by Blue Native PAGE, for example as described in WO 2018 / 007492. Alternatively, the rapeseed protein isolate may comprise at least 50, 60, 70, 80% (w / w), preferably at least 85% (w / w), preferably at least 90% (w / w), more preferably at least 95% (w / w) cruciferins as verified by Blue Native PAGE, for example as described in WO 2018 / 007492. Alternatively, the rapeseed protein isolate may comprise at least 50, 60, 70, 80% (w / w), preferably at least 85% (w / w), preferably at least 90% (w / w), more preferably at least 95% (w / w) napins as verified by Blue Native PAGE, for example as described in WO 2018 / 007492. More preferably the rapeseed protein isolate comprises 10-40% (w / w) napins and 40-60% (w / w) cruciferins, preferably as verified by Blue Native PAGE, for example as described in WO 2018 / 007492. More preferably the rapeseed protein isolate comprises 25-35% (w / w) napins and 40-55% (w / w) cruciferins, preferably as verified by Blue Native PAGE, for example as described in WO 2018 / 007492. Most preferably, the rapeseed protein isolate comprises 35-60% (w / w) napins and 40-65% (w / w) cruciferins, preferably as verified by Blue Native PAGE, for example as described in WO 2018 / 007492.

[0098] Preferably, the present rapeseed protein comprises 40 to 65 wt. % 12S and 35 to 60 wt. % 2S. Preferably, the present rapeseed protein comprises 40 to 55 wt. % 12S and 45 to 60 wt. % 2S.

[0099] In a preferred embodiment, the present rapeseed protein comprises 60 to 80 wt. % 12S and 20 to 40 wt. % 2S. Preferably, the present rapeseed protein comprises 65 to 75 wt. % 12S and 25 to 35 wt. % 2S.

[0100] In a preferred embodiment, the present rapeseed protein comprises 0 to 20 wt. % 12S and 80 to 100 wt. % 2S. Preferably, the present rapeseed protein comprises 0 to 10 wt. % 12S and 90 to 100 wt. % 2S. Preferably, the present rapeseed protein comprises 1 to 5 wt. % 12S and 95 to 100 wt. % 2S. Preferably, the present rapeseed protein comprises around 15 wt. % 12S and around 85 wt. % 2S. An example of such a rapeseed protein is Puratein® HS. Preferably, the amounts of 12S and 2S is determined by sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis. Preferably, the amounts of 12S and 2S is determined by sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis using the following test: samples of protein isolate are dissolved in a 3.0% (or 500 mM) NaCI saline solution and amounts determined using interference optics.

[0101] In a preferred embodiment, the present rapeseed protein comprises 0 to 20 wt. % cruciferins and 80 to 100 wt. % napins. Preferably, the present rapeseed protein comprises 0 to 10 wt. % cruciferins and 90 to 100 wt. % napins. Preferably, the present rapeseed protein comprises 1 to 5 wt. % cruciferins and 95 to 100 wt. % napins. Preferably, the present rapeseed protein comprises around 15 wt. % cruciferins and around 85 wt. % napins. An example of such a rapeseed protein is Puratein® HS.

[0102] In a preferred embodiment, the present rapeseed protein (does not) comprises 70 to 95 wt. % cruciferins and 5 to 30 wt. % napins. Preferably, the present rapeseed protein (does not) comprises 80 to 90 wt. % cruciferins and 10 to 20 wt. % napins, such as around 90% cruciferins and 10% napins.

[0103] In an embodiment the rapeseed protein isolate is low in anti-nutritional factors and contains less than 1 .5% (w / w) phytate, preferably less than 0.5% (w / w) phytate and is low in glucosinolates (<5 pmol / g) and low in phenolics (<10 mg / g).

[0104] In one embodiment, the used rapeseed protein has a solubility (in water) of at least 88%, preferably at least 90%, more preferably at least 94% and most preferably at least 96% when measured over a pH range from 3 to 10 at a temperature of 23±2°C. This is also known as the soluble solids index (SSI).

[0105] The above-described method suitably results in a rapeseed protein hydrolysate. Such a hydrolysate may suitably comprise hydrolysed cruciferins and hydrolysed napins. Preferably, the vast majority (preferably at least 80% of the napins, more preferably at least 85, 90 or 95% of the napins) of the napins which were present in the starting material have been hydrolysed by the combination of endoproteases. That is, preferably the rapeseed protein hydrolysate is a rapeseed protein hydrolysate wherein at least 80% of the napins, more preferably at least 85, 90 or 95% of the napins have been hydrolysed.

[0106] The presence of remaining (if any) intact napins can be observed by different techniques, for example by NMR, such as described by Rico et al. Biochemistry 1996 35 p15672-156821H NMR Assignment and Global Fold of Napin Bnlb, a Representative 2S Albumin Seed Protein, and Pantoja-Uceda et al. Biochemistry 2004, 43, p16036-16045 Solution Structure and Stability against Digestion of rproBnlb, a Recombinant 2S Albumin from Rapeseed: Relationship to Its Allergenic Properties. Such a method would enable the determination of effectiveness of napin hydrolysis.

[0107] Preferably, the rapeseed protein hydrolysate is prepared, obtained and / or derived from a process which does not comprise, apply or use subtilisin. Without wishing to be bound by any kind of theory, it is believed that the senolytic effect may at least partly be due to the generation of peptides which may have an improved penetration grade through the skin barrier (epidermis).

[0108] As mentioned above, the rapeseed protein hydrolysate suitably comprises protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa.

[0109] Preferably the rapeseed protein hydrolysate is further obtained by a process for preparing a hydrolyzed rapeseed proteins comprising the application of an aspergillopepsin I.

[0110] That is, preferably the rapeseed protein hydrolysate is a rapeseed protein hydrolysate obtained from, produced by or derived from a hydrolysis of rapeseed protein with the help of an aspergillopepsin I enzyme.

[0111] Without wishing to be bound by any kind of theory, aspergillopepsin I, an aspartic protease from Aspergillus species, is believed to cleave peptide bonds preferentially at specific sites within protein sequences and to have a preference for hydrophobic amino acids at the P1 position and the PT position. This cleavage specificity means that the resulting fragments will often have hydrophobic amino acids at their termini. By a hydrophobic amino acid is herein preferably understood an amino acid with a hydrophobic side chain. Amino acids with a hydrophobic side chain include Alanine (Ala), Valine (Vai), Isoleucine (He), Leucine (Leu), Methionine (Met), Phenylalanine (Phe), Tyrosine (Tyr) and Tryptophan (Trp). It is believed that the protein fragments resulting from hydrolysis with Aspergillopepsin I are therefore likely to be peptides having such amino acids with such hydrophobic side chain at one or both of its termini.

[0112] Therefore, the rapeseed protein hydrolysate preferably contains protein fragments, wherein preferably at least 5% (w / w), more preferably at least 10% (w / w), even more preferably at least 20% (w / w), still more preferably at least 30% (w / w), yet more preferably at least 40% (w / w), yet still more preferably at least 50% (w / w) and most preferably at least 60% (w / w) of such protein fragments, based on the total weight of such protein fragments, are peptides having an hydrophobic amino acid at one or both of its termini, preferably an hydrophobic amino acid selected from the group consisting of Leucine (Leu), Phenylalanine (Phe), Tyrosine (Tyr) and Tryptophan (Trp), more preferably an hydrophobic amino acid selected from the group consisting of, Phenylalanine (Phe), Tyrosine (Tyr) and Tryptophan (Trp).

[0113] Hence, more preferably the rapeseed protein hydrolysate as described herein is a rapeseed protein hydrolysate comprising protein fragments, wherein, based on the total weight of the protein fragments: at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa; and at least 5% (w / w), more preferably at least 10% (w / w), even more preferably at least 20% (w / w), still more preferably at least 30% (w / w), yet more preferably at least 40% (w / w), yet still more preferably at least 50% (w / w) and most preferably at least 60% (w / w) of the protein fragments, are peptides having an hydrophobic amino acid at one or both of its termini, preferably an hydrophobic amino acid selected from the group consisting of Leucine (Leu), Phenylalanine (Phe), Tyrosine (Tyr) and Tryptophan (Trp), more preferably an hydrophobic amino acid selected from the group consisting of, Phenylalanine (Phe), Tyrosine (Tyr) and Tryptophan (Trp).

[0114] The cosmetic composition

[0115] The composition according to the invention is preferably a cosmetic composition. More preferably the composition is a composition for cosmetic application, for example for moisturisation, anti- ageing and / or energizing. Preferably the composition is a cosmetic composition, more preferably a topical composition. More preferably the composition is a skincare composition, a haircare composition or a suncare composition, most preferably the composition is a skincare and / or haircare composition.

[0116] Preferably the composition, more preferably the cosmetic composition, is a composition, respectively a cosmetic composition comprising or consisting of a rapeseed protein hydrolysate comprising protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa; and a cosmetically acceptable further ingredient, preferably a cosmetically acceptable carrier, more preferably water and / or oil.

[0117] Preferably the rapeseed protein hydrolysate in such a composition has a degree of hydrolysis of between 1 and 30%, preferably between 2 and 20%, more preferably between 5 and 10%. Preferably the degree of hydrolysis is determined by determining the weight of hydrolyzed proteins based on the total weight of all proteins in the composition.

[0118] Preferably the rapeseed protein hydrolysate in such a composition has a protein content of 40 to 100% (w / w), suitably based on the total weight of the composition.

[0119] As outlined above, the invention suitably provides the use of rapeseed protein hydrolysate as an active ingredient in, and for the manufacture of, cosmetic compositions for treating signs of skin and / or hair aging. Preferences for the rapeseed protein hydrolysate are as described above.

[0120] In a further embodiment, the present invention also provides rapeseed protein hydrolysate as an active ingredient for use in the selective elimination of senescent (skin and / or hair) cells. Preferences for the rapeseed protein hydrolysate are as described above (in this and the previous sections).

[0121] The invention also provides the use of rapeseed protein hydrolysate as an active ingredient in, and for the manufacture of, cosmetic compositions for the selective elimination of senescent (skin and / or hair) cells. Preferences for the rapeseed protein hydrolysate are as described above (in this and the previous sections).

[0122] The invention also provides a cosmetic method for treating signs of skin and / or hair aging, comprising topically applying to an external surface of the human body a cosmetic composition containing rapeseed protein hydrolysate, wherein rapeseed protein hydrolysate preferably comprises at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa. The present invention also refers to a cosmetic skin and / or hair care method for selective removal of senescent cells in the skin preferably of senescent human dermal fibroblasts and / or hair follicle dermal papilla cells by applying, onto the skin (including the scalp and / or hair) in need, at least an effective amount of an extract of rapeseed protein hydrolysate, or a composition comprising rapeseed protein hydrolysate, and optionally appreciating the effect. Preferably, the effective amount of rapeseed protein hydrolysate (based on dry matter) applied on said skin or hair cells is selected in the range of 0.001 to 1 wt.% / cm2of skin. Again, preferences for the rapeseed protein hydrolysate are as described above (in this and the previous sections).

[0123] As indicated above, as used herein, the term ‘cosmetic’ preferably refers to a treatment which does not cure, treat, or prevent a disease or disorder, but instead serves as a skincare or hair care product intended to beautify or improve the appearance of the skin or hair, e.g., the colour or texture of the skin or hair. In the context of this invention, the term ‘treating’ preferably denotes reducing, preventing, or eliminating. The expression ‘signs of skin aging’ as used herein preferably denotes one or more characteristics of intrinsic or chronological skin aging such as thin skin, fine wrinkles, decreased elasticity and aberrant pigmentation. These signs affect everyone, whatever their skin type or state of health. Such process may be amplified by extrinsic factors such as exposure to sunlight, pollutants, and cigarette smoke. Accordingly, the term ‘treating the signs of skin aging’ preferably includes the prevention, reduction or treatment of fine lines, wrinkles, crow’s feet, sagging, skin thinning, age spots as well as improving skin elasticity or skin firmness without being limited thereto. The term ‘prevention, reducing or treating skin aging’ also preferably encompasses smoothening of wrinkles and fine lines as well as decreasing their volume and depth in a person in need thereof.

[0124] The expression ‘signs of hair aging’ as used herein preferably denotes one or more characteristics of intrinsic or chronological hair aging such as hair loss and baldness. As noted above, the process may be amplified by extrinsic factors such as exposure to sunlight, pollutants, and cigarette smoke. Accordingly, the term ‘treating the signs of hair aging’ includes the prevention, reduction or treatment of hair loss and baldness. The term also includes stimulating hair growth as well as retaining hair.

[0125] In all embodiments of the present invention that the term ‘skin’ respectively the term ‘the external surface of the human body’ preferably includes all of the skin of a human body including oral cavities as well as the scalp. Preferably, in all embodiments of the present invention the skin respectively the external surface of the human body treated according to the present invention is the scalp, the face, neck and / or body skin, most preferably the face (including the (lateral) cheek, forehead, nose, chin) skin or the scalp.

[0126] The term ‘skin cells’ as used herein may suitably refer to any skin resident cell type (such as dermal fibroblasts, melanocytes, and epidermal keratinocytes). Preferably in all embodiments of the present invention, the term refers to (human) dermal fibroblasts. The term ‘hair cells’ as used herein may suitably refer to any hair follicle resident cell type (such as hair follicle dermal papilla cells). Preferably in all embodiments of the present invention, the term refers to (human) hair follicle dermal papilla cells (HFDPCs).

[0127] In the context of this invention, the term ‘senescent’ preferably refers to a state of permanent cell cycle arrest in which cells remain metabolically active and adopt characteristic phenotypic changes. The establishment of this phenotype is believed to be either the result of telomere shortening after several cell divisions (replicative senescence) or a response to stress stimuli (stress-induced senescence). One of the defining features of senescent cells is their stable cell cycle arrest. This cell cycle exit is controlled by activation of the p53 / p21 and p16INK4a / Rb tumour suppressor pathways. Unlike quiescent cells, senescent cells are nonresponsive to mitogenic or growth factor stimuli; thus, they are unable to re-enter the cell cycle even in advantageous growth conditions. Senescent cells are also distinct from terminally differentiated cells, which are also irreversibly withdrawn from the cell cycle. While terminal differentiation is the result of a defined developmental programme, which turns undifferentiated precursors into specialized effector cells, senescence is mainly implemented as a cellular stress response.

[0128] Senescent cells can unambiguously be identified in vitro and in vivo since they exhibit a number of characteristics that allow their explicit identification.

[0129] For example, senescent cells often appear multinucleated, large and extended, and exhibit spindle and vacuolisation features. They also display modifications in the organisation of chromatin that can help identify them. In normal cells, DNA staining reveals completely uniform colour outlines, whereas senescent cells usually show dot-like patterns, known as senescence-associated heterochromatic foci (SAHF). This phenomenon is due to intensive remodelling in the chromatin, which results in less susceptibility for digestion by nucleases.

[0130] Furthermore, senescence-related chromatin remodelling leads to profound transcriptional changes. Among the assortment of upregulated genes is a prominent subset of genes that encode secreted proteins, including cytokines and chemokines with proinflammatory properties, as well as various growth factors and proteases that together alter tissue structure and function, collectively known as SASP. The SASP is one of the key characteristics that distinguish senescent cells from quiescent, terminally differentiated, and other types of non-proliferating cells.

[0131] A distinctive measurable feature of senescent cells is the presence of p-galactosidase enzymatic activity. This enzyme normally displays activity at pH 4.0 within lysosomes, but in senescent cells it is also active at pH 6.0. This phenomenon is termed senescence associated-p- galactosidase (SA- p-gal) activity and is thought to be due to an enlargement in the structure of lysosomes in senescent cells. SA-p-gal activity is detectable by histochemical staining by using X- gal as a substrate for SA-p-gal. Since SA-p-gal activity is detected in most senescent settings, both in vitro and in vivo, it is considered a de facto hallmark of senescence. SA-p-gal can be visualized in the cells to quantify the senescence levels using flow cytometry.

[0132] The term ‘senolytic activity’ as used herein preferably refers to specifically (also: selectively) inducing or promoting cell death of senescent cells. This may also be considered as ability for eliminating selectively senescent cells, for example, by inducing or promoting apoptosis. It may also be the capability to selectively eliminate senescent cells found in a tissue with a reduced or even no harmful effect on the normal resident (i.e., non-senescent) cells of the said tissue. Such senolytic property may be beneficial, for example, for preventing or attenuating the increase of the number of age-related senescent cells and / or stress-related senescent cells within a tissue.

[0133] Selective elimination of senescent cells may suitably be determined by the change in ratio of senescent (decreasing) versus proliferating cells (increasing) based on SA-p-gal activity in the cell population model after incubation with a fixed concentration of a test compound in a suitable medium as illustrated in the examples. Such a cell population model may contain a set percentage ratio of senescent versus proliferating cells. By comparing with and without added test compound conditions, senolytic activity can be evaluated.

[0134] Compounds may suitably be determined as capable of selectively eliminating senescent cells if the calculated senescent cell ratio (based on SA-p-gal activity) is reduced in a statistically significant manner versus the control (no compound added during incubation).

[0135] In one preferred embodiment, preferably the number of senescent cells is reduced by at least 10%, preferably by at least 15%, more preferably by at least 20%, most preferably by at least 30% such as by at least 50 %, based on the initial number of senescent cells present. It is well understood, that concomitantly, the number of normal cells is increased by the same factor.

[0136] In another embodiment, preferably, the number of senescent cells is reduced by at least a statistical difference in percentage between the control and treated conditions, more preferably by at least 50% in difference (half of original senescent cells removed), most preferably by at least 90% in difference (90% of original senescent cells removed), based on the initial number of senescent cells present. It is well understood, that concomitantly, the number of normal cells is increased by the same factor.

[0137] In all embodiments of the present invention the rapeseed protein hydrolysate is preferably administered in the form of a cosmetic composition comprising an effective amount of the rapeseed protein hydrolysate and a physiologically acceptable carrier.

[0138] The term 'an effective amount' refers to an amount necessary to obtain the desired physiological effect. The physiological effect may be achieved by one application dose or by repeated applications. The dosage administered may, of course, vary depending upon known factors, such as the physiological characteristics of the particular composition comprising the rapeseed protein hydrolysate and its mode and route of administration; the age, health and weight of the recipient; the kind of concurrent treatment; the frequency of treatment; and the effect desired and can be adjusted by a person skilled in the art.

[0139] The term “physiologically acceptable carrier” means a carrier that is compatible with an administration to a human subject, suited to the desired administration route of the composition i.e., in the case of oral administration, a medium that is compatible with the digestive system and in the case of topical administration compatible with the skin, and / or mucous membranes, and compatible with the form in which the composition is intended to be packaged, in particular solid or fluid at ambient temperature and atmospheric pressure. Such carriers are well-known to one of ordinary skill in the art and can include one or more compatible liquid(s) or solid filler diluent(s), excipient(s), additive(s) or vehicle(s) which are suitable for oral or topical application.

[0140] Preferably the composition, more preferably the cosmetic composition comprises water and / or oil. In an advantageous embodiment, the topical cosmetic compositions according to the present invention comprise from about 50% to about 99%, preferably from about 60% to about 98%, more preferably from about 70% to about 98%, such as in particular from about 80% to about 95% of a carrier, preferably water and / or oil, based on the total weight of the composition. In a particular advantageous embodiment, the carrier of such topical cosmetic compositions consists furthermore of at least 40 wt.%, more preferably of at least 50 wt.%, most preferably of at least 55 wt.-% of water, such as in particular of about 55 to about 90 wt.% of water, based on the total weight of the composition.

[0141] The composition, preferably cosmetic composition, of the invention may preferably also comprise one or more further cosmetically active ingredients, such as for example skin lightening agents; UV-filters, agents for the treatment of hyperpigmentation; agents for the prevention or reduction of inflammation; firming, moisturizing, soothing, and / or energizing agents as well as agents to improve elasticity and skin barrier.

[0142] In an especially preferred embodiment the composition, preferably cosmetic composition, of the invention, comprises one or more further senolytic agents.

[0143] Therefore, the invention also provides a composition, preferably a cosmetic composition, comprising:

[0144] - a first senolytic agent comprising or consisting of a rapeseed protein hydrolysate, preferably comprising protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa; and

[0145] - one or more further senolytic agents, preferably chosen from the group consisting of myricitrin and / or Epilobrium fleicheri extract.

[0146] Preferences for such myricitrin and / or an Epilobrium fleicheri extract are as respectively described in EP4400091A1 and WO2024149898.

[0147] Without wishing to be bound by any kind of theory it is believed that a combination of such ingredients leads to an improved senolytic effect.

[0148] The composition, preferably cosmetic composition, of the invention may further comprise other acceptable ingredients. Preferably the composition, preferably cosmetic composition, comprises further ingredients, preferably conventional (cosmetic) adjuvants and additives, preferably selected from the group consisting of preservatives / antioxidants, fatty substances / oils, water, organic solvents, silicones, thickeners, softeners, emulsifiers, antifoaming agents, aesthetic components such as fragrances, aroma ingredients, surfactants, fillers, anionic, cationic, non-ionic or amphoteric polymers or mixtures thereof, propellants, acidifying or basifying agents, dyes, colourings / colorants, abrasives, absorbents, chelating agents and / or sequestering agents, essential oils, skin sensates, astringents, pigments or any other ingredients usually formulated into such compositions.

[0149] Use as an active ingredient

[0150] As illustrated by the examples, the rapeseed protein hydrolysate or a composition comprising such rapeseed protein hydrolysate may advantageously be used as an active ingredient in a cosmetic composition. More preferably the rapeseed protein hydrolysate as described herein may advantageously be used as an anti-oxidant, a collagen booster or a senolytic agent.

[0151] Hence the invention also provides a use, preferably a non-therapeutic use, of a rapeseed protein hydrolysate as described herein or a composition comprising such rapeseed protein hydrolysate as described herein, preferably as an active ingredient, for the reduction of damage to DNA in the presence of oxygen; and / or for the boosting of Collagen 1 production by human dermal cell; and / or for the treatment of the signs of aging; and / or for reducing or eliminating senescent skin cells.

[0152] Preferably the invention provides a use, preferably a non-therapeutic use, of a rapeseed protein hydrolysate or a composition comprising such rapeseed protein hydrolysate as described herein as an active ingredient as an active ingredient for the treatment of the signs of aging by, preferably selectively, reducing and / or eliminating senescent skin cells. Preferably the treatment of any signs of aging comprises or consists of the treatment of fine lines, wrinkles, crow’s feet, sagging, skin thinning, age spots as well as the improvement of skin elasticity and skin firmness.

[0153] More preferably the invention provides a use, preferably a non-therapeutic use, of a rapeseed protein hydrolysate as described herein or a composition comprising such rapeseed protein hydrolysate as described herein, preferably as an active ingredient, for reducing or eliminating senescent skin cells. Preferably the reduction or elimination of the senescent skin cells is a selective reduction or elimination of senescent skin cells Preferably the senescent skin cells are human dermal fibroblasts. Preferably the senescent cells are age-related senescent cells, stress-related senescent cells, or both.

[0154] In addition, the invention advantageously provides a combination of :

[0155] - a first senolytic agent comprising or consisting of a rapeseed protein hydrolysate, preferably comprising protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa; and

[0156] - one or more further senolytic agents, preferably chosen from the group consisting of myricitrin and / or Epilobrium fleicheri extract.

[0157] Preferences for the rapeseed protein hydrolysate are as described above and preferences for the composition are as described above.

[0158] Cosmetic skin and scalp care method

[0159] In addition to the above, the invention provides a cosmetic skin and scalp care method, said method encompassing the step of applying, onto the skin or the scalp in need, at least an effective amount of a rapeseed protein hydrolysate as described herein or a composition as described herein. Suitably such method can be a method for the reduction of damage to DNA in the presence of oxygen; and / or for the boosting of Collagen 1 production by human dermal cell; and / or for the treatment of the signs of aging; and / or for reducing or eliminating senescent skin cells. Preferably such method is a method for the reduction and / or removal of senescent cells in the skin and / or hair follicle.

[0160] More preferably such reduction and / or removal comprises or consists of the selective removal of senescent cells in the skin and / or hair follicle. Preferably the senescent cells are senescent human dermal fibroblasts. Preferably the senescent cells are age-related senescent cells, stress-related senescent cells, or both.

[0161] Preferences for the rapeseed protein hydrolysate are as described above and preferences for the composition are as described above.

[0162] The invention is further illustrated by the non-limiting examples below.

[0163] Examples

[0164] Materials and methods

[0165] Rapeseed protein isolate (RPI) was prepared from cold-pressed rapeseed oil seed meal as described in WO 2018 / 007492; the protein content was 90% (w / w). The resultant RPI comprised in the range of from 40 to 65% (w / w) cruciferins and 35 to 60% (w / w) napins, contained less than 0.26% (w / w) phytate and had a solubility of at least 88% when measured over a pH range from 3 to 10 at a temperature of 23±2°C. The following enzymes of table 1 were used.

[0166] Table 1 : Enzymes

[0167] Analysis methods

[0168] SDS electrophoresis

[0169] As such: 65 pl sample (protein solution) was added to 25 pl NuPAGE LDS sample buffer (4x) Invitrogen and 10 pl NuPAGE Sample Reducing Agent (10x) Invitrogen. Samples were heated for 10 min at 70°C. The samples were 5 times diluted in sample buffer (65 milliQ water : 25 LDS sample buffer : 10 Reducing agent). Gel electrophoresis

[0170] SDS-PAGE was performed as follows. 10 pl of the sample solutions and 5-10 pl marker M12 (Invitrogen) were applied on NuPage 9-12% Bis Tris 10 wells gel. The gel was run at 200 V for 43 minutes, using the Xcell Surelock, with 600 ml 20x diluted MES SDS running buffer in the outer chamber and 200 ml 20x diluted MES SDS running buffer in the inner buffer chamber. After running, the gels were stained with 50 ml InstantBlue staining. And decolored for a duration of 1 to 3 nights.

[0171] Determination of primary amino groups by o-Phthaldialdehyde (OPA)

[0172] Primary amino groups were determined using what is commonly known as the “OPA” method, with the reagent o-Phthaldialdehyde. This is needed to calculate the degree of hydrolysis of a protein (see next). The degree of hydrolysis (DH (%)) is defined as the percentage of cleaved peptide bonds after hydrolysis. Analysis is always performed in combination with Kjeldahl Nitrogen; necessary for calculation “degree of hydrolysis”. o-Phthaldialdehyde (OPA) reacts in the presence of 1 ,4-Dithiothreitol (DTT) with primary amino groups of the cleaved protein fragments at ambient temperature. Color development of the reaction products is measured spectrophotometrically at 340 nm.

[0173] Degree of hydrolysis

[0174] The degree of hydrolysis was determined from the Kjeldahl Nitrogen (A10327) and amino groups (A2343).

[0175] First, the primary NH2 per g protein (h) need to be calculated: h = ((106x F) / (NTKN x N factor) - p) / a

[0176] Where: h = mmol primary NH2 per g protein

[0177] F = mmol primary NH2 per g sample

[0178] NTKN = g total Kjeldahl nitrogen per g sample

[0179] 106= conversion factor NTKN mg / kg -> g / g

[0180] N factor= conversion factor N to protein (see table 2) a = slope of the hydrolysis curve (no dimension) (see table 2)

[0181] B = intercept of the hydrolysis curve (mmol primary NH2 per g protein)

[0182] The degree of hydrolysis can be calculated:

[0183] DH (%) = h / htot *100% where: h = mmol primary NH2 per g protein htot = mmol primary NH2 theoretically available per g protein (see table 2) Table 2 Constants for some frequently used proteins

[0184] * Calculated based on amino acid composition of theoretical sequence of cruciferin and napin

[0185] Analysis of molecular weight distribution by HP SEC

[0186] Several samples were analysed on molecular weights by using high performance sizeexclusion chromatography, HP-SEC. After homogenization on a vortex stirrer (Scientific Industries G-560E), 100 pl of each sample (protein hydrolysates in approximately 5% solution) were mixed with 900 pl of a 0.5% sodium dodecyl sulphate (SDS) solution in purified water, to obtain a 10 times (v / v) dilution of each sample. The mixtures were denatured for 15 minutes at 70°C and 1000 rpm in a thermomixer (Eppendorf ThermoMixer C). 1 pl of the clear solution was injected in a Waters ACQUITY HCIass-Bio UPLC system, equipped with a pump (Waters 186015041), autosampler (Waters 186015040), column heater (Waters 186015011) and photodiode array detector (Waters 186015032). The running buffer was 0.5% SDS solution in purified water. Separation was performed on a Phenomenex BioZen SEC-3, 1.8 pm, 4.6 mm X 150 mm column (Phenomenex 00F-4772-E0) at 50°C and 0.35 ml / min. Size exclusion chromatography (SEC) profiles were recorded using ultraviolet (UV) detection at 214 nm. The following standards were used: bovine serum albumin (Sigma P0914), chicken ovalbumin (Bio-Rad 1511901), equine myoglobin (Bio-Rad 1511901), lysozyme (Sigma, L6876), insulin (Sigma, I5500), glucagon (Sigma, G2044), [Glu1] Fibrinopeptide B (Sigma, F3261), glutathione oxidized (Sigma G4376), and glutathione reduced (G4251). These were dissolved in 0.5% SDS solution, denatured according to sample protocol, and injected. Their corresponding monomeric molecular weights and elution times were used to assign molecular weight fractions of >66 kDa, 66-44 kDa, 44-17 kDa, 17-14.4 kDa, 14.4-5.7 kDa, 5.7-3.5 kDa, 3.5-1 .6 kDa, 1 .6-0.6 kDa, 0.6-0.3 kDa and <0.3 kDa. The peak areas and peak percentages of each molecular weight fraction of the samples were exported and evaluated.

[0187] Example 1

[0188] Proteolysis of rapeseed protein isolate with standard proteases

[0189] Rapeseed protein isolate was incubated with standard proteases: ALCALASE™ (subtilisin) and Flavourzyme (both Novozymes); and Delvoplant PSP, a proline-specific fungal protease produced by DSM Firmenich. The rapeseed protein was dissolved at 5 or 20% concentration in process water, and the pH was set and controlled (using 4N H3PO4 or 4N NaOH) and temperature were set as indicated in the table below. A sample was taken at t=0. Then enzyme solutions were added, see table below. Samples (40 ml) were taken every 60 min for a period of 4 hr. Proteolytic activity was inactivated by placing the tubes in a water bath at 90°C for 5 min. For the experiment with Flavourzyme, the pH was decreased to pH 5 after two hours, since Flavourzyme exhibits more endoprotease activity at this pH. At pH 7, exo-protease activity is more abundant.

[0190] The degree of hydrolysis was measured using the method above, figures are given in the table 3 below.

[0191] Table 3 Degree of hydrolysis

[0192] Samples were heat treated to inactivate the protease, and subsequently analyzed using SDS-Page. The results for untreated and ALCALASE™-treated rapeseed protein isolate are shown in figure 1 , 2 and 3.

[0193] The figures 1-3 all show the intact bands of cruciferin (36, 31 and 21 kDa) and the napin bands (at 9 and 6 kDa, commonly poorly resolved in this SDS Page set up). Only little or no indications of substantial hydrolysis appear from the analyses.

[0194] The total amount of free amino acids was also measured and for none of the samples this exceeded 1 % free amino acids (on total protein), except for the treatment with Flavourzyme, showing more than 25% free amino acids (on total protein) - indicative of its endo-protease activity Conclusion: Despite high enzyme-to-substrate ratios and optimum conditions for the various enzymes, only limited hydrolysis of RPI was observed. Especially the low hydrolysis levels by ALCALASE™ were surprising. Flavourzyme appeared to show good hydrolysis but that was due to its exo-protease preference at pH7, that led to high level of free amino acids, but not to substantial breakdown of the core of the rapeseed proteins.

[0195] Example 2

[0196] Hydrolysis of rapeseed protein isolate with aspergillopepsin 1

[0197] In this example hydrolysis of rapeseed protein hydrolysate is described using 5% RPI under the following conditions:

[0198] A. Pretreatment at pH 1.8 using nitric acid followed by proteolysis with ALCALASE™ at pH 8

[0199] B. Proteolysis using fungal endoprotease aspergillopepsin 1 (MAXIPRO™ AFP) at pH

[0200] 3.5 C. Proteolysis using fungal endoprotease aspergillopepsin 1 (MAXIPRO™ AFP) at pH 4 followed by second incubation with proline-specific endoprotease (Delvoplant PSP) (pH 4)

[0201] Table 4 Hydrolysis conditions

[0202] *Doses in reaction mixture, not on protein or dry matter

[0203] These runs were executed under food-grade conditions. 1 .5 L solution of rapeseed protein isolate (5% w / w) in process water was made and the pH was adjusted (using 4N H3PO4 or 4N NaOH) and temperature were set. Sample 2A was first brought to pH2 using HCI solution and then brought back to 8 using 4N NaOH (effectively producing around 4g / L NaCI after pH swing). Samples at t=0 were taken for analysis. The enzyme solutions were added - see table. Samples (40 ml) were taken every 60 min over a period of 4 hr. Samples were deactivated by placing the tubes in a water bath at 90°C for 5 min. At the end of the incubation time, the final mixtures were heated 90°C for 5 min to inactivate the enzyme. For sample C, after incubation with MAXIPRO™ AFP, the temperature was reduced to 50°C and Delvoplant PSP was added, the incubation was continued for 18 hr to obtain final the sample C, followed by heated 90°C for 5 min to inactivate the enzyme.

[0204] Samples were characterized by SDS Page as is shown in figure 4 and 5.

[0205] The SDS Page gels all showed substantial hydrolysis, however a relatively pronounced band at around 6kDa remained visible in samples 2A and 2B which was due to one of the napin subunits. In the final sample 2C (lane 4 in figure 5) only a small trace of this band was still visible.

[0206] These samples at the end of full incubation were further analyzed with HP SEC. In this analysis also PeptoPro casein hydrolysate (DSM) was taken along. The samples were separated in various molecular weight classes, for convenience here grouped into three classes: >17 kDa: larger than napin; 17 - 3.5 kDa: fraction containing unhydrolyzed napin and its subunits; <3.5 kDa: oligopeptides and amino acids, hydrolyzed fragments. In Table 5 it can be seen that in hydrolysate 2A and 2C a substantial part of the napins (having a molecular weight of 14 kDa) had been hydrolyzed, and that in 2B still a substantial portion of the napin was intact.

[0207] Without wishing to be bound by any kind of theory, it is further believed that the type of peptides in Hydrolysate 2A differs from those in Hydrolysate 2B and 2C. According to Brenda (Enzyme database compiled by the National Institutes of Health) Aspergillopepsin I (such as MAXIPRO™ AFP) is quite specific and generally favours hydrophobic residues in P1 and P1 ', whereas subtilisin (such as ALCALASE™) cleaves in a rather random manner and is understood to have a broad specificity for many types of peptide bonds. As a result it may be expected that Hydrolysate 2B and 2C may comprise more peptides (herein also referred to as protein fragments) having a hydrophobic amino acid at one or both termini.

[0208] Table 5 Hydrolysis results

[0209] Final products of the protein hydrolysate after dilution to a protein content of around 2% were tasted at room temperature without further adjustment of pH or sweetness, by an internal group of tasters. Overall, compared to standard rapeseed protein isolate in solution, the astringency was found substantially reduced or absent, with sample 2C as the least astringent, and the ‘typical hydrolysate’ tastes were also modest. The sample 2A (ALCALASE™ after acidification and neutralization) led to a salty product.

[0210] Conclusion: Only after heavy pre-treatment by pH swing to very low pH and back to pH 8, subtilisin (ALCALASE™) was capable of hydrolysing rapeseed protein isolate. Aspergillopepsin I (MAXIPRO™ AFP), however, could hydrolyse rapeseed protein isolate already directly at its optimum pH of 3.5 to 4. After subsequent hydrolysis with a proline-specific protease (Delvoplant PSP) napin was further broken down, resulting in a fairly well tasting hydrolysate.

[0211] Example 3

[0212] Use of rapeseed protein hydrolysate in a growth medium

[0213] The hydrolysates from example 2 were used for the growth of Bacillus subtilis (168 RUG2010A), E. coll (RV308), Pichia pastoris (SMD1168) and Saccharomyces cerevisiae CEN.PK113-7DAA) cells. A Verduyn growth medium was used without urea, with a pH of 6 adjusted with KOH. The Verduyn medium further contained KH2PO4, 0.3%, K2SO4, 0.66%, MgSO4.7H2O, 0.05%, urea (optional - only in one case) 0.23%, liquid vitamin mix, 0.1 %, liquid spore elements mix 0.1 %, glucose 2%, the remaining part being demi water. The medium was filter sterilized and stored at 4°C.

[0214] The following experiments shown in table 6 were carried out. Table 6 Experiments

[0215] The growth media were prepared as follows. For experiment 1 : 10 ml growth medium, 5 ml demi water, 0.8 ml 50% glucose, 0.4 gram rapeseed protein isolate, 4.2 ml demi water to a final volume of 20 ml, and pH adjusted to pH 6.06. For experiments 2, 3 and 4: 10 ml growth medium, 0.8 ml 50% glucose, 8 ml 5% hydrolysate, and pH adjusted to 6, 1.2 ml demi water was added to a final volume of 20 ml. For experiments 5 and 6, 10 ml growth medium, 0.8 ml 50% glucose, 0.092 gram urea (only experiment 5), pH adjusted to 6 and 9.2 ml demi water was added to a final volume of 20 ml.

[0216] To pre-culture the 4 strains, 75 pl of the cells were added to a 100 ml non-baffled shake flask containing 15 ml yeast extract pepton dextrose growth medium and incubated overnight at 30°C with 280 RPM. Thereafter, 4 ml of the cultures was centrifuged for 5 minutes at 4000g. Supernatant was discarded, and cells were resuspended in 8 ml demi water.

[0217] The growth media were inoculated with 0.05 ml of the resuspended cells in a Biolector using 48 plates containing 1 ml media. Growth conditions were 30°C, 900 RPM and every 10 minutes a measurement of the optical density [Arbitrary Units] was taken.

[0218] The results are shown in figure 6.

[0219] Conclusion: Taking Optical Density as measure for growth of microorganisms it is clear that for all species tested, growth is better on a hydrolysate compared to unhydrolyzed rapeseed protein isolate. Comparing the hydrolysates amongst each other, the hydrolysate obtained after hydrolysis using a general fungal protease Aspergillopepsin 1 [MAXIPRO™ AFP] + a proline-specific endo protease [Delvoplant PSP] performed best.

[0220] Example 4 Use of rapeseed protein hydrolysate in topical application

[0221] The hydrolysates from example 2, Hydrolysate 2A (CanolaPRO™ A), Hydrolysate 2B (CanolaPRO™ B) and Hydrolysate 2C (CanolaPRO™ C), were used to test their efficacy upon topical application focusing on specific assays to evaluate the anti-oxidant activity and protection from DNA damage (gH2AX staining upon oxidative stress induced DNA damage), increase in Collagen I production and effect as senolytic compound. 1) qH2AX staining upon oxidative stress induced DNA damage:

[0222] The assay was carried out by seeding Human Dermal Fibroblasts (HDF) at high confluency in 96 well plates. 48 hours (h) after seeding cells were incubated with the indicated doses of rapeseed protein hydrolysate samples for additional 24h, together with the control (treatment with N-acetyl-cystein (NAC) 1 mM). Then the cells were stimulated (stressed) with 1 hour H2O2 500uM treatment to induce DNA damage. Cells were then stained with gH2AX antibody following the manufacturer’s instructions and acquired at fluorescence microscope. The nuclear signal was then quantified, which was directly proportional to the amount of DNA damage in the cells. The experiment was performed in 3 independent plates (biological replicates), with 3 replicate per samples (technical replicates) (See Figure 7).

[0223] The results of this experiment showed that a trend toward protection of DNA damage was present for all the three rapeseed protein hydrolysate batches.

[0224] 2) Collagen I production

[0225] In this experiment the Collagen I production by human dermal cells was evaluated. The experiment was performed once with 4 technical replicates (n=4) (See Figure 8). The test was performed with the 3 batches of rapeseed protein hydrolysate, e.g. for each of Hydrolysate 2A (CanolaPRO™ A), Hydrolysate 2B (CanolaPRO™ B) and Hydrolysate 2C (CanolaPRO™ C). In addition, a benchmark was evaluated as comparison.

[0226] The results showed a Collagen I boosting activity for the three rapeseed protein hydrolysate batches (See Figure 8). In this experiment all rapeseed protein hydrolysate batches showed an increase in Collagen I production similar to the benchmark or even slightly more (Hydrolysate 2B (CanolaPRO™ B)).

[0227] 3) Senolytic effect

[0228] This experiment was performed by seeding a defined percentage of Human Dermal Fibroblasts under homeostatic conditions (70%) or in a senescent state (30%). Upon treatment with the compounds of interest the percentage of remaining senescent cells is evaluated (with the help of a B-gal senescence assay). The lowest percentage of remaining senescent cells is the most advantageous I represents the best result. The experiment was performed once with n=4 replicates (See Figure 9).

[0229] The results showed that Hydrolysate 2B (CanolaPRO™ B) and Hydrolysate 2C (CanolaPRO™ C) displayed at higher concentrations a senolytic activity that was not present instead with Hydrolysate 2A (CanolaPRO™ A). As explained before, the use of a different enzyme for hydrolysis may lead to a different type of peptides (also referred to herein as protein fragments) being present in the hydrolysate formed .

[0230] Hydrolysate 2B (CanolaPRO™ B) and Hydrolysate 2C (CanolaPRO™ C) were prepared in a hydrolysis process wherein an Aspergillopepsin I (MAXIPRO™ AFP) was applied, whereas Hydrolysate 2A (CanolaPRO™ A) was prepared in a hydrolysis process wherein subtilisin (ALCALASE™) was applied.

[0231] According to Brenda (Enzyme database compiled by the National Institutes of Health) Aspergillopepsin I (such as MAXIPRO™ AFP) is quite specific and generally favours hydrophobic residues in P1 and P1', whereas subtilisin (such as ALCALASE™) cleaves in a rather random manner and is understood to have a broad specificity for many types of peptide bonds.

[0232] As a result it may be expected that Hydrolysate 2B (CanolaPRO™ B) and Hydrolysate 2C (CanolaPRO™ C) may comprise more peptides having a hydrophobic amino acid at one or both termini than Hydrolysate 2A (CanolaPRO™ A).

[0233] Conclusions:

[0234] From in vitro tests on Human Dermal Fibroblasts, we concluded that rapeseed protein hydrolysates displayed a certain DNA protective function upon oxidative stress together with a moderate increase in Collagen I production. In addition, Hydrolysate 2B (CanolaPRO™ B) and Hydrolysate 2C (CanolaPRO™ C) showed an additional senolytic effect in vitro.

Claims

CLAIMS1 . A composition, preferably a cosmetic composition, comprising- a rapeseed protein hydrolysate comprising protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa; and- optionally a cosmetically acceptable further ingredient.

2. The composition according to claim 1 , wherein the composition is a cosmetic composition, more preferably a topical composition.

3. The composition to any one of the preceding claims, wherein the composition is a skincare composition, a haircare composition or a suncare composition.

4. The composition according to any one of the preceding claims, wherein the rapeseed protein hydrolysate has a degree of hydrolysis of between 1 and 30%, preferably between 2 and 20%, more preferably between 5 and 10%.

5. The composition according to any one of the preceding claims, wherein the rapeseed protein hydrolysate has a protein content of 40 to 100% (w / w).

6. The composition according to any one of the preceding claims, wherein the protein hydrolysate is is present in the composition in a dissolved, emulsified or dispersed form.

7. The composition according to any one of the preceding claims, wherein the rapeseed protein hydrolysate comprises:1 to 10% (w / w) of protein fragments having a molecular weight of more than 17 kDa;10 to 30% (w / w) of protein fragments having a molecular weight of 3,5 to 17 kDa;60 to 89% (w / w) of protein fragments having a molecular weight of less than 3.5 kDa.

8. The composition according to any one of the preceding claims, wherein the rapeseed protein hydrolysate contains protein fragments and wherein at least 5% (w / w), more preferably at least 10% (w / w), even more preferably at least 20% (w / w), still more preferably at least 30% (w / w), yet more preferably at least 40% (w / w), yet still more preferably at least 50% (w / w) and most preferably at least 60% (w / w) of such protein fragments, based on the total weight of such protein fragments, are peptides having an hydrophobic amino acid at one or both of its termini.

9. The composition according to any one of the preceding claims,wherein the rapeseed protein hydrolysate is a rapeseed protein hydrolysate obtained from, produced by or derived from a hydrolysis of rapeseed protein with the help of an aspergillopepsin I enzyme and preferably a proline-specific endoprotease.

10. The composition according to any one of the preceding claims, wherein the composition comprises water and / or oil.11 . The composition according to any one of the preceding claims, wherein the composition comprises one or more further ingredients selected from the group consisting of preservatives / antioxidants, fatty substances / oils, water, organic solvents, silicones, thickeners, softeners, emulsifiers, antifoaming agents, aesthetic components such as fragrances, aroma ingredients, surfactants, fillers, anionic, cationic, non-ionic or amphoteric polymers or mixtures thereof, propellants, acidifying or basifying agents, dyes, colourings / colorants, abrasives, absorbents, chelating agents and / or sequestering agents, essential oils, skin sensates, astringents and / or pigments.

12. Use of a rapeseed protein hydrolysate or a composition according to any one of claims 1 to 11 for the reduction of damage to DNA in the presence of oxygen; and / or for the boosting of Collagen 1 production by human dermal cell; and / or for the treatment of the signs of aging; and / or for reducing and / or eliminating senescent skin cells.

13. The use according to claim 12 for reducing or eliminating senescent skin cells, wherein the senescent skin cells are human dermal fibroblasts.

14. The use according to any one of claims 12 to 13, wherein the senescent cells are age- related senescent cells, stress-related senescent cells, or both.

15. A cosmetic skin and scalp care method for the reduction and / or removal of senescent cells in the skin and / or hair follicle, said method encompassing the step of applying, onto the skin or the scalp in need thereof, at least an effective amount of a rapeseed protein hydrolysate or a composition according to any one of claims 1 to 11 .

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