The present invention relates to a hair care composition comprising an aqueous composition comprising a functional silk protein, its use and applications.

Hair washing, conditioning, and styling is a common ritual in all parts of the world. Beyond the usual cleaning of the hair fiber, secondary benefits are expected by consumers to enhance their experience, such as an easier detangling, an easy styling, a different color, a greater shine or a better smoothness of their hair. Some of these hair treatments involve harsh conditions that may damage the hair.

Human hair can be damaged in many ways. Frequently in everyday life, these natural fibers come into contact with heat or chemical agents, which attack and damage their structure and can impair their visual appearance. Repeated and excessive use of hair styling tools, such as curling wand, straightener or blow dryer, can damage the hair and cause moisture loss, frizz, dryness, split ends and hair breakage. Chemical treatments (e.g. hair dying or bleaching) can also negatively impact the hair texture and quality. In fact, these chemical treatments are the leading cause of changes in hair structure and ultimately hair damage. Environmental conditions such as UV radiation and air pollution can also have an impact on hair quality.

Damaged hair has less shine and smoothness, thus directly indicating poor hair health. In addition, damaged hair also has reduced elasticity and breaks easily under tension. In recent decades, various substances have been identified and developed by the cosmetics industry to minimize such influences. For example, silicones are commonly used in order to form a protective layer on the hair fibers. However, as it can be difficult to wash silicones out of the hair entirely, they can cause a “build-up effect” on hair. Other protective and widely applied conditioning agents include protein derived actives, such as hydrolysed keratin, wheat protein and wheat starch, which have been found to enhance the tensile strength of damaged hair (Teglia, A. & Secchi, G., 1999. Proteins in Cosmetics. In: Principles of Polymer Science and Technology in Cosmetics and Personal Care. New York: pp. 433-453).

The effect of such substances is mostly based on their molecular composition. In theory, some of these proteins have a similar amino acid composition as human hair, which supposedly results in an increased affinity of the molecules to the cuticle of the hair. However, conventional protein-based conditioners are mostly hydrolysates of animal or vegetable origin. In such chemically and/or physically processed and partially degraded peptide mixtures breakdown of proteins, usually including loss of cysteine, serine, and other amino acids occurs, which greatly impairs their binding capacity and affinity to human hair (Secchi, G., 2008. Role of proteins in cosmetics. Clinics in Dermatology, Issue 26, pp. 321 - 325).

Since hydrolysates have no defined composition and molecular structure, the components of the hydrolysed protein mixtures can interact only to a limited extent with the surface structure of the hair. In addition, the keratin present in natural hair is an irreplaceable protein in respect to its mechanical and protective properties, and the use of amino acids or peptide fragments does not replace or restore the damaged molecule structure (Villa, A. L. V. et al., 2013. Feather keratin hydrolysates obtained from microbial keratinases: effect on hair fiber. BMC Biotechnol, Issue 13:15).

Silk is a natural protein-based material which allows many cosmetic applications, including hair care. However, the traditional method of silk production is not compatible with nowadays’ sustainability requirements, as it consumes high quantity of raw materials (like the mulberry leaves favored by caterpillars) and supervision. Approximately 200 kg of feedstock are needed to produce 1 kg of silk. In addition, respect of nature is more and more important for consumers, resulting in increased vegetarian or even vegan lifestyles.

There is, therefore, a need for a sustainable and effective hair repair treatment.

In a first aspect, the present invention provides a leave-in hair care composition comprising an aqueous composition comprising at least one functional silk protein. The at least one functional silk protein is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and mixtures thereof.

SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 are shown in Figures 1 , 2, 3, and 4, respectively.

Cosmetic or dermatological compositions comprising natural or recombinant spider silk proteins are generally known from US 2005/0019297 A1 , for instance, and have also been described for other applications (e.g. WO 2006/008163 A2).

Surprisingly, it has now been found that the leave-in hair care composition of the present invention is able to improve the breakage strength of hair and even to repair damaged hair.

Therefore, in a second aspect, the present invention refers to the use of the leave-in hair care composition of the present invention for improving the breakage strength of hair. In a third aspect, the present invention refers to the use of the leave-in hair care composition of the present invention for repairing damaged hair.

In a fourth aspect, the present invention provides a method of improving the breakage strength of hair, said method comprising the step of topically applying the leave-in hair care composition of the present invention to human hair.

In a sixth aspect, the present invention provides a method of repairing damaged hair, said method comprising the step of topically applying the leave-in hair care composition of the present invention to human hair.

The hair care composition of the present invention comprises an aqueous composition comprising at least one functional silk protein selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and mixtures thereof. These recombinant proteins are based on the repetitive core sequence of the dragline silk protein ADF4 ( Araneus diadematus fibroin 4) of the European garden spider ( Araneus diadematus ).

The biotechnological production of these functional silk proteins delivers them in high amounts and with consistent quality (Heidebrecht, A.; Scheibel, T. Recombinant production of spider silk proteins. Adv Appl Microbiol 2013, 82, 115-153). Typically, bacteria are cultivated in a natural growth medium using sugar from plant sugar as feed. The proteins are not hydrolysed.

A suitable method for the preparation of the functional silk proteins of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 is described in example 1 of WO 2019/092073, with the C ₈ protein corresponding to SEQ ID NO:1 , the Ci ₆ protein corresponding to SEQ ID NO:2, the C32 protein corresponding to SEQ ID NO:3, and the C ₄₈ protein corresponding to SEQ ID NO:4 of the present disclosure, respectively.

The functional silk proteins are biodegradable and can be produced in a highly sustainable and cruelty-free manner.

The functional silk proteins used in the hair care composition of the present invention have structure- or film-forming properties. In contrast to the hydrolysates used in conventional protein-based conditioners, the biotechnologically produced functional silk proteins have a precisely defined amino acid composition and corresponding weight. Thanks to their large size, their defined composition and their ability to self-assemble, the functional silk proteins are able to form a protective and conditioning film on the surface of the hair.

It has been found that the hair care composition of the present invention is able to increase the breakage strength of hair, thereby protecting the hair from future damage. Moreover, the hair care composition of the present invention is able to repair already damaged hair. For instance, the hair may have been damaged by heat (e.g. drying or styling) or by chemicals (e.g. coloring).

Detailed results can be found in the examples below.

The hair care composition of the present invention provides a physical and breathable protection for the hair. It is light weight and does not have a build-up effect.

Furthermore, the hair care composition of the present invention also improves the texture of the hair, providing a silky feeling, smoothness and shine.

Another benefit of the hair care composition of the present invention is that it is able to inhibit bacterial adhesion and thus prevent biofilm formation. In contrast to conventional raw materials such as silicones and micro plastics, which are commonly used for hair conditioning and protection, the functional silk proteins represent a truly ecological and natural alternative with an excellent protective performance against thermally or chemically induced damages.

In contrast to natural silk-derived hydrolysates, the functional silk proteins used in the present invention have a defined chain length and molecular weight and thus uniform characteristics. They are also much easier to incorporate into cosmetic formulations thanks to their higher solubility in alcoholic bases. Furthermore, the functional silk proteins used in the present invention are odourless.

The term “hair care composition” includes both leave-in products and wash-out. For example, there are hair cleansing compositions, hair conditioning compositions, and hair styling compositions, such as shampoos, conditioners, sprays, treatments, masks, strengtheners, pre-shampoos, lotions, serums, creams, foams, mousses, and gels. Many of these compositions that are known are water-based formulations. Hair cleansing compositions are generally effective to remove soil from hair. The soil includes natural exudations from the scalp, environmental agents, and styling products. The soil can coat or deposit on the hair and scalp. Hair coated with such soil is typically greasy in feel and appearance, heavy to the touch, possibly malodorous, and generally unable to maintain a desired style. Known cleansing compositions typically include a combination of water and surface-active ingredients, such as soap or synthetic surfactants, and may also include a non-aqueous blend of starches. The combination of water and surface -active agents emulsifies the soil from the hair and scalp, allowing it to be rinsed away.

Cleansing compositions may also contain conditioning agents that deposit on the hair and scalp during rinsing with water. Such conditioning agents can include polymers, oils, waxes, protein hydrolysates, silicones, and mixtures and derivatives thereof. In addition, the conditioning composition can be a separate and different product from the cleansing composition.

Conditioning compositions that are known in the art are typically water-based formulations. However, there are also known conditioning compositions, which include at least one of silicones; animal, mineral or vegetable oils; waxes; petrolatums; and greases. The water- based conditioning compositions typically include substituted cationic waxes, fatty alcohols, cationic polymers, hydrolysed proteins and derivatives thereof, and fragrances. Such conditioning formulations impart combability and manageability to the treated hair, thereby minimizing breakage during the styling process and resulting in shiny, healthy, and manageable hair. Conditioning compositions may also be effective to moisturize the hair. Subsequent drying and styling processes can include air drying or heating.

Best results are achieved if the at least one functional silk protein is formulated in a leave-in hair care composition.

However, it would also be possible to formulate the at least one functional silk protein in a rinse-off hair care composition.

Typically, the hair care composition of the present invention will further comprise a suitable carrier. Alternatively, it is also possible to apply the aqueous composition comprising the at least on functional silk protein neat.

The suitable carrier must be cosmetically acceptable. “Cosmetically acceptable”, as used herein, means that the carrier is suitable for use in contact with human keratinous tissue without undue toxicity, incompatibility, instability, allergic response, and the like. All compositions described herein, which have the purpose of being directly applied to keratinous tissue, are limited to those being cosmetically acceptable.

Hair care compositions typically comprise the carrier at a level from about 20 wt% to about 99 wt%. The carrier may comprise water, organic solvents (miscible or non-miscible with water) silicone solvents, and/or mixtures thereof. The solvents should be dermatologically acceptable. Carriers usually do not comprises more than about 2 wt% of non-volatile solvent, as significantly higher concentrations will increase hair weigh -down and greasy feel. Water, organic and silicone solvents that have boiling points below or equal to 250 °C are considered volatile solvents. Suitable carriers typically include water and water solutions of lower alkyl alcohols, such as monohydric alcohols, having 1 to 6 carbons (e.g. ethanol and/or isopropanol), and polyhydric alcohols, such as glycols, glycerine, and other diols. The carrier is preferably selected from the group consisting of water, methanol, ethanol, propanol, isopropanol, DMSO, and mixtures thereof.

The hair care composition according to the present invention may further comprise one or more materials selected from the group consisting of solvents, surfactants, thickeners, styling polymers, anti-dandruff actives, antimicrobial materials, skin and scalp actives, vitamins, salts, buffers, hair growth agents, conditioning materials, hair-fixative polymers, fragrances, colorings/colorants, dyes, pigments, opacifiers, pearlescent aids, oils, waxes, preservatives, sensates, sunscreens, medicinal agents, antifoaming agents, antioxidants, binders, biological additives, buffering agents, bulking agents, chelating agents, chemical additives, film formers or materials, pH adjusters, propellants, oxidizing agents, and reducing agents.

All additives should be physically and chemically compatible with the essential components of the hair care composition, and should not otherwise unduly impair stability, aesthetics or performance. Most importantly, they should also be cosmetically acceptable. The CTFA Cosmetic Ingredient Handbook, Tenth Edition (published by the Cosmetic, Toiletry, and Fragrance Association, Inc., Washington D.C., 2004) describes a wide variety of non-limiting materials that can be added to the hair care composition of the present invention.

The amount or concentration of the aqueous composition comprising the at least one functional silk protein in the hair care composition of the present invention may depend on the desired end use and/or the other active ingredients and/or adjuvants, solvents and other commonly used ingredients added to the hair care composition. Also, it has been found that the positive effects of the hair care composition of the present invention can be increased by increasing the silk protein content.

The hair care composition of the present invention may consist entirely of the aqueous composition comprising the at least one functional silk protein. However, the herein described positive effects are already achieved at much lower doses.

In an embodiment, the hair care composition of the present invention comprises from 0.5 to 50 wt%, more preferably from 1 to 20 wt%, even more preferably from 1 to 10 wt%, and most preferably from 2 to 5 wt%, of the aqueous composition. These concentrations guarantee a good miscibility, easy formulation and stability, as well as the desired activity.

Also all the other active ingredients, adjuvants and/or solvents may be used in the hair care composition of the present invention in any suitable concentration range and ratio. In particular, it is desirable to adapt the concentration ranges in order to guarantee sufficient activity, solubility, stability, and ease of formulation. The term “aqueous composition”, as used throughout the present application refers to any kind of formulation comprising water, and in particular to aqueous solutions, suspensions, hydrogels, and mixed forms of these.

The aqueous composition present in the hair care composition of the present invention mainly consists of water. Apart from the at least one functional silk protein, it may comprise other ingredients, such as other, active ingredients, solvents and/or adjuvants. The aqueous composition is preferably transparent and colorless; but it may be slightly turbid or brownish.

In an embodiment of the present invention, the aqueous composition comprising the at least one functional silk protein is a hydrogel.

It has been found that such a hydrogel is highly robust, non-sticky, thixotropic, and sprayable. It is also not thermally sensitive and thus suitable for cold as well as hot production processes, up to about 85 °C. This offers a great flexibility for formulation.

The hydrogel can be prepared as described in examples 1 and 3 of WO 2019/092073, for instance. The same document also discloses the preparation of an aqueous solution of functional silk proteins. The thus obtained hydrogel is thixotropic, non-occlusive, and sprayable and has excellent biocompatibility.

The amount or concentration of the at least one functional silk protein in the aqueous composition may depend on the preparation process, the addition of other active ingredients and/or adjuvants, solvents and other commonly used ingredients, and/or the desired end use.

In an embodiment of the present invention, the aqueous composition, and in particular the hydrogel, comprises from 1 to 10wt%, more preferably from 1 to 5 wt%, and most preferably from 1 to 3 wt%, of the at least one functional silk protein, for example 2 wt% or 3 wt%. This guarantees the desired activity, stability, ease of formulation and rheological properties. If the at least one functional silk protein is prepared biotechnologically, it is possible to obtain a vegan product. Therefore, in an embodiment of the present invention, the at least one functional silk protein is vegan. More preferably, the hair care composition of the present invention as a whole is vegan, i.e. all ingredients used in the composition comply with vegan standards. Without being bound by theory, it is believed that smaller functional silk proteins may reach into small pores or cavities in the hair, whereas larger functional silk proteins have better film forming properties.

Therefore, in an embodiment of the present invention, the aqueous composition comprises the functional silk proteins of SEQ ID NO:1 and of SEQ ID NO:4. In this embodiment, the aqueous composition may optionally further comprise one or more of the other functional silk proteins of SEQ ID NO:2 and/or SEQ ID NO:3.

In an embodiment, the aqueous composition further comprises 1 ,2-hexanediol. Preferably, the aqueous composition comprises from 0.05 to 5 wt%, more preferably from 0.2 to 1 wt%, and most preferably from 0.4 to 0.6 wt%, of 1 ,2-hexanediol. In an embodiment, the aqueous composition further comprises 1 ,2-octanediol. Preferably, the aqueous composition comprises from 0.05 to 5 wt%, more preferably from 0.2 to 1 wt%, and most preferably from 0.4 to 0.6 wt%, of 1 ,2-octanediol.

Both 1 ,2-hexanediol and 1 ,2-octanediol serve as “preservation boosters”, allowing for the reduction of the amount of preservatives in the final hair care composition. Preferably, the aqueous composition further comprises both 1 ,2-hexanediol and 1 ,2- octanediol. For instance, the hydrogel may comprise Symdiol 68 (by Symrise AG), an assumed 1 :1 mixture of 1 ,2-hexanediol and 1 ,2-octanediol. Preferably, the aqueous composition further comprises 1 ,2-hexanediol and 1 ,2-octanediol in the above described concentrations.

Alternatively or in addition, the hair care composition of the present invention may also comprise 1 ,2-hexanediol and/or 1 ,2-octanediol that was added independently of the aqueous composition.

Therefore, in an embodiment, the hair care composition further comprises 1 ,2-hexanediol. Preferably, the hair care composition comprises from 0.05 to 5 wt%, more preferably from 0.2 to 1 wt%, and most preferably from 0.4 to 0.6 wt%, of 1 ,2-hexanediol.

Therefore, in an embodiment, the hair care composition further comprises 1 ,2-octanediol. Preferably, the hair care composition comprises from 0.05 to 5 wt%, more preferably from 0.2 to 1 wt%, and most preferably from 0.4 to 0.6 wt%, of 1 ,2-octanediol. Preferably, the hair care composition further comprises both 1 ,2-hexanediol and 1 ,2- octanediol, for instance Symdiol 68. Preferably, the hair care composition further comprises 1 ,2-hexanediol and 1 ,2-octanediol in the above described concentrations.

Alternatively or in addition, the hair care composition of the present invention may comprise other known preservatives. In an embodiment, the leave-in hair care composition is provided in the form of a spray. This allows for simple and thorough application to the hair. Alternatively, the leave-in hair care composition of the present invention may also be provided in the form of an emulsion, a milk or a gel.

In a further aspect, the present invention relates to the use of the leave-in hair care composition of the present invention for improving the breakage strength of hair.

It has been found that leave-in hair care composition of the present invention provides an equivalent or even better protection against thermal hair damage than conventional conditioning agents, such as collagen, keratin and wheat hydrolysates. Furthermore, as can be seen from the results in examples 4 and 5 below, the hair care composition of the present invention is able to improve the hair surface with a smoothing effect. Without being bound by theory, it is believed that their film forming properties enable the compositions of the present invention to protect the hair fibers against external stress. In a further aspect, the present invention relates to the use for repairing damaged hair.

In a further aspect, the present invention relates to a method of improving the breakage strength of hair, said method comprising the step of topically applying the leave-in hair care composition of the present invention to human hair.

In a further aspect, the present invention relates to a method of repairing damaged hair, said method comprising the step of topically applying the leave-in hair care composition of the present invention to damaged human hair.

The hair care composition of the present invention may be applied to either wet or dry hair, depending on formulation.

The above beneficial effects of the hair care composition of the present invention have been confirmed by ex vivo studies, some of which are described in the examples below.

The present invention is thus further illustrated by means of the following non-limiting examples:

Example 1 : Hair Strengthening before Thermal Treatment (ex vivo)

Overview Four blond hair tresses (remis, color 9/0; Kerling International Flaarfabrik GmbFI) were tested. Three of these hair tresses (samples 2-4) were subjected to thermal treatment with a hair straightener, whereas the fourth tress (sample 1) was used as an untreated negative control. Prior to the thermal treatment, samples 2 and 3 were treated with different hair care compositions to provide heat protection: Samples 1 through 4 were washed with a standard shampoo (FA Fiji dream) and dried with a towel. Afterwards, the selected hair care composition (untreated, got2be or Composition 1 ) was sprayed into damp hair (3x spraying, corresponding to approximately 300 pi). Subsequently, the samples were gently blow-dried at 50 °C. This was followed by thermal treatment with the hair straightener for 1 min at 220 °C.

This treatment was repeated 22 times at irregular intervals over 20 days. At the end of the 20 days, the samples were subjected to tensile testing.

Hair Care Compositions

As a benchmark product, the commercially available silicone-containing heat protection spray “got2be” by Schwarzkopf was used. The manufacturer advertises this product as having a heat protection effect up to 220 °C.

Composition 1 was a leave-in hair care composition according to the present invention. It was prepared by adding 1.62 g (about 3 wt%) of a silk protein hydrogel and 0.26 g (about 0.5 wt%) of Symdiol 68 (ex Symrise; 1 ,2-hexanediol / 1 ,2-octanediol) to 48.25 g (about 96.5 wt%) of water and homogenizing the mixture for 3-4 min at 12Ό00-15Ό00 rpm. The silk protein hydrogel consisted of 3 wt% of the functional silk protein of SEQ ID NO:2, 1 .0 wt% of Symdiol 68 in 96 wt% of water.

Tensile Testing

In order to minimize diameter dependent fluctuations, the measured breaking force values (= non-normalized values) were standardized relative to the diameter of each measured individual hair.

Therefore, the diameter of each hair was determined prior to the tensile test using a precision micrometer (ID-H0530) from Mitutoyo GmbFI. As each hair becomes thinner from the shaft to the tip, an average of several measurements was taken. The individual hair diameters varied between 45 and 70 mhi.

The hair diameters were then used in the calculation of the normalized breaking force: F _N normalized maximum breaking force

F _M measured maximum breaking force r radius of the hair sample Afterwards, 10 individual hairs per sample were subjected to the tensile test using a Zwick BT1-FK0.5N.D14 from Zwick / Roell GmbFI & Co. KG tensile tester.

The samples were tested following essentially the DIN EN ISO 2062 standard originating from the textile industry, but using a pulling speed of 250 mm/min and an effective clamping length of 80 mm. The generated data was analyzed using 1 -way Analysis of Variance (ANOVA), followed by a post-hoc analysis Tukey Flonest Significance Difference (TFISD) test, using the R programming language. Probability values p<0.05 were considered statistically significant. For probability values p<0.01 , the term “highly significant” was used, and probability values p<0.001 were considered to be “most significant”. Results

The normalized breaking forces for the four samples tested are shown in Figure 5.

It was found that the thermal treatment leads to a reduction of the normalized breaking force by -41% (sample 2 relative to sample 1 ; non-normalized: -30%).

Compared to sample 2, the two samples treated with got2be (sample 3) and Composition 1 (sample 4) had a significantly increased normalized breaking force by +111% (non- normalized: +56%) and +99% (non-normalized: +47%), respectively (p<0.001).

Thus, the hair care composition of the present invention was able to significantly improve the breakage strength of the hair, similar to a silicone-based benchmark product.

For comparison: Nicholson et al. (“Nicholson, S., Daniels, G., Grant-Ross, P. & Tamburic, S., 2016. An ex vivo comparison of the tensile strengthening properties of protein derivates on damaged hair. IFSCC, pp. 1 -6.) observed for the use of protective collagen, keratin und wheat hydrolysates non-normalized tensile strength increase percentages between 18 to 29%. Thus, the present testing not only confirmed the protective effect of silk proteins against thermal damage in general, but also their increased effectiveness compared to conventional conditioning agents, such as collagen, keratin and wheat hydrolysates.

Example 2: Hair Repair after Thermal T reatment ( ex vivo ) Overview

Six blond hair tresses (remis, color 9/0; Kerling International Haarfabrik GmbH) were tested. Five of these hair tresses (samples 6-10) were subjected to thermal treatment with a hair straightener, whereas the sixth tress (sample 5) was used as an untreated negative control. After repeated thermal treatment, samples 7 through 10 were treated with different hair care compositions for repair:

Samples 6 through 10 were washed with a standard shampoo (FA Fiji dream) and dried with a towel. They were than subjected to the thermal treatment with the hair straightener for 1 min at 220 °C. This treatment was repeated 20 times at irregular intervals over 20 days. At the end of the 20 days, all samples were washed with a standard shampoo (FA Fiji dream) and dried with a towel. Afterwards, the selected hair care composition (untreated, Express Repair Conditioner or composition 1 , 2 or 3) was sprayed onto the samples (3x spraying, corresponding to approximately 300 pi). Subsequently, the samples were gently blow-dried at 50 °C. This treatment was repeated 14 times at irregular intervals over 14 days. All samples were then subjected to tensile testing.

Hair Care Compositions

As a benchmark product, the commercially available “Express Repair Conditioner” by Schwarzkopf, which contains hydrolyzed silk, was used. Composition 1 was the same as that used in example 1.

Compositions 2 and 3 were both leave-in hair care compositions in the form of an emulsion, with Composition 2 being another comparative example and Composition 3 being a leave-in hair care composition according to the present invention. The two compositions consisted of essentially the same ingredients, the only difference being that Composition 3 did comprise a silk protein hydrogel, whereas Composition 2 did not:

The silk protein hydrogel consisted of 3 wt% of the functional silk protein of SEQ ID NO:2, 1 .0 wt% of Symdiol 68 in 96 wt% of water. Composition 2 was prepared as follows: Water was heated up to 80 °C. The oil phase containing Glycerol, Olivem 1000, Jojoba oil and Castor oil was heated up to 80 °C under stirring until it was homogenize in a separate vessel. The oil phase was then added under stirring to the water phase and homogenized for 1 min at 12Ό00 rpm. The thus obtained mixture was cooled down to less than 60 °C. Xanthan gum was added under stirring, followed by homogenization at 12Ό00 rpm for 1-2 min. Finally, Symdiol 68 was added and the formulation was homogenized at 12Ό00 rpm for 1-2 min to obtain Composition 2.

Composition 3 was prepared analogously, using a slightly lower amount of water (see table above). The silk protein hydrogel, which consisted of 3 wt% of the functional silk protein of SEQ ID NO:2, 1 .0 wt% of Symdiol 68 in 96 wt% of water, was added to the mixture and the resulting formulation was homogenized at 12Ό00 rpm for 1-2 min prior to the final addition of Symdiol 68.

Tensile Testing The tensile testing was performed as described in example 1 above.

Results

The normalized breaking forces for the six samples tested are shown in Figure 6.

It was found that the thermal treatment leads to a reduction of the normalized breaking force by -40% (sample 6 relative to sample 5). Compared to sample 6, the two samples used as comparative examples, namely samples 7 (Express Repair Conditioner) and 9 (Composition 2), both showed a slight, but non-significant increase in normalized breaking force.

On the other hand, the two samples treated with Composition 1 (sample 8) and composition 3 (sample 10) had a significantly increased normalized breaking force by +55% (P<0.01) and +72% (p<0.001), respectively.

Thus, the hair care composition of the present invention was able to repair hair damaged by heat treatment.

Example 3: Hair Repair after Dvina (ex vivo)

Overview Five blond hair tresses (remis, color 9/0; Kerling International Flaarfabrik GmbFI) were tested. Four of these hair tresses (samples 12-15) were treated with a commercial ammonia based red hair polish (Brilliance Intensive-Color-Creme, kaschmir-rot 842 (Schwarzkopf)), whereas the fifth tress (sample 11) was used as an untreated negative control. After dying, samples 13 through 15 were treated with different hair care compositions for repair (3x spraying, corresponding to approximately 300 pi):

Hair Care Compositions

As a benchmark product, the commercially available “Nourishing Protection Milk” by Gamier was used. This product is advertised as “providing a silky feeling” and has a similar composition as the hair care composition of the present invention, also comprising natural conditioning (e.g. rice starch).

Composition 3 was a leave-in hair care composition according to the present invention. It was prepared by adding 1.08 g (about 2 wt%) of a silk protein hydrogel and 0.26 g (about 0.5 wt%) of Symdiol 68 (ex Symrise; 1 ,2-hexanediol / 1 ,2-octanediol) to 48.66 g (about 97.5 wt%) of water and homogenizing the mixture for 3-4 min at 12Ό00-15Ό00 rpm. The silk protein hydrogel consisted of 3 wt% of the functional silk protein of SEQ ID NO:2 and 1 .0 wt% of Symdiol 68 in 96 wt% of water.

Composition 4 was a leave-in hair care composition according to the present invention, in the form of a spray:

Composition 4 was prepared as follows: Water and xanthan gum were mixed under stirring and homogenized together with the silk protein hydrogel, which consisted of 3 wt% of the functional silk protein of SEQ ID NO:2, 1.0 wt% of Symdiol 68 in 96 wt% of water, at 12Ό00 rpm for 2 to 3 minutes. The mixture was heated up to 75 °C to obtain the water phase. The oil phase (Glyceryl Stearate Citrate, Sodium Stearoyl Lactylate, Caprylic/Capric Triglycerides, Cetearyl Alcohol, Simmondsia Chinensis Seed Oil and Isoamyl Laurate) was prepared by mixing the respective ingredients and heating up to 75 °C. The oil phase was then added to the water phase. Afterwards, the thus obtained mixture was homogenized for 1 to 2 minutes at 12Ό00 rpm and cooled down to room temperature. Finally, propylene Glycol and Symdiol 68 were added to the formulation and the mixture was homogenized for 1 to 2 minutes at 12Ό00 rpm to obtain Composition 4.

Tensile Testing

The tensile testing was performed as described in example 1 above. Results

The normalized breaking forces for the five samples tested are shown in Figure 7.

It was found that the dying procedure leads to a reduction of the normalized breaking force by -28% (sample 12 relative to sample 11 ).

Compared to sample 12, the hair treated with Nourishing Protection Milk (sample 13) showed in increase in normalized breaking force by +14% (P<0.05).

On the other hand, the two samples treated with Composition 3 (sample 14) and Composition 4 (sample 15) had a significantly increased normalized breaking force by +22% and +35%, respectively (P<0.01). Thus, the hair care composition of the present invention was able to repair hair damaged by dying.

Example 4: Hair Repair after Bleaching (ex vivo)

Overview Twelve blond hair tresses (human natural blond hair lock) were tested. Two groups of these hair tresses (samples 17 and 18) were bleached using 9% (v/v) hydrogen peroxide and 3% (w/v) ammonium persulfate solution for 1 hour at 40 °C, whereas the third group (sample 16) was used as an untreated negative control. The hair tresses were rinsed 3 times with water and blow dried for 1 hour. After bleaching, samples 17 and 18 were straightened using a hair straightener at 220 °C for 1 minute to increase the breakage of disulphide bonds and increase porosity (3 passages).

In a final step, sample 17 was treated with a placebo composition (99.48 wt% of water and 0.52 wt% of Symdiol 68) and sample 18 was treated with 0.5 ml of Composition 5, each followed by a gentle massage and incubation overnight. Composition 5 was a leave-in hair care composition according to the present invention. It was prepared by combining 2.0 wt% of a silk protein hydrogel and 0.5 wt% of Symdiol 68 (ex Symrise; 1 ,2-hexanediol / 1 ,2-octanediol) with 97.5 wt% of water and homogenizing the mixture for 3-4 min at 12Ό00-15Ό00 rpm. The silk protein hydrogel consisted of 3 wt% of the functional silk protein of SEQ ID NO:2 and 1.0 wt% of Symdiol 68 in 96 wt% of water. X-Ray Nanotomography

To visualize and measure the efficacy of the hair care composition of the present invention as repair agent, x-ray nanotomography was conducted after the chemical and thermal treatment of hair wefts, and the 3D volume fraction and area increase were determined.

More particularly, hair porosity analysis was performed using X-ray nanotomography. A resolution of 800 nm was applied to generate 3D images of all samples. Quantification of the damage caused by the treatments was performed using image analysis techniques. Two parameters were analyzed: the 3D volume fraction of the damage out of the total volume of the sample and the increase of 3D area created by pores/openings due to damage. Four tresses each per condition were analyzed. Results

Figure 8 shows 3D representative images of samples 16 (untreated), 17 (bleached control), and 18 (Composition 5). The top row shows the entire hair, whereas the bottom row shows the hair cut in half. It was found that the bleaching procedure induced a lightening of the hair tresses and a rough-touch effect. Indeed, the grey color represents a healthy hair and in contrary, the black color reflects damages. The hair fibers of the bleached control were reduced in diameter and more damaged areas were observed in comparison to samples treated with Composition 5.

Figure 9 shows hair porosity quantification for 3D volume fraction and area increase after bleaching, comparing samples 17 (bleached control) and 18 (Composition 5). The results are expressed in percentage of the untreated control (sample 16, set to 0%).

The results align with the findings from the 3D representative images: The quantification of the damages in sample 18 demonstrated a lower degree of damages in comparison with the bleached control sample 17. The 3D volume fraction of the damage is reduced by 56% and the 3D area by 63% (p<0.1 ).

These results confirmed that a single application of the hair care composition of the present invention is enough to restore hair damages and to strongly reduce the hair porosity caused by bleaching.

Example 5: Flair Musicalitv (ex vivo) Overview

18 blond hair tresses (human natural blond hair lock) were tested.

Two groups of nine human natural hair locks were exposed to a device programmed to operate blow drying and repeated brushing in order to mimic 1800 brush strokes. After the hair damage, the hair locks were washed with neutral shampoo and treated with a leave-on placebo lotion (sample 20) or with Composition 5 (sample 21). The treatment was repeated six times. Before and after the treatments, the hair locks were combed ten times and the sound during the combing was caught by a specific probe. The mean amplitude (dB) of specific frequency (800 to lOOOFIz) was measured. Composition 5 was the same as described in example 4 above.

Results

The Results are shown in Figure 10.

The mean amplitude after damage was significantly increased in both groups by about +30% (sample 19).

After the treatment with the placebo leave-on lotion (sample 20) and Composition 6 (sample 21), respectively, the evolution of the mean amplitude was decreased in each group by 25% and 49%, respectively. Thus, the decrease after treatment with the composition of the present invention was significantly higher than after treatment with the placebo (p<0.05). The present example demonstrates the repair effect of the composition of the present invention after damage caused by 1800 brush strokes.