LOW MOLECULAR WEIGHT KERATIN HYDROLYSATES

Abstract

The subject of the application is keratin hydrolyzates which are characterized by a minimum amount of protein fragments having molecular weights of less than 1000 Da, enzymatic methods for the preparation thereof and also the use thereof in hair treatment compositions and also in cleansing compositions for skin and hair.

Claims

1. A keratin hydrolyzate comprising protein fragments, wherein at least 30% of the protein fragments present have a molecular weight of less than 1000 Da.

2. The keratin hydrolyzate according to claim 1, wherein at least 35% of the protein fragments present have a molecular weight of less than 1000 Da.

3. The keratin hydrolyzate according to claim 1, wherein at least 50% of the protein fragments present having a molecular weight of less than 1000 Da have a molecular weight of greater than 100 Da.

4. The keratin hydrolyzate according to claim 1, wherein at least 15% of the protein fragments have a molecular weight of less than 500 Da.

5. The keratin hydrolyzate according to claim 4, wherein at least 50% of the protein fragments present having a molecular weight of less than 500 Da have a molecular weight of greater than 100 Da.

6. A method for preparing a keratin hydrolyzate according to claim 1, wherein firstly a) keratin is oxidatively pretreated and b) enzymatically hydrolyzed in the presence of at least one endopeptidase, wherein subsequently c) it is enzymatically hydrolyzed once again in the presence of at least one exopeptidase.

7. The method for preparing the keratin hydrolyzates according to claim 6, wherein b) is enzymatically hydrolyzed in the presence of endopeptidases selected from Bacillus strains.

8. The method for preparing the keratin hydrolyzate according to claim 6, wherein c) is enzymatically hydrolyzed in the presence of exopeptidases obtainable from Aspergilllus oryzae.

9. (canceled)

10. A conditioner in a hair treatment composition, comprising the keratin hydrolyzate of claim 1.

11. (canceled)

12. A cosmetic composition comprising the keratin hydrolyzate according to claim 1, wherein the composition comprises the keratin hydrolyzate in an amount of 0.002 to 0.4 wt % based on the active substance content.

13. A cosmetic composition conditioning hair treatment, comprising the keratin hydrolyzate according to claim 1 and additionally comprising cationic polymers or cationic or pseudo-cationic surfactants.

14. A cosmetic composition for cleansing skin and/or hair, comprising the keratin hydrolyzate according to claim 1 and additionally comprising anionic surfactants and amphoteric or zwitterionic surfactants.

Description

EXAMPLES

A) Preparation Examples of the Keratin Hydrolyzates

Inventive Example 1: Low Molecular Weight Keratin Hydrolyzate

[0171] 100 g of water were initially charged, and adjusted with a base to pH 10. Subsequently, 10 g of keratin wool were stirred in and, by addition of 5 g of a 30% strength hydrogen peroxide (H.sub.2O.sub.2) solution, the oxidative pretreatment of the wool was started (duration: ˜10 hours). Thereafter, the pH was adjusted to pH 8.5 and firstly 0.15 g of protease (from Bacillus licheniformis; e.g. Alcalase® from Novozymes) was added. After complete hydrolysis with this enzyme, the pH was lowered to pH 7 and subsequently 0.15 g of exopeptidase (from Aspergillus oryzae; e.g. Flavourzyme® from Novozymes) was added, and likewise again complete hydrolysis was carried out. Subsequently, the remaining water-insoluble wool residues were separated off using filtration and the resulting product was adjusted to pH 4.2 for stabilization.

[0172] The keratin hydrolyzate was formulated as a 20 wt % strength aqueous solution by concentration.

Comparative Example: Keratin Hydrolyzate (not According to the Invention)

[0173] 100 g of water were initially charged, and adjusted with a base to ˜pH 9.4. Subsequently, 3.75 g of keratin wool were stirred in and, by addition of 1.9 g of a 30% strength hydrogen peroxide (H.sub.2O.sub.2) solution, the oxidative pretreatment of the wool was started (duration: ˜10 hours). Subsequently, 0.10 g of a protease (e.g. from Bacillus licheniformis; Alcalase from Novozymes), was added. After complete hydrolysis, the remaining water-insoluble wool residues were separated off using filtration and the resulting product was adjusted to pH 4.2 for stabilization. The keratin hydrolyzate was formulated as a 20 wt % strength aqueous solution by concentration.

[0174] Molecular Weight of the Keratin Hydrolyzates:

[0175] The molecular weight and also the distribution of the molar masses were determined using size exclusion chromatography (SEC), which is liquid chromatography in which the molecules of dissolved substances are separated based on their size. SEC is a customary method for proteins (see also the Wikipedia article relating to gel permeation chromatography, dated 06.17.2016). The Superdex Peptide chromatography column from GE Healthcare Life Sciences was chosen for high-resolution separation of proteins and peptides, which is particularly well-suited to determining protein fragments having molecular weights in the range of 100 to 7000.

TABLE-US-00001 Comparative example Example 1 Average molecular ~3600 Da ~2000 Da weight Proportion <500 Da <15% ~28% Proportion <1000 Da ~25% ~38% Proportion <3500 Da ~55% ~70% Proportion >3500 Da ~45% ~30% Proportion >20 000 Da  ~1% <0.01%  

[0176] The percentage figures relate to the total number of protein fragments, i.e. for example 1, for example, this means that 38% of all protein fragments have a molecular weight of less than 1000 Da.

B) Use Examples

[0177] B1) Tensile Strength Method for Analyzing the Influence of the Keratin Hydrolyzates on the Strength and Elasticity of the Hair

[0178] The tensile strength method can be used to determine the mechanical stability of the hair, especially the cortex region. To this end, the force required to pull an individual hair lengthwise is measured. The measured forces are subsequently standardized to the cross section of the hair, and this gives what is referred to as a stress-strain graph, by means of which the properties of the hair can be determined.

[0179] A fully automated DiaStron MTT680 from Diastron, UK, with a laser measuring device was used for 4-point cross-sectional measurement of the hair and a measuring device was used for the force measurement.

[0180] In each case, 3 cm long individual hairs that had been clamped in metal plates were tested. After determining the cross section of the hair, the hair clamped in metal plates was covered with demineralized water for 60 minutes prior to the measurement. The hair tresses also remained in the water during the measurement.

[0181] A 5% strength keratin hydrolyzate solution according to example 1, or comparative example in water, were tested.

[0182] Statistical significance (p=0.05), calculated according to Tukey's HSD test.

[0183] Results:

TABLE-US-00002 Comparative example Example 1 Tensile strength vs. +5.8% +18.7% placebo Elasticity vs. placebo −1.47 .Math. 10.sup.7 N/mm.sup.2 5.23 .Math. 10.sup.7 N/mm.sup.2

[0184] The hair treated with the inventive keratin hydrolyzate demonstrates significantly better tensile strength and elasticity than the untreated hair (placebo). However, it also demonstrates significantly better results than hair treated with the comparative example, for example 3 times better tensile strength and better elasticity, which indicates strengthening of the hair from the inside out.

[0185] B2) Method for Determining Hair Breakage (Hair Breakage Test):

[0186] The resistance of the hair to mechanical stress was determined by what is referred to as the “hair breakage test”. To this end, the hair tresses were treated with a conditioner comprising keratin hydrolyzate (composition, see below). After leaving to act for 3 minutes, rinsing was carried out for 1 minute and the process was repeated once. Subsequently, the hair tresses were placed on a grid and dried for 1 hour with a blowdryer. The hair tresses were then combed 50 000 times with a combing machine. The broken hair fragments were weighed and related to the weight of the hair tresses.

[0187] Statistical significance (p=0.05), calculated according to Tukey's HSD test.

[0188] Composition of the tested conditioner: the values are in weight %.

TABLE-US-00003 Comparative Inventive INCI Placebo example example Lanette ® O Cetyl Stearyl Alcohol 4.5 4.5 4.5 Myritol ® 312 Caprylic/Capric Triglyceride 2 2 2 Dehyquart ® A CA Cetyl Trimethyl Ammonium 2 2 2 Chloride Dehyquart ® F 75 Distearoylethyl Hydroxyethylmonium 1.4 1.4 1.4 Methosulfate (and) Cetearyl Alcohol Comparative 1 example 1 Example 1 1 Sodium benzoate 0.3 0.3 0.3 Perfume 0.3 0.3 0.3 water 89.5 88.5 88.5

[0189] Results:

TABLE-US-00004 Comparative example Example 1 Hair breakage vs. No improvement 50% less hair breakage placebo

[0190] The hair treated with the inventive keratin hydrolyzate demonstrates significantly less hair breakage than the hair without keratin hydrolyzate in the tested conditioner (placebo) and than the hair tresses treated with the conditioner comprising the comparative example. The result shows the strengthening effect of the inventive keratin hydrolyzates for the interior of the hair.

[0191] B3) Conditioner with Protein Hydrolyzate

[0192] Conditioners with the following composition were prepared (values in wt %):

TABLE-US-00005 Conditioner without Conditioner keratin according hydrolyzate Conditioner to the INCI (placebo) comparative invention Cosmedia ® Triple C Polyquaternium 37(and) 0.40 0.40 0.40 Dicaprylylcarbonate(and) Lauryl Glucoside Dehyquart ® F 75/ Distearoylethyl Hydroxyethylmonium 2.50 2.50 2.50 Conditioning Methosulfate (and) Cetearyl Alcohol Lanette ® O Cetyl Stearyl alcohol 3.50 3.50 3.50 Cutina ® HVG Hydrogenated Vegetable 1.00 1.00 1.00 Glycerides Cetiol ® C 5 Coco Caprylate 2.00 2.00 2.00 Cetiol ® SB 45 Butyrospermum Parkii 0.70 0.70 0.70 Butter Lipofructyl ® Argan Argania Spinosa Kernel 0.20 0.20 0.20 LS 9779 Oil water 85.90 83.90 83.90 Dehyquart ® A-CA Cetyl Trimethyl Ammonium 3.00 3.00 3.00 Chloride Comparative 0 2.00 example Example 1 0 2.00 Perfume 0.30 0.30 0.30 Sodium benzoate 0.50 0.50 0.50 Citric acid q.s. q.s. q.s.

[0193] The conditioners were subject to a sensory test. As standard, 11 testers take part in the sensory test, one after the other, in a climate-controlled room (T=22° C., relative air humidity=40%), assessing hair tresses treated with conditioners in pairwise comparisons of conditioners with comparative keratin hydrolyzate to placebo conditioners and conditioners with inventive keratin hydrolyzate to placebo, in the wet and in the dry state. The testers had previously been intensively trained in the attributes to be assessed, such as combability of the hair tresses, shine, and volume. For statistical analysis, a calculation adapted to the experimental protocol, the Wilcoxon test, was used, in order to determine significances in the profiles for the products.

[0194] After 3 minutes exposure time, 10 out of 11 testers assessed the caring character of the conditioner with the inventive keratin hydrolyzate as better than that of the placebo conditioner. In addition, well over half of the testers assessed the volume of the hair locks under investigation as fuller, and the conditioner with the inventive keratin hydrolyzate was better accepted by the testers. After 15 minutes exposure time, the testers assessed the volume of the hair tresses that had been treated with the conditioner with the inventive protein hydrolyzate as better than the hair treated with the placebo conditioner. The conditioner with the keratin hydrolyzate according to comparative example demonstrated no difference to the placebo conditioner in terms of volume.

[0195] B4) Improved Sensory Properties for a Leave-in Serum Conditioner for Dry Hair

TABLE-US-00006 Without keratin hydrolyzate INCI (placebo) comparison Inventive water 93.53 91.53 91.53 comparative example 2.00 Example 1 2.00 Melhydran ® LS 9876 Aqua(and)Butylene 1.00 1.00 1.00 Glycol(and)Mel Extract (and) Glycerol (and) Urea D-Panthenol ® 75 W Panthenol 0.67 0.67 0.67 Glycerol Glycerol 3.00 3.00 3.00 Sodium benzoate 0.5 0.5 0.5 Eumulgin ® CO 40 PEG-40 Hydrogenated 0.5 0.5 0.5 Castor Oil Copherol ® 1250 C Tocopheryl Acetate 0.5 0.5 0.5 Perfume 0.3 0.3 0.3 Citric acid q.s q.s q.s

[0196] The sensory assessment was carried out analogously to B3). The leave-in serum conditioner with the inventive protein hyrdolyzate was assessed by significantly more than half the testers to be better than the placebo conditioner in terms of shine and also the silkiness of the hair tresses. This effect was not detected for the formulation with the keratin hydrolyzate according to the comparative example.

[0197] B5) Application in Hair Shampoos

[0198] The following formulations were prepared. The values are in wt %.

[0199] Shampoo 1

TABLE-US-00007 Ingredients INCI % Texapon ® N 70 Sodium Laureth Sulfate 14.30 Dehyton ® PK 45 cocamidopropylbetaine 5.40 Dehyquart ® Guar N Guar Hydroxypropyltrimonium 0.20 Chloride Lamesoft ® Care PEG-4 Distearyl Ether 3.00 (and) Sodium Laureth Sulfate (and) Distearyl Ether (and) Dicaprylyl Ether Dehydol ® LS 2 Deo N Laureth-2 1.00 Water, demin. Aqua 74.60 Example 1 0.50 Sodium Chloride Sodium Chloride 0.50 Sodium Benzoate Sodium Benzoate 0.50 Citric Acid Citric Acid qs

[0200] Shampoo 2

TABLE-US-00008 Ingredients INCI % Texapon ® N 70 Sodium Laureth Sulfate 14.30 Dehyton ® PK 45 cocamidopropylbetaine 5.40 Water, demin. Aqua 77.30 Example 1 0.50 Sodium Chloride Sodium Chloride 2.00 Sodium Benzoate Sodium Benzoate 0.50 Citric Acid Citric Acid qs

[0201] Shampoo 3

TABLE-US-00009 Ingredients INCI % Texapon ® N 70 Sodium Laureth Sulfate 14.30 Dehyton ® PK 45 cocamidopropylbetaine 5.40 Dehyquart ® Guar N Guar Hydroxypropyltrimonium 0.20 Chloride Water, demin. Aqua 76.70 Example 1 1.00 Sodium Chloride Sodium Chloride 1.60 Perfume Cotton Touch Perfume 0.30 Sodium Benzoate Sodium Benzoate 0.50 Citric Acid Citric Acid qs

[0202] Comb tests on dry hair demonstrated a reduction in the required combability of 20% compared to the placebo shampoo without inventive keratin hydrolyzate.

[0203] Shampoo 4 “Extra Care”

TABLE-US-00010 Ingredients INCI % Water Water 68.12 Dehyquart ® Guar N Guar Hydroxypropyltrimonium 0.20 Chloride Dehyton ® PK 45 cocamidopropylbetaine 2.70 Luviquat ® UltraCare Polyquaternium-44 1.15 Texapon ® N 70 Sodium Laureth Sulfate 12.90 Plantapon ® LC 7 Laureth-7 Citrate 1.00 Plantacare ® 818 UP Coco-Glucoside 0.40 Lamesoft ® PO 65 Coco-Glucoside, Glyceryl Oleate 5.00 Cetiol ® LDO Dicaprylyl Ether, Lauryl Alcohol 0.70 Example 1 2.00 D-Panthenol 75 W Panthenol 1.33 Euperlan ® PK 710 Benz Glycol Distearate, 3.00 Sodium Laureth Sulfate, Cocamide MEA Sodium Benzoate Sodium Benzoate 0.50 Perfume Perfume qs Magnesium Chloride Magnesium Chloride 1.00 Hexahydrate Citric Acid (50% solution) Citric Acid qs Sodium Chloride Sodium Chloride qs

[0204] Shampoo 5 Thickening Shampoo

TABLE-US-00011 Ingredients INCI % Texapon ® N 70 Sodium Laureth Sulfate 14.30 Dehyton ® PK 45 cocamidopropylbetaine 5.40 Plantapon ® LC 7 Laureth-7 Citrate 2.00 Gluadin ® WQ PP Laurdimonium Hydroxypropyl 0.50 Hydrolyzed Wheat Protein Example 1 2.00 D-Panthenol 75 W Panthenol 1.33 Euperlan ® PK 710 Benz Glycol Distearate, 3.00 Sodium Laureth Sulfate, Cocamide MEA Perfume Perfume qs Water Water 68.97 Sodium Benzoate Sodium Benzoate 0.50 Magnesium Chloride Magnesium Chloride 1.00 Hexahydrate Dehydol LS 2 Deo N Laureth-2 1.00 Citric Acid (50% solution) Citric Acid qs Sodium Chloride Sodium Chloride qs

[0205] B6) Conditioner as Gel

[0206] Conditioning Gel

TABLE-US-00012 Ingredients INCI % Water, demin. Aqua 80.90 Example 1 1.50 Ethanol Alcohol 15.00 Dehyquart ® Guar HP Guar Hydroxypropyltrimonium 1.50 Chloride Citric Acid Citric Acid 0.30 Luviquat ® Supreme AT 1 Polyquaternium-68 2.00 Perfume Cotton Touch Perfume 0.30

[0207] B7) Styling Mousse

TABLE-US-00013 Ingredients INCI % Water, demin. Aqua 82.70 Example 1 2.00 Luviquat ® Supreme AT 1 Polyquaternium-68 7.50 Luviquat ® PQ 11 AT 1 Polyquaternium-11 7.50 Cremophor ® A 25 Ceteareth-25 0.50 Perfume Cotton Touch Perfume 0.10 Sodium Benzoate Sodium Benzoate 0.30 Citric Acid (20% solution) Citric Acid 1.40

[0208] B8) Conditioner for Natural Cosmetics

TABLE-US-00014 Ingredients INCI % Plantaquat ® NC Cetearyl Alcohol (and) 9.0 Lecithin (and) Sodium Cetearyl Sulfate (and) Olus Oil [EU] Lanette ® O Cetearyl Alcohol 2.0 Cetiol ® C5 Coco-Caprylate 2.0 Cegesoft ® SB Butyspermum Parkii Butter 2.0 Example 1 2.0 Sodium Benzoate Sodium Benzoate 0.5 Perfume Perfume q.s. Water Water ad 100

[0209] B9) Hair and Body Wash

[0210] Hair and Body Wash—Cleansing Composition 1 for Natural Cosmetics

TABLE-US-00015 Ingredients INCI % Water, demin. Water 68.20 Sulfopon ® 1216 G Sodium Coco-Sulfate 8.00 Plantacare ® 818 UP Coco-Glucoside 13.30 Euperlan ® GREEN Lauryl Glucoside 2.00 (and) Stearyl Citrate Lamesoft ® PO 65 Coco-Glucoside 2.00 (and) Glyceryl Oleate Example 1 0.50 Sodium Benzoate Sodium Benzoate 0.50 Perfume Perfume q.s. Apple juice 100%, Pyrus Malus (Apple) Juice 3.00 direct juice Citric Acid (50%) Citric Acid 1.20 Sodium Chloride Sodium Chloride 1.30

[0211] Hair and Body Wash—Cleansing composition 2 for natural cosmetics

TABLE-US-00016 Ingredients INCI % Water, demin. Water 70.10 Sulfopon ® 1216 G Sodium Coco-Sulfate 8.00 Plantacare ® 818 UP Coco-Glucoside 13.30 Lamesoft ® PO 65 Coco-Glucoside 2.00 (and) Glyceryl Oleate Example 1 0.50 Sodium Benzoate Sodium Benzoate 0.50 Perfume Perfume q.s. Apple juice 100%, Pyrus Malus (Apple) Juice 3.00 direct juice Citric Acid (50%) Citric Acid 1.20 Sodium Chloride Sodium Chloride 1.30