Indigo-based dye containing fungal or bacterial beta-glucosidase

Abstract

The invention relates to an agent, a kit and a method for the non-oxidative dyeing of keratin-containing fibers, in particular human hair, using an indicane-containing component which, depending on its origin, was pretreated at a temperature in the range of 60°C to 200°C, and a beta-glucosidase from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum.

Description

[0001] Indigo-based dye with fungal or bacterial beta-glucosidase

[0002] The invention relates to agents, kits and methods for coloring keratin-containing fibers, in particular human hair, using an indican-containing component which, depending on the origin, has been pretreated at a temperature in the range of 60 °C to 200 °C, and a component which contains at least one beta-glucosidase (EC 3.2.1.21).

[0003] The desire to change one's hair color is a major need for many consumers. To satisfy this need, the cosmetics industry offers a diverse range of products. Hair dyes that achieve particularly long-lasting color with high coverage are usually oxidation dyes. These use oxidizing agents that can damage the hair structure. Certain cationic direct azo dyes are also capable of achieving hair color changes with excellent color fastness properties. However, these azo dyes are synthetic dyes.

[0004] A growing number of consumers are demanding hair dyes and hair coloring processes based on natural dyes, even though these products and processes are often inferior to the aforementioned products and processes in terms of authenticity, coverage and color variety.

[0005] In addition to dyeing with henna, which is obtained from the plant Lawsonia inermis, dyeing hair with plants from which indigo can be produced has also been known for a long time.

[0006] State of the art

[0007] The production of storage-stable cosmetic products containing natural plant material is often difficult. The plant material contains enzymes that can react with other ingredients of the plant material or the cosmetic, even during storage. Therefore, there has been no lack of efforts in the prior art to increase the storage stability of cosmetics containing plant material. When coloring with indigo-producing plant materials, it is often observed that the color changes constantly over a period of up to two weeks after application of the colorant, and the final color only persists after this period. At a concentration of 2% by weight of indigo-producing plant powder in water, the color on the hair changes in the first few days after coloring from an initial turquoise-blue tone to a redder and darker shade, creating a purple color impression.Therefore, there has been no shortage of efforts in the prior art to improve the coloring of keratin fibers, especially human hair, with indigo. According to the invention, "improvement" means that the formation of a reddish and a purple shade is suppressed as completely as possible, or at least to a high degree. Furthermore, "improvement" means that the final blue shade is achieved as quickly as possible after the end of the coloring process.

[0008] US20040055096A1 discloses a dyeing agent for the production of which powdered leaves of an indigo-producing plant, such as Polygonum tinctorium, Strobilanthes cusia, Indigofera tinctoria or Isatis indigotica, whose natural enzyme content has been deactivated by heat treatment, are mixed shortly before use with a beta-glucosidase-containing component (e.g. fresh leaves of an indigo-producing plant which have not been heat-treated, or the pressed juice from these non-heat-treated leaves, optionally in freeze-dried form, or extracts of various mushrooms, such as enokitake (Flammulina velutipes) and shiitake (Lentinus Edodes), or extracts of corn leaves, apricot kernels or almonds). By separating the indigo precursor indican, which is present in the plants in the form of 3-indioxyl-ß-D-glucopyranoside, from the plant's own enzyme beta-glucosidase, an improved storage stability of the natural dye is achieved.US20040055096A1 also deals with suppressing the red hue and achieves this through the addition of saponins or saponin-containing plants, particularly in powder form. The addition of saponins in the form of saponin-containing plant parts is particularly disadvantageous for the commercial production of indigo-based dyes, as these are natural products with fluctuating saponin content, making it difficult to produce products of consistent, standardized quality. Furthermore, saponins cause the dye to foam, which impairs its ability to absorb dyes and is therefore undesirable.

[0009] In PCT application W02014104301A1 a similar approach to improved storage stability is disclosed as in US20040055096A1, namely the combination of two different indigo powders, whereby the natural enzyme content in the first leaf powder component was deactivated by heat treatment, while the second leaf powder component was dried at temperatures below 50 °C and thus contained active enzyme.

[0010] From WO2016007521A2, transgenic organisms, preferably E. coli and Agrobacterium tumefaciens, are known which, through their modified DNA, produce biotechnologically increased amounts of indole derivatives, including indican.

[0011] WO2015082482A1 discloses a method for coloring hair with powdered plant parts from plants that produce indigo. After the indigo dyeing, which can be performed in the presence of hydrogen peroxide, the hair is treated with a strongly alkaline composition. This is intended to accelerate the development of the final color and prevent color change after the actual dyeing process. However, the alkaline pH and the oxidizing agent damage the hair.

[0012] An object of the present invention was to provide agents, kits and methods for the non-oxidative coloring of keratin-containing fibers, in particular human hair, using plants that contain indican and produce indigo, with which the final color result of the indigo dyeing is obtained as soon as possible after completion of the actual dyeing process.

[0013] A further object of the present invention was to provide agents, kits and methods for the non-oxidative coloring of keratin-containing fibers, in particular human hair, using plants containing indican and producing indigo, with which the formation of a red tone and a purple tone is suppressed as completely as possible or at least to a high degree.

[0014] A further object of the present invention was to provide saponin-free or low-saponin agents, kits and methods for the non-oxidative coloring of keratin-containing fibers, in particular human hair, using plants containing indican and producing indigo, with which the formation of a red tone and a purple tone is suppressed as completely as possible or at least to a high degree.

[0015] A further object of the present invention was to provide agents, kits and methods for the non-oxidative coloring of keratin-containing fibers, in particular human hair, using plants containing indican and producing indigo, with improved storage stability.

[0016] Surprisingly, it has been found that the dyeing agents, dyeing processes, and dyeing kits described in the patent claims, using parts or extracts of plants containing indican and producing indigo, or using synthetically obtained indican, can produce dyeings that achieve the final indigo color result as quickly as possible after completion of the actual dyeing process. The final indigo color result achieved according to the invention has only a low red content, even without the addition of saponins or saponin-containing plant parts. The dyeing agents and dyeing kits according to the invention also exhibit high storage stability.

[0017] A first subject of the present invention is therefore an agent for the non-oxidative coloring of keratinic fibers, in particular human hair, comprising i) at least one indican-containing component, furthermore ii) at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum and iii) water iv) characterized in that saponins are present in a total amount of zero to less than 0.01 wt.%, preferably from zero to 0.005 wt.%, particularly preferably from zero to 0.001 wt.%, in each case based on the weight of the ready-to-use coloring agent, wherein the indican-containing component has been pretreated at a temperature in the range of 60°C to 200°C, provided that it comprises plant parts of an indigo-producing plant or an extract from plant parts an indigo-producing plant.

[0018] The indigo precursor indican occurs naturally in plants. Indican is a glucoside of indoxyl and is found in some plants known as indigo plants, such as / satis tinctoria (woad) or Indigofera tinctoria. Synonyms for indican are 3-indoxyl-ß-D-glucopyranoside, indoxyl-ß-D-glucoside, 3-(ß-D-glucosido)indole, or plant indican.

[0019] It is also possible to use synthetic indican or biotechnologically obtained indican according to the invention. In this case, pretreatment of the indican-containing component at a temperature in the range of 60°C to 200°C can be omitted. The indican used according to the invention has preferably been obtained by a biotechnological production process in which, in particular, bacteria, yeasts or fungi form the indican. Corresponding processes for producing indican are known in the art, for example from WO 84 / 01787 A1 and W02016007521A2. The indican used according to the invention can, for example, be extracted from animals, fungi, algae or bacteria. The indican used according to the invention can also be obtained from genetically modified fungi, algae or bacteria. Petrochemically or oleochemically synthesized indican is also encompassed within the scope of the present invention.

[0020] Before indican is converted into the dye indigo, it must be cleaved into indoxyl and the glucoside residue. This cleavage preferably occurs enzymatically. Cleavage should occur in situ, i.e., shortly before application to the keratin fibers to be dyed.

[0021] Accordingly, the indican-containing component, which, if it comprises plant parts of an indigo-producing plant or is an extract from plant parts of an indigo-producing plant, has been pretreated at a temperature in the range of 60°C to 200°C, and the enzyme mixture ii) according to the invention should be physically separated from each other until use. When using biotechnologically or petrochemically or oleochemically synthesized indican, no natural enzymes are present; therefore, thermal pretreatment can be dispensed with when using them.

[0022] A further subject of the present invention is therefore a kit for the non-oxidative coloring of keratinic fibers, in particular human hair, with natural dyes, which comprises the following compartments which are physically separate from one another: i) a composition (Ind) which contains at least one indican-containing component which, if it comprises plant parts of an indigo-producing plant or is an extract from plant parts of an indigo-producing plant, has been pretreated at a temperature in the range from 60°C to 200°C, and ii) an aqueous composition (E) which contains at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum, iii) wherein saponins are present in a total amount of from zero to less than 0.01% by weight, preferably from zero to 0.005% by weight, particularly preferably from zero to 0.001 wt.-%, each based on the weight of the ready-to-use dye.

[0023] "Non-oxidative" is understood according to the invention to mean that no oxidizing agents other than atmospheric oxygen are used in the agents, kits, and methods according to the invention. In particular, the oxidizing agents hydrogen peroxide, persulfates, perbromates, percarbonates, perborates, and percarbamides are excluded. Atmospheric oxygen does not constitute an oxidizing agent in the context of the invention.

[0024] Preferred agents and kits for non-oxidative dyeing according to the invention are characterized in that the indican-containing component i) is selected from comminuted parts of an indigo-producing plant and from indican-containing extracts of an indigo-producing plant and mixtures of these indican-containing components i).

[0025] Particularly preferred agents and kits for non-oxidative dyeing according to the invention are characterized in that the indican-containing component i) is selected from indican-containing extracts of an indigo-producing plant.

[0026] Further agents and kits for non-oxidative coloring preferred according to the invention are characterized in that the extraction agent used to obtain the at least one indican-containing extract from an indigo-producing plant is selected from water, furthermore C1-C4 alkanols and C2-C4 polyols, in particular ethanol, isopropanol, n-propanol, ethylene glycol, 1,2-propanediol, glycerol and 1,3-butylene glycol, and mixtures of these extraction agents, preferably selected from water, ethanol, mixtures of water and at least one C1-C4 alkanol, mixtures of water and at least one C2-C4 polyol and water / ethanol mixtures, particularly preferably selected from water, ethanol and water / ethanol mixtures.

[0027] Preferred agents and kits for non-oxidative dyeing according to the invention are characterized in that the indigo-producing plant(s) is / are selected from at least one species of the following genera:

[0028] - Indigofera, in particular Indigofera tinctoria, Indigo suffruticosa, Indigofera articulata, Indigofera arrecta, Indigofera heterantha “Gerardiana”, Indigofera argentea or Indigofera longiracemosa;

[0029] - Isatis, especially Isatis tinctoria (woad);

[0030] - Persicaria, especially Persicaria tinctoria (dyer's knotweed);

[0031] - Wrightia, especially Wrightia tinctoria;

[0032] - Calanthe, especially Calanthe veratrifolia; and

[0033] - Baphicacanthus cusia, synonym Strobilanthes cusia.

[0034] Particularly preferred plants producing indigo according to the invention are selected from the genus Indigofera and in particular from Indigofera tinctoria.

[0035] The plants of the aforementioned genera and species contain no or only small amounts of indigo. Instead, the plants contain glycosidically bound indican, which, after enzymatic cleavage of the glycoside residue, represents a precursor to indigo. The parts of the aforementioned plant genera and species that are rich in indican and also contain indigo are the leaves of these plants. To obtain the indican-containing component i) according to the invention, the plant leaves of the aforementioned plant genera and species are dried and ground and used as plant powder.

[0036] Plant powders preferred according to the invention have a particle size of less than 500 pm, particularly preferably in the range of 120 pm - 200 pm, extremely preferably in the range of 150 pm - 180 pm. Further plant powders preferred according to the invention have a bulk density in the range of 0.20 - 0.60 g / cm 3, particularly preferably in the range of 0.25 - 0.40 g / cm 3 Other plant powders preferred according to the invention have, based on their weight, a moisture content of less than 10% by weight, preferably 0.1-6.0% by weight, particularly preferably 0.2-3.0% by weight. The moisture content is determined after 3 hours of drying at 105°C.

[0037] According to the invention, it is preferred that the powder of the indigo-producing plant, based on its weight, consists of at least 50 wt.%, preferably at least 80 wt.%, particularly preferably more than 80-100 wt.%, of the leaves of the plant. Further preferred embodiments according to the invention are characterized in that the powder of the indigo-producing plant has a particle size of less than 500 pm, particularly preferably in the range of 120 pm-200 pm, extremely preferably in the range of 150 pm-180 pm, and also a bulk density in the range of 0.20-0.60 g / cm 3, particularly preferably in the range of 0.25 - 0.40 g / cm 3 and, based on its weight, has a moisture content of less than 10 wt.%, preferably 0.1-6.0 wt.%, particularly preferably 0.2-3.0 wt.%. Further preferred agents and kits according to the invention are characterized in that the powder of the indigo-producing plant contains indican in an amount of 2-5 wt.%, preferably 2.5-4.5 wt.%, particularly preferably 3-4.1 wt.%, based on the weight of the powder, wherein the indican content is converted to pure indican.

[0038] Deactivation of the natural enzyme content in the indican-containing component i), provided that this component i) comprises parts of an indigo-producing plant or is an extract from parts of an indigo-producing plant

[0039] Further agents and kits for non-oxidative dyeing preferred according to the invention are characterized in that the pretreatment of the at least one indican-containing component i), provided that this comprises plant parts of an indigo-producing plant or is an extract from plant parts of an indigo-producing plant, is carried out at a temperature in the range from 60 °C to 200 °C by heating, in particular by heating in a solvent, further by treatment with steam or by dry heating, preferably for a period of 1 to 120 minutes, particularly preferably 2 to 60 minutes, extraordinarily preferably 5 to 10 minutes.

[0040] Accordingly, the indican-containing component, which, if it comprises plant parts of an indigo-producing plant or is an extract from plant parts of an indigo-producing plant, has been pretreated at a temperature in the range of 60°C to 200°C, and the enzyme mixture ii) according to the invention should be physically separated from each other until use. When using biotechnologically or petrochemically or oleochemically synthesized indican, no natural enzymes are present; therefore, thermal pretreatment can be dispensed with when using them.

[0041] Option 1 : Indican-containing component i) is an extract

[0042] Particularly preferred agents and kits for non-oxidative dyeing according to the invention are characterized in that the indican-containing component i) is selected from at least one indican-containing extract of an indigo-producing plant.

[0043] Agents and kits for non-oxidative coloring which are extraordinarily preferred according to the invention are characterized in that the indican-containing component i) is selected from at least one indican-containing extract of an indigo-producing plant, wherein the extraction agent used to obtain the at least one indican-containing extract from an indigo-producing plant is selected from water, furthermore C1-C4 alkanols and C2-C4 polyols, in particular ethanol, isopropanol, n-propanol, ethylene glycol, 1,2-propanediol, glycerol and 1,3-butylene glycol, and mixtures of these extraction agents, preferably selected from water, ethanol, mixtures of water and at least one C1-C4 alkanol, mixtures of water and at least one C2-C4 polyol and water / ethanol mixtures, particularly preferably selected from water, ethanol and water / ethanol mixtures.

[0044] The indican can be extracted using these aforementioned polar solvents. Since the extraction can be easily carried out at a temperature in the range of 60°C to 200°C by heating the solvent, this preferred procedure not only achieves the isolation of the indican but also the simultaneous deactivation of the plant's own enzymes. The deactivation of the plant's own enzymes is desirable according to the invention, because it was surprisingly discovered that the resulting coloration can be significantly improved if the plant's own enzymes, which as a natural product are subject to a multitude of qualitative and quantitative fluctuations in composition, are replaced by at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis, or Bifidobacterium bifidum.

[0045] In a further preferred embodiment of the invention, the extracted indican is not separated from the solvent after completion of the extraction procedure and separation of the solid plant parts, for example by filtration or decantation, but is used directly as indican-containing component i), optionally after concentration by evaporation of the solvent or after dilution, wherein further cosmetic adjuvants, such as preservatives, thickeners, perfumes, pH regulators, or hair conditioners, can optionally be added. In another, likewise preferred embodiment of the invention, the extracted indican is also separated from the solvent after completion of the extraction procedure and separation of the solid plant parts, for example by filtration or decantation, for example by freeze-drying or by distilling off the solvent, so that the indican-containing component i) is obtained in powder form.To produce the preferred colorant according to the invention, the powdered indican-containing component i) can be dissolved in water and then mixed with the beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis, or Bifidobacterium bifidum and, if appropriate, additional water. In a further preferred embodiment, to produce the preferred colorant according to the invention, the powdered indican-containing component i) can be mixed with the beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis, or Bifidobacterium bifidum and with water.

[0046] Option 2: dried and crushed plant parts of an indiqo-producing plant as indican-containing component i)

[0047] According to the invention, dried and comminuted plant parts of an indigo-producing plant which have been pretreated at a temperature in the range from 60°C to 200°C can also be used as the indican-containing component i). The heat pretreatment of this embodiment of the invention is characterized in that the pretreatment of the at least one indican-containing component i) is carried out at a temperature in the range from 60°C to 200°C by heating, in particular by heating without solvent, further by treatment with steam or by dry heating, preferably for a period of 1 to 120 minutes, particularly preferably 2 to 60 minutes, extraordinarily preferably 5 to 10 minutes.

[0048] Further dyeing agents preferred according to the invention are characterized in that as at least one indican-containing component i) dried and comminuted parts of at least one indigo-producing plant, which have been pretreated at 60 - 200°C, are contained in a total amount of 1 - 80 wt.%, preferably 2 - 50 wt.%, particularly preferably 3 - 25 wt.%, extraordinarily preferably 4 - 15 wt.%, further extraordinarily preferably 5 - 10 wt.%, further extraordinarily preferably 6 - 7 wt.%, based on the weight of the agent.

[0049] Further colorants preferred according to the invention are characterized in that as at least one indican-containing component i) powdered leaves of Indigofera tinctoria are contained in an amount of 1-80 wt.%, preferably 2-50 wt.%, particularly preferably 3-25 wt.%, extraordinarily preferably 4-15 wt.%, further extraordinarily preferably 5-10 wt.%, further extraordinarily preferably 6-7 wt.%, based on the weight of the agent.

[0050] Preferred non-oxidative coloring agents according to the invention are characterized in that the indican-containing component i) contains, based on its weight, 0.01-6 wt.% indican. Preferred indican-containing components i) contain, based on their weight, 0.02-5 wt.%, preferably 0.05-4.5 wt.%, particularly preferably 0.1-4 wt.%, extremely preferably 0.2

[0051] - 3.9 wt.% indican. Depending on the type of indican-containing component i), different subranges from the previously mentioned broad ranges of indican content may be particularly preferred.

[0052] If the indican-containing component i) is dried and crushed plant parts of an indigo-producing plant, its indican content, in each case based on its weight, is preferably 1 - 6 wt.%, particularly preferably 2 - 5 wt.%, particularly preferably 3 - 4.5 wt.%, extremely preferably 3.5 - 4.1 wt.%.

[0053] If the indican-containing component i) is the solvent-containing extract from dried plant parts of an indigo-producing plant, its indican content, based on its weight, is preferably 0.01 - 2 wt.%, particularly preferably 0.02

[0054] - 1 wt.%, particularly preferably 0.1 - 0.5 wt.%, extraordinarily preferably 0.2 - 0.4 wt.%.

[0055] If the indican-containing component i) comprises biotechnologically, petrochemically or oleochemically synthesized indican, its content of indican, in each case based on its weight, is in a first embodiment preferably 0.01 - 6 wt.%, more preferably 0.02 - 5 wt.%, more preferably 0.05 - 4.5 wt.%, particularly preferably 0.1 - 4 wt.%, extraordinarily preferably 0.2 - 3.9 wt.%.

[0056] If the indican-containing component i) comprises biotechnologically, petrochemically or oleochemically synthesized indican, its content of indican, in each case based on its weight, is in a further embodiment preferably 30-100 wt.%, more preferably 40-98 wt.%, more preferably 50-90 wt.%, particularly preferably 60-85 wt.%, extraordinarily preferably 70-80 wt.%.

[0057] The dyes according to the invention also contain water. The water essentially serves as a solvent for the enzymes and the at least one indican. If dried and ground plant parts of an indigo-producing plant, which have previously been subjected to heat treatment, are used as the indican-containing component i), these are preferably dispersed in water to extract the indican and make it accessible for reaction to indigo.

[0058] If the indican-containing component i) is the solvent-containing extract from dried parts of an indigo-producing plant, it preferably contains water as a solvent.

[0059] If the indican-containing component i) comprises indican synthesized by biotechnological, petrochemical or oleochemical means, it preferably contains water as a solvent.

[0060] Water in the entire ready-to-use dye

[0061] Ready-to-use colorants preferred according to the invention are characterized in that they contain, in each case based on their weight, water in an amount of 19.9 - 95.0 wt.%, preferably 30.0 - 90.0 wt.%, particularly preferably 50.0 - 88.0 wt.%, extraordinarily preferably 60.0 - 85.0 wt.%.

[0062] Water in the indican-containing extract

[0063] If the indican-containing component i) is the solvent-containing extract from dried plant parts of an indigo-producing plant, it preferably contains water in an amount of 0.5 - 99.5 wt.%, more preferably 10.0 - 95.0 wt.%, particularly preferably 30.0 - 90.0 wt.%, extraordinarily preferably 50.0 - 85.0 wt.%, more extraordinarily preferably 65 - 80 wt.%, based on its weight.

[0064] Water in the indican-containing plant powder dispersion

[0065] If dried and comminuted plant parts of an indigo-producing plant which have previously been subjected to a heat treatment are used as indican-containing component i), these are preferably dispersed in water, this dispersion preferably containing water in an amount of 15.0 - 99.5 wt.%, more preferably 30.0 - 95.0 wt.%, particularly preferably 50.0 - 90.0 wt.%, extraordinarily preferably 60.0 - 85.0 wt.%, more extraordinarily preferably 70 - 80 wt.%, based on its weight.

[0066] Water in the indican-containing dry extract dispersion

[0067] If the dried extract from the plant parts of an indigo-producing plant is used as the indican-containing component i), this is preferably dispersed or dissolved in water, wherein this dispersion or solution, in each case based on its weight, preferably contains water in an amount of 15.0 - 99.5 wt.%, more preferably 30.0 - 95.0 wt.%, particularly preferably 50.0 - 90.0 wt.%, extraordinarily preferably 60.0 - 85.0 wt.%, more extraordinarily preferably 70 - 80 wt.%.

[0068] Water in the indican-containing component i) with biotechnologically, petrochemically or oleochemically synthesized indican

[0069] If the indican-containing component i) comprises indican synthesized by biotechnology, petrochemicals or oleochemicals, this is preferably dispersed or dissolved in water, wherein this dispersion or solution, in each case based on its weight, preferably contains water in an amount of 15.0 - 99.5 wt.%, more preferably 30.0 - 95.0 wt.%, particularly preferably 50.0 - 90.0 wt.%, extraordinarily preferably 60.0 - 85.0 wt.%, more extraordinarily preferably 70 - 80 wt.%.

[0070] Water in composition (E) containing at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum ii)

[0071] The enzyme preparation ii) used according to the invention, comprising at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis, or Bifidobacterium bifidum, can preferably be dispersed or dissolved in water before being mixed with the indican-containing component i) to form a colorant according to the invention. This enzyme dispersion preferably contains, in each case based on its weight, water in an amount of 15.0-99.5 wt.%, more preferably 30.0-95.0 wt.%, particularly preferably 50.0-90.0 wt.%, exceptionally preferably 60.0-80.0 wt.%, even more exceptionally preferably 64-75 wt.%.

[0072] Water as part of an optional third compartment

[0073] The water content of the colorant according to the invention can also be controlled via an optional third compartment that contains neither an indican-containing component i) nor a beta-glucosidase. Ready-to-use colorant is understood to mean the colorant obtained by mixing the obligatory components i) and ii) and, if appropriate, further optional compartments.

[0074] The coloring agent according to the invention is further characterized in that it contains at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum.

[0075] Surprisingly, it was discovered that using a beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis, or Bifidobacterium bifidum, used according to the invention, dyeings can be achieved in which the final color result of the indigo dyeing is achieved shortly after completion of the actual dyeing process and does not change over time. However, using an indican-containing component with its natural enzyme content results in a very unstable dyeing result, with a significant shift in color within two weeks of dyeing.

[0076] The beta-glucosidase (EC 3.2.1.21) contained in the enzyme preparation ii) used according to the invention originates from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum.

[0077] The term "beta-glucosidase", as used herein, refers to enzymes that catalyze the hydrolysis of 1,4-beta-D-glucoside bonds, which are present, for example, in cellulose (cellobiose).

[0078] The term "beta-glucosidase activity" is defined here as an enzyme that catalyzes the hydrolysis of 1,4-beta-D-glucoside bonds in disaccharides, e.g., cellobiose, oligosaccharides, and glucose-substituted molecules, e.g., flavonoids and anthocyanidins. Enzyme activity is measured using a standard method, e.g., as follows: beta-glucosidases release glucose from CMC (carboxymethylcellulose). Samples are incubated with a substrate (1.25 wt% CMC) under defined reaction conditions (100 mM sodium phosphate buffer pH 7.5, 40°C, 15 min). Reaction with p-hydroxybenzoic acid hydrazide (PAHBAH) in the presence of bismuth produces a yellow dye that can be determined photometrically at 410 nm. An alkaline pH value is required during the color reaction. The amount of sugar released corresponding to the color is a measure of enzyme activity (Lever, Anal. Biochem., 1972, 47 and 1977, 81).

[0079] The beta-glucosidases (EC 3.2.1.21) used according to the invention do not originate from plants, but are of bacterial or fungal origin, more specifically from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis, or Bifidobacterium bifidum. For the production of the beta-glucosidases particularly preferably used according to the invention, fungi selected from Aspergillus spp., in particular Aspergillus niger, Aspergillus niger SOI017, Aspergillus niger AS 3.4309, Aspergillus niger HDF 05, Aspergillus oryzae, Aspergillus strain SA 58, Aspergillus terreus EMOO 6-4, and mixtures of Aspergillus niger and Aspergillus oryzae, are used. For the production of other beta-glucosidases particularly preferred according to the invention, fungi selected from Trichoderma spp., in particular Trichoderma reesei, are used. Other beta-glucosidases particularly preferred according to the invention originate from Penicillium spp., in particular Penicillium simplicissimum H-11, Penicillium citrinum YS40-5, and Penicillium miczynskii. Other beta-glucosidases particularly preferably used according to the invention originate from Pseudomonas, in particular Pseudomonas ZD-8. Beta-glucosidases from Trichoderma reesei and Aspergillus niger are extremely preferred. Chemically modified or protein-engineered mutants are included.

[0080] A further particularly preferred embodiment of the invention is characterized in that the agent for non-oxidative coloring does not contain 1,4-beta-D-glucanases.

[0081] A further particularly preferred embodiment of the invention is characterized in that the agent for non-oxidative coloring contains, based on its weight, at least one beta-glucosidase (EC 3.2.1 .21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum in a total amount of 0.0001 - 1 wt.%, preferably 0.001 - 0.1 wt.%, more preferably 0.002 - 0.009 wt.%, particularly preferably 0.003 - 0.007 wt.%.

[0082] The precursor to the indigo dye is indican. Indican, which occurs naturally in plants, is a glycoside, or more precisely, a glucoside, which is cleaved into indoxyl and the glycoside residue before the indican is converted into the indigo dye. This cleavage occurs primarily enzymatically. Cleavage should occur in situ, i.e., shortly before application to the keratin fibers to be dyed.

[0083] The above-discussed agent for non-oxidative coloring of keratin fibers is therefore preferably presented as a kit comprising at least two components (Ind) and (E), which are physically separate from each other.

[0084] A further subject of the present invention is therefore a kit for the non-oxidative coloring of keratinic fibers, in particular human hair, with natural dyes, which comprises the following compartments which are physically separate from one another: i) a composition (Ind) which contains at least one indican-containing component which, if it comprises plant parts of an indigo-producing plant or is an extract from plant parts of an indigo-producing plant, has been pretreated at a temperature in the range from 60°C to 200°C, and ii) an aqueous composition (E) which contains at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum, iii) wherein saponins are present in a total amount of from zero to less than 0.01% by weight, preferably from zero to 0.005% by weight, particularly preferably from zero to 0.001 wt.-%, each based on the weight of the ready-to-use dye.

[0085] The beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis, or Bifidobacterium bifidum used according to the invention can preferably be dispersed or dissolved in water before being mixed with the indican-containing component i) to form a colorant according to the invention. This enzyme dispersion, which represents component (E) of the kit according to the invention, preferably contains, based on its weight, water in an amount of 15.0-99.5 wt.%, more preferably 30.0-95.0 wt.%, particularly preferably 50.0-90.0 wt.%, exceptionally preferably 60.0-80.0 wt.%, and even more exceptionally preferably 64-78 wt.%.

[0086] The enzyme active substance content of beta-glucosidase (EC 3.2.1 .21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum in the aqueous composition (E) is preferably, in each case based on its weight, 1 - 70 wt.%, particularly preferably 5 - 50 wt.%, further particularly preferably 10 - 35 wt.%, extraordinarily preferably 15 - 25 wt.%, further extraordinarily preferably 18 - 22 wt.%.

[0087] The colorants according to the invention are further characterized in that saponins are contained in a total amount of zero to less than 0.01 wt.%, preferably from zero to 0.005 wt.%, particularly preferably from zero to 0.001 wt.%, in each case based on the weight of the ready-to-use colorant.

[0088] With regard to further preferred embodiments of the dyeing kits according to the invention, what has been said regarding the dyeing agents according to the invention applies mutatis mutandis.

[0089] The present invention further provides a process for the non-oxidative coloring of keratinic fibers, in particular human hair, which process comprises the following process steps in the given order: a) providing a composition (Ind) which contains at least one indican-containing component which, if it comprises plant parts of an indigo-producing plant or is an extract from plant parts of an indigo-producing plant, has been pretreated at a temperature in the range from 60°C to 200°C, and providing an aqueous composition (E) which contains at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum, wherein saponins are present in a total amount of from zero to less than 0.01% by weight, preferably from zero to 0.005% by weight, particularly preferably from zero to 0.001% by weight.-%, in each case based on the weight of the ready-to-use colorant, b) mixing the compositions (Ind) and (E) with one another to produce a colorant, c) applying the colorant obtained in step b) to the keratin fibers, d) leaving to act for a time of 30 seconds to 60 minutes, preferably 5 to 45 minutes, particularly preferably 15 to 30 minutes, e) rinsing the keratin fibers with water, f) optionally drying the keratin fibers.

[0090] The ready-to-use dye obtained in step b) is applied to the keratin fibers. The ready-to-use dye obtained in step b) is preferably applied to the keratin fibers within 1 second to 30 minutes, particularly preferably within 10 seconds to 15 minutes, after completion of process step b), i.e., immediately after the preparation of the ready-to-use dye.

[0091] Surprisingly, it was found that the temporal development of the coloring result on the keratin fibers can be further accelerated if the ready-to-use coloring agent according to the invention is allowed to act under the influence of heat, for example by means of a heat lamp or a drying hood or a hair dryer.

[0092] A particularly preferred embodiment of the dyeing process according to the invention is therefore characterized in that the ready-to-use dyeing agent obtained in step b) is allowed to act under the influence of heat, preferably at a temperature of 25 - 60 °C, particularly preferably at a temperature of 30 - 50 °C, extremely preferably at a temperature of 35 - 40 °C.

[0093] Conventional dyes and dyeing processes using natural dyes often use compounds, particularly salts, of metals other than alkali metals to improve the adhesion of the natural dye to the keratin fibers. Such compounds are also referred to as "mordants." For example, US20040055096A1 discloses a pretreatment or posttreatment of keratin fibers with metals or metal salts selected from aluminum, iron, zinc, nickel, calcium, and magnesium. Other mordants include transition metals and lanthanides, as well as their salts and complexes. The content of metal ions other than alkali metal ions, which is typically present in the water used, e.g., tap water or city water, is not taken into account here. For example, tap water can contain an average of 2 mg of copper ions per liter, or approximately 0.0002 wt% copper ions.Tap water contains approximately 20–120 mg calcium ions per liter, or approximately 0.002–0.012 wt% calcium ions. Tap water contains approximately 2–25 mg magnesium ions per liter, or approximately 0.0002–0.0025 wt% magnesium ions. At such low concentrations, no perceptible mordant effect occurs. For the purposes of the present invention, the agents according to the invention are "free of mordants" if the mordant(s) is / are present at a maximum concentration that is naturally common for the ingredients used, such as water, enzymes, and plant parts.

[0094] The dyeing agents, dyeing kits and dyeing processes according to the invention can dispense with the use of such mordants.

[0095] Preferred dyeing agents and dyeing processes according to the invention are therefore characterized in that they are free from mordants.

[0096] In order to preserve the hair-protecting potential of natural dyes, preferred methods according to the invention are limited to those methods in which the keratin fibers were not treated with a mordant in a period of up to 7 days before and up to 7 days after the application of a coloring agent according to the invention.

[0097] Further preferred dyes, dye kits, and dyeing processes according to the invention are characterized by being free of hydrogen peroxide. Further preferred dyes, dye kits, and dyeing processes according to the invention are characterized by being free of mordants and hydrogen peroxide.

[0098] With regard to further preferred embodiments of the dyeing methods according to the invention, what has been said regarding the dyeing agents and dyeing kits according to the invention applies mutatis mutandis.

[0099] The keratin fibers to be colored, to which the colorant prepared in step b) by mixing the compositions (Ind) and (E) is applied in process step c), are preferably dry.

[0100] In order to preserve the hair-protecting potential of natural dyes, the claimed method is preferably limited to those methods in which the keratin fibers have not been treated with an oxidizing agent within a period of up to 7 days prior to the application of the coloring agent according to the invention.

[0101] Oxidizing agents commonly used in hair cosmetics, but which are not intended for hair treatment according to the invention, even as a pretreatment, include hydrogen peroxide, persulfates, perbromates, percarbonates, perborates, and percarbamides. The oxygen contained in ambient air does not constitute an oxidizing agent in the context of the invention.

[0102] In order to preserve the hair-protecting potential of natural dyes, preferred methods according to the invention are limited to those methods in which the keratin fibers have not been treated with a keratin-reducing compound within a period of up to 7 days prior to the application of a coloring agent according to the invention.

[0103] The keratin fibers are preferably dried after rinsing out the colorant prepared from compositions (Ind) and (E). Drying can be carried out without actively applying heat. However, drying can also be carried out with the application of heat at a temperature of 25-120°C, particularly preferably at a temperature of 30-80°C, and extremely preferably at a temperature of 35-60°C. Heat is preferably applied using a heat lamp, a drying rod, a hair dryer, a straightening iron, or a hair dryer.

[0104] A further feature of the dyeing process according to the invention is that the dyeing agent prepared from the compositions (Ind) and (E) is allowed to act on the keratin fibers after application thereto for a time of 30 seconds to 60 minutes, preferably 5 to 45 minutes, particularly preferably 20 to 35 minutes, extraordinarily preferably 25 to 30 minutes.

[0105] After the dye has been applied, the keratin fibers are rinsed with water to wash out the dye.

[0106] Optionally, the keratin fibers can be dried after this rinsing step. This can be done with an absorbent cloth, such as a towel. Towel-dried hair can also be partially or completely dried with a hairdryer or other heat source. Allowing the keratin fibers to air dry is also possible.

[0107] Further dyeing processes preferred according to the invention are characterized in that no oxidation dye precursors are used in them. Typical oxidation dye precursors are p-aminophenol, 4-amino-3-methylphenol, 4-amino-2-aminomethylphenol, 4-amino-2-(1,2-dihydroxyethyl)phenol, 4-amino-2-(diethylaminomethyl)phenol, 2-(2,5-diaminophenyl)ethanol, 2-(1,2-dihydroxyethyl)-p-phenylenediamine, N,N-bis-(2-hydroxyethyl)-p-phenylenediamine, N-(4-amino-3-methylphenyl)-N-[3-(1H-imidazol-1-yl)propyl]amine, N,N'-bis-(2-hydroxyethyl)-N,N'-bis-(4-aminophenyl)-1,3-diamino-propan-2-ol, Bis-(2-hydroxy-5-aminophenyl)methane, 1,3-bis-(2,5-diaminophen- oxy)propan-2-ol, N,N'-bis-(4-aminophenyl)-1,4-diazacycloheptane, 1,10-bis-(2,5-diaminophenyl)-1,4,7,10-tetraoxadecane, 2,4,5,6-tetraaminopyrimidine, 4-Hydroxy-2,5,6-triaminopyrimidine, 2-hydroxy-4,5,6-triaminopyrimidine, 2,3-diamino-6,7-dihydro-1H,5H-pyrazolo-[1,2-a]-pyrazol-1-one, 3-amino-phenol, 5-amino-2-methylphenol,3-Amino-2-chlor-6-methylphenol, 2-Hydroxy-4-aminophenoxy- ethanol, 5-Amino-4-chlor-2-methylphenol, 5-(2-Hydroxyethyl)-amino-2-methylphenol, 2,4-Dichlor-3- aminophenol, 2-Aminophenol, 3-Phenylendiamin, 2-(2,4-Diaminophenoxy)ethanol, 1 ,3-Bis(2,4-di- aminophenoxy)propan, 1-Methoxy-2-amino-4-(2-hydroxyethylamino)benzol, 1 ,3-Bis(2,4-diamino- phenyl)propan, 2, 6-Bis(2'-hydroxyethylamino)-1 -methylbenzol, 2-({3-[(2-Hydroxyethyl)amino]-4- methoxy-5-methylphenyl}amino)ethanol, 2-({3-[(2-Hydroxyethyl)amino]-2-methoxy-5-methyl- phenyl}amino)ethanol, 2-({3-[(2-Hydroxyethyl)amino]-4,5-dimethylphenyl}amino)ethanol, 2-[3-Mor- pholin-4-ylphenyl)amino]ethanol, 3-Amino-4-(2-methoxyethoxy)-5-methylphenylamin, 1-Amino-3- bis-(2-hydroxyethyl)aminobenzol, Resorcin, 2-Methylresorcin, 4-Chlorresorcin, 1 ,2,4-Trihydroxy- benzol, 2-Amino-3-hydroxypyridin, 3-Amino-2-methylamino-6-methoxypyridin, 2,6-Dihydroxy-3,4-di- methylpyridin, 3,5-Diamino-2,6-dimethoxypyridin, 1-Phenyl-3-methylpyrazol-5-on, 1-Naphthol,1,5-dihydroxynaphthalene, 2,7-dihydroxynaphthalene, 1,7-dihydroxynaphthalene, 1,8-dihydroxynaphthalene, 2-(1,3-benzodioxole-5-ylamino)ethanol (1-beta-hydroxyethyl-3,4-methylenedioxyaniline), 4-hydroxyindole, 6-hydroxyindole, 7-Hydroxyindole, 4-Hydroxyindoline, 6-Hydroxyindoline and 7-Hydroxyindoline.

[0108] The compositions (Ind) and (E) used according to the invention and preferably according to the invention may, independently of one another, optionally contain further additives that can optimize their application properties. Preferred additives are, in particular, thickeners, which ensure that the coloring agent according to the invention or preferred according to the invention prepared from the compositions (Ind) and (E) remains better on the hair during application.

[0109] Compositions (Ind) and (E) used particularly preferably according to the invention contain - independently of one another - at least one or more hydrophilic thickeners other than cellulose, which are preferably selected from polysaccharides that may be chemically and / or physically modified. Compounds from the group of polysaccharides are particularly preferred as hydrophilic thickeners according to the invention, since the basic structures of the polysaccharides are of natural origin and biodegradable. Preferred hydrophilic polysaccharide thickeners are selected from xanthan gum, alginic acids (and their corresponding physiologically acceptable salts, the alginates), agar agar (with the polysaccharide agarose present in agar agar as the main constituent), starch fractions and starch derivatives such as amylose, amylopectin and dextrins, karaya gum, locust bean gum, gum arabic, pectins, dextrans and guar gum, and mixtures thereof.

[0110] In preferred embodiments, xanthan gum is included as a hydrophilic thickener with a view to reliable viscosity adjustment and residue-free application to keratin fibers and the scalp.

[0111] Compositions (Ind) and (E) which are particularly preferably used according to the invention contain, in each case based on the weight, independently of one another, at least one hydrophilic thickener in a total amount of from 0.1 to 5% by weight, preferably from 0.5 to 4% by weight, more preferably from 1 to 3.5% by weight and most preferably from 1.2 to 2% by weight.

[0112] In a further preferred embodiment of the present invention, the compositions (Ind) and (E) used according to the invention contain, in each case based on their weight, independently of one another, 0.1 to 3 wt.%, preferably 0.5 to 2.5 wt.%, more preferably 1.2 to 2.0 wt.%, of xanthan gum.

[0113] Compositions (Ind) and (E) used particularly preferably according to the invention contain, independently of one another, at least one organic solvent having a phenyl group in the molecule. This solvent is preferably selected from phenoxyethanol, benzyl alcohol, and mixtures thereof. Surprisingly, it has been found that such aromatic solvents can have a positive effect on the dyeing results of the dyeing process according to the invention; this has been observed particularly with aromatic solvents.

[0114] In a further preferred embodiment of the present invention, the compositions (Ind) and (E) used according to the invention contain, in each case based on their weight, independently of one another, 0.1 to 3% by weight, preferably 0.5 to 2.5% by weight, more preferably 0.8 to 1.0% by weight, of at least one organic solvent which has a phenyl group in the molecule. In a further preferred embodiment of the present invention, the compositions (Ind) and (E) used according to the invention contain, in each case based on their weight, independently of one another, 0.1 to 3% by weight, preferably 0.5 to 2.5% by weight, more preferably 0.8 to 1.0% by weight, of at least one organic solvent selected from phenoxyethanol, benzyl alcohol and mixtures thereof.

[0115] Compositions (Ind) and (E) which are particularly preferably used according to the invention contain, independently of one another, at least one aliphatic solvent selected from C1-C4 alkanols and C2-C4 polyols, in particular selected from ethanol, isopropanol, n-propanol, ethylene glycol, 1,2-propanediol, glycerol and 1,3-butylene glycol, and mixtures of these solvents, but only in a total amount of 0.01-8% by weight, preferably 0.1-6% by weight, particularly preferably 0.5-4% by weight, in each case based on the weight of the compositions (Ind) or (E).

[0116] Other compositions (Ind) and (E) which are particularly preferably used according to the invention contain, independently of one another, no aliphatic solvent selected from C1-C4 alkanols and C2-C4 polyols.

[0117] Powders from the leaves of Indigofera tinctoria, which are preferably used according to the invention, react approximately neutrally in aqueous dispersion, with slight deviations between a slightly acidic and a slightly alkaline pH range. A 5 wt. % dispersion of ground leaves of Indigofera tinctoria in deionized water has a pH of 6.6 to 7.4, preferably 6.8 to 7.2, particularly preferably 6.9 to 7.1, each measured at 20°C.

[0118] Colorants preferred according to the invention have a pH of 2.0 to 10.0, preferably from 4.0 to 9.0, particularly preferably from 5.0 to 8.0, extraordinarily preferably from 6.6 to less than 7.5, in each case measured at 20°C.

[0119] In a preferred embodiment of the dyeing process according to the invention, the desired pH is adjusted using an acid or a base. Preferred acids are selected from citric acid, lactic acid, gluconic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, galactaric acid (mucous acid), tartaric acid, malic acid, sulfuric acid, and phosphoric acid, as well as mixtures of these acids. Preferred bases are sodium hydroxide, potassium hydroxide, arginine, lysine, monoethanolamine, triethanolamine, 2-amino-2-methylpropan-1-ol, and mixtures of these bases.

[0120] In a further preferred embodiment of the dyeing process according to the invention, the desired pH is adjusted using a buffer system selected from a mixture of a medium-strength or weak acid with its conjugate or corresponding base (or the respective salt) and a mixture of a medium-strength or weak base with its conjugate or corresponding acid.

[0121] According to the invention, preferred suitable corresponding acid-base pairs are those which stabilize the compositions used according to the invention in the pH range from 2.0 to 10.0, preferably from 4.0 to 9.0, particularly preferably from 5.0 to 8.0, extraordinarily preferably from 6.6 to less than 7.5, in each case measured at 20°C.

[0122] Particularly preferred buffer systems according to the invention are selected from

[0123] - Ammonia / ammonium salt mixtures, wherein the ammonium salt is preferably selected from ammonium chloride, ammonium bromide, ammonium hydrogen sulfate, ammonium sulfate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium phosphate, ammonium bicarbonate, ammonium carbonate, ammonium nitrate, ammonium acetate, ammonium glycolate, ammonium gluconate, ammonium tartrate, ammonium lactate, and mixtures of these ammonium salts, particularly preferably selected from ammonium chloride, ammonium hydrogen sulfate, ammonium sulfate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium phosphate, ammonium bicarbonate, and ammonium carbonate, extremely preferably selected from ammonium chloride,

[0124] - mixtures of hydrogen phosphate and dihydrogen phosphate, in particular the alkali metal salts of hydrogen phosphate and dihydrogen phosphate, particularly preferably the sodium salts and / or the potassium salts of hydrogen phosphate and dihydrogen phosphate, - mixtures of alkali metal hydrogen carbonate with alkali metal carbonate, in particular mixtures of sodium or potassium hydrogen carbonate with sodium or potassium carbonate,

[0125] - mixtures of citric acid and its salts, in particular the alkali metal citrates, in particular the sodium salts, in particular trisodium citrate,

[0126] - mixtures of tartaric acid and its salts, in particular alkali metal tartrates, in particular potassium salts, in particular potassium hydrogen tartrate,

[0127] - mixtures of phthalic acid and its salts, in particular potassium salts, in particular potassium hydrogen phthalate,

[0128] - mixtures of lactic acid and its salts, in particular lactic acid / sodium lactate mixtures,

[0129] - mixtures of gluconic acid and its salts, in particular gluconic acid / sodium gluconate mixtures,

[0130] - mixtures of succinic acid and its salts, in particular the sodium salts, in particular sodium hydrogen succinate and disodium succinate, and

[0131] - mixtures of malic acid and its salts, in particular the sodium salts, in particular sodium hydrogen malate and disodium malate, and

[0132] - Ammonia / ammonium salt mixtures, wherein the ammonium salt is preferably selected from ammonium chloride, ammonium bromide, ammonium hydrogen sulfate, ammonium sulfate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium phosphate, ammonium bicarbonate, ammonium carbonate, ammonium nitrate, ammonium acetate, ammonium glycolate, ammonium gluconate, ammonium tartrate, ammonium lactate, and mixtures of these ammonium salts, particularly preferably selected from ammonium chloride, ammonium hydrogen sulfate, ammonium sulfate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium phosphate, ammonium bicarbonate, and ammonium carbonate, extremely preferably selected from ammonium chloride.

[0133] Other buffer systems, such as acetic acid / sodium acetate, are also suitable in principle according to the invention. However, due to the vinegar odor, such a buffer is not acceptable for the production of a commercial cosmetic product.

[0134] In principle, other buffer systems are also suitable.

[0135] The specialist can obtain the appropriate weights of buffer salts to adjust the desired pH value from the relevant manuals.

[0136] The subject matter of the present application is summarized in the following points: Agent for the non-oxidative coloring of keratinic fibers, in particular human hair, containing i) at least one indican-containing component, furthermore ii) at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum and iii) water, iv) characterized in that saponins are contained in a total amount of zero to less than 0.01 wt.%, preferably from zero to 0.005 wt.%, particularly preferably from zero to 0.001 wt.%, in each case based on the weight of the ready-to-use coloring agent, wherein the indican-containing component has been pretreated at a temperature in the range of 60°C to 200°C, provided that it comprises plant parts of an indigo-producing plant or a Extract from parts of an indigo-producing plant.Agent for non-oxidative coloring according to point 1, characterized in that the indican-containing component i) is selected from crushed parts of an indigo-producing plant and from indican-containing extracts of an indigo-producing plant and mixtures of these indican-containing components i). Agent for non-oxidative coloring according to point 2, characterized in that the extraction agent used to obtain the at least one indican-containing extract from an indigo-producing plant is selected from water, furthermore C1-C4 alkanols and C2-C4 polyols, in particular ethanol, isopropanol, n-propanol, ethylene glycol, 1,2-propanediol, glycerol and 1,3-butylene glycol, and mixtures of these extraction agents, in particular mixtures of water and at least one C1-C4 alkanol, mixtures of water and at least one C2-C4 polyol, particularly preferably water / ethanol mixtures.Agent according to one of points 2 or 3, characterized in that the indigo-producing^) plant(s) is / are selected from at least one species of the following genera:.

[0137] - Indigofera, in particular Indigofera tinctoria, Indigo suffruticosa, Indigofera articulata, Indigofera arrecta, Indigofera heterantha “Gerardiana”, Indigofera argentea or Indigofera longiracemosa;

[0138] - Isatis, especially Isatis tinctoria (woad);

[0139] - Persicaria, especially Persicaria tinctoria (dyer's knotweed);

[0140] - Wrightia, especially Wrightia tinctoria;

[0141] - Calanthe, especially Calanthe veratrifolia; and

[0142] - Baphicacanthus cusia, synonym Strobilanthes cusia, wherein the indigo-producing plant(s) is / are preferably selected from the genus Indigofera and in particular from Indigofera tinctoria. Agent according to any one of points 1 to 4, characterized in that the pretreatment of the at least one indican-containing component i) is carried out at a temperature in the range of 60°C to 200°C by heating, in particular by heating in a solvent, further by treatment with steam or by dry heating, preferably for a period of 1 to 120 minutes, particularly preferably 2 to 60 minutes, extraordinarily preferably 5 to 10 minutes. Agent according to any one of points 1 to 5, characterized in that the extracted indican is separated from the solvent after completion of the extraction procedure and separation of the solid plant parts, so that the indican-containing component i) is in powder form.Agent according to item 1, characterized in that the indican-containing component i) comprises biotechnologically, petrochemically, or oleochemically synthesized indican. Agent according to any one of items 1 to 7, characterized in that the indican-containing component i) contains 0.01-6 wt.% indican, based on its weight. Agent according to any one of items 1 or 7, characterized in that the indican-containing component i) comprises 30-100 wt.% biotechnologically, petrochemically, or oleochemically synthesized indican. Agent according to any one of items 1 to 9, characterized in that no 1,4-beta-D-glucanases are included. Agent for non-oxidative coloring according to one of points 1 to 10, characterized in that the at least one beta-glucosidase (EC 3.2.1 .21) from Aspergillus spp., in particular Aspergillus niger, Aspergillus niger SOI017, Aspergillus niger AS 3.4309, Aspergillus niger HDF 05, Aspergillus oryzae, Aspergillus strain SA 58, Aspergillus terreus EMOO 6-4, and mixtures of Aspergillus niger and Aspergillus oryzae, further selected from Trichoderma spp., in particular Trichoderma reesei, further selected from Penicillium spp., in particular Penicillium simplicissimum H-11, Penicillium citrinum YS40-5 and Penicillium miczynskii, further selected from Pseudomonas, in particular Pseudomonas ZD-8, with beta-glucosidases from Trichoderma reesei and Aspergillus niger being extremely preferred. Agent according to one of points 1 - 11, characterized in that it contains, based on its weight, at least one beta-glucosidase (EC 3.2.1 .21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum in a total amount of 0.0001 - 1 wt.%, preferably 0.001 - 0.1 wt.%, more preferably 0.002 - 0.009 wt.%, particularly preferably 0.003 - 0.007 wt.-%, contains. Agent according to one of points 1 - 12, characterized in that it contains, based on its weight, 19.9 - 95.0 wt.%, preferably 30.0 - 90.0 wt.%, particularly preferably 50.0 - 88.0 wt.%, extraordinarily preferably 60.0 - 85.0 wt.%, water. Kit for the non-oxidative coloring of keratinic fibers, in particular human hair, with natural dyes, comprising the following compartments which are physically separate from one another: i) a composition (Ind) which contains at least one indican-containing component which, if it comprises plant parts of an indigo-producing plant or is an extract from plant parts of an indigo-producing plant, has been pretreated at a temperature in the range from 60°C to 200°C, and ii) an aqueous composition (E) which contains at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum, iii) wherein saponins are present in a total amount of zero to less than 0.01 wt.%, preferably from zero to 0.005 wt.%, particularly preferably from zero to 0.001 wt.%, in each case based on the weight of the ready-to-use colorant, characterized in that the compositions i) (Ind) and ii) (E) are designed as described in any one of points 1 - 13.A method for the non-oxidative coloring of keratinic fibers, in particular human hair, comprising the following method steps in the specified order: a) providing a composition (Ind) which contains at least one indican-containing component which, if it comprises plant parts of an indigo-producing plant or is an extract from plant parts of an indigo-producing plant, has been pretreated at a temperature in the range from 60 °C to 200 °C, and providing an aqueous composition (E) which contains at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum, wherein saponins are present in a total amount of from zero to less than 0.01% by weight, preferably from zero to 0.005% by weight, particularly preferably from zero to 0.001% by weight.-%, in each case based on the weight of the ready-to-use colorant, b) mixing the compositions (Ind) and (E) with one another to produce a colorant, c) applying the colorant obtained in step b) to the keratin fibers, wherein the application of the colorant obtained in step b) to the keratin fibers preferably takes place within 1 second to 30 minutes, particularly preferably within 10 seconds to 15 minutes after completion of step b), d) leaving to act for a time of 30 seconds to 60 minutes, preferably 5 to 45 minutes, particularly preferably 15 to 30 minutes, e) rinsing the keratin fibers with water, f) optionally drying the keratin fibers.

[0143] 16. Dyeing process according to item 15, characterized in that the process is free from hydrogen peroxide and / or free from mordants and / or characterized in that the keratin fibers have not been treated with a mordant in a period of up to 7 days before and up to 7 days after the application of a dyeing agent according to the invention.

[0144] 17. Dyeing process according to one of points 14 or 15, characterized in that the dyeing agent obtained in step b) is an agent according to one of points 1 - 13.

[0145] Implementation examples

[0146] The exemplary embodiments presented below are intended to explain the subject matter of the invention in more detail without limiting it thereto.

[0147] The dyeing for this invention was carried out on strands of buffalo belly hair (brush body tied, approx. 8 cm of free hair).

[0148] Preparation of the indican-containing component i) (Composition (Ind)

[0149] 95 ml of demineralized water was heated to approximately 95 °C. Subsequently, 5 grams of ground leaves of Indigofera tinctoria L* were added (5 wt% of the total dispersion) and stirred for 5 minutes at approximately 95 °C.

[0150] * The following raw materials were used:

[0151] “C30-Indigo Leaves Powder” (Indigofera tinctoria L.) powdered leaves of Indigofera tinctoria pH of the 5 wt.% dispersion in water: 6.8 to 7.2 (20°C) or “Indigo Powder” (Indigofera tinctoria L.) powdered leaves of Indigofera tinctoria pH of the 5 wt.% dispersion in water: 6.8 to 7.2 (20°C)

[0152] The dispersion was then cooled to room temperature (20°C) and filtered (filter: Whatman 10340810, viscose, pore size 100 pL, approx. 5 to 10 min, vacuum pump at approx. 500 mbar).

[0153] The filtrate was a brown, opaque solution without precipitate, with a pH of approximately 7.7 (20°C). This indican-containing component i) is referred to below as "Composition (Ind)".

[0154] Treating hair strands with composition (Ind) does not produce color under standard dyeing conditions (50 ml of composition (Ind) per 1 g of hair strand, dyeing for 30 minutes at room temperature (20°C) while stirring, rinsing for 30 seconds under running deionized water, combing 20 times at 20°C, drying with a hairdryer, distance: d = 10 cm, temperature: 80 ± 5 °C, combing 20 times).

[0155] Coloring according to the invention

[0156] The aqueous composition (E) according to the invention contains beta-glucosidase (EC 3.2.1 .21) from Trichoderma reesei.

[0157] The enzyme active substance content of the beta-glucosidase from Trichoderma reesei used, which was available as an aqueous composition (E), was 70 pg / μl, i.e., 7 wt.%. Furthermore, composition (E) contained, based on its weight, 78 wt.% water and 15 wt.% of a sucrose / D-glucose mixture.

[0158] To prepare the dyeing composition according to the invention, 49.95 ml of the inventive composition (Ind) were mixed with 50 μL of the inventive composition (E). The dyeing composition according to the invention thus obtained had a total enzyme content of 0.007 wt.% based on its weight.

[0159] A buffalo belly strand was then added to this inventive dyeing composition and dyed for 30 minutes at room temperature (20°C) while stirring (liquor ratio: 50 ml for 1 g of hair strand; pH: approx. 7.7, measured at 20°C). The dye solution was then washed off the strand (see above) and dried (see above).

[0160] The addition of beta-glucosidase from Trichoderma reesei resulted in a blue hue being visible on the hair fiber after dyeing (see Table 1). Control dyeing was performed with a non-inventive indican-containing component that had not been heat-treated.

[0161] To prepare a non-inventive control dye composition, 2.5 g of indigo powder were dispersed in 47.5 grams of demineralized water (5% by weight of the total dispersion). After stirring the dispersion for 2 minutes, a strand of buffalo belly hair (brush body tied round, approximately 8 cm of free hair) was placed in the control dye composition for 30 minutes while stirring (liquor ratio: 50 grams of dye composition to 1 gram of hair strand, pH: 6.46-7.29 (20°C)). The control dye composition was then washed off the strands by rinsing the strands under running deionized water for 30 seconds, followed by 20 combing cycles (20°C). The hair strands were then dried with a commercially available hair dryer at a defined distance (d = 10 cm) and a defined temperature (T = 80 ± 5 °C) with 20 combs.

[0162] The following plant raw materials were used:

[0163] “C30-Indigo Leaves Powder” (Indigofera tinctoria L.) powdered leaves of Indigofera tinctoria pH of the 5 wt% dispersion in water: 6.8 to 7.2 (20°C) or

[0164] Indigo Powder (Indigofera tinctoria L.) powdered leaves of Indigofera tinctoria. pH of the 5 wt.% dispersion in water: 6.8 to 7.2 (20°C)

[0165] Colorimetric measurements

[0166] All colorimetric measurements were performed using the Spectraflash SF 600 colorimetric device from Datacolor.

[0167] The color difference, also known as dE or AE, can be easily determined colorimetrically using a colorimeter that measures colors in the L*, a*, b* color space, for example a Datacolor Spectraflash SF 600 colorimeter.

[0168] The L*,a*,b* color space refers to the CIELAB color space. The L value represents the brightness of the color (black-white axis); the higher the L value, the brighter the color. The a value represents the red-green axis of the system; the higher this value, the more the color is shifted toward red. The b value represents the yellow-blue axis of the system; the higher this value, the more the color is shifted toward yellow.

[0169] The color shift AE, i.e. the color difference between two (hair) colors, for each of which an L*, a*, b* value combination has been determined, is calculated according to the following formula:

[0170] AE = (AL 2 + Aa 2 + From 2 ) 05

[0171] The larger the value for AE, the more pronounced the color difference.

[0172] A D65 illuminant and a diffuse / 8° optical configuration were used for the spectrophotometer measurements. The spectral reflectance data for each sample from 380 nm to 700 nm were converted to colorimetric data using DCI Color software. Reflectance measurements were determined for each hair sample, with the average of four measurements recorded.

[0173] The color difference (AE) between two strands with different coloring treatments was calculated according to the following formula:

[0174] Lv, av, bv: colorimetric values ​​for dyed strands

[0175] Ln, an, bn: Colorimetric values ​​for undyed strands

[0176] The differences between the control strand and the strands with heat-deactivated and subsequently enzymatically reactivated indican-containing extract, corresponding to the mixture of composition according to the invention (Ind) and composition (E) directly after coloring (day 0), are shown in Table 1 (mean values).

[0177] Table 1: Color comparison of indigo dyeing control and heat-treated composition (Ind) with reactivation by beta-glucosidase addition 0 days after dyeing

[0178] The conversion of the L a b values ​​into Pantone values ​​and color names was carried out using the website https: / / www.e-paint.co.uk / convert-lab.asp.

[0179] Immediately after coloring, the mean values ​​of the color difference between both strands differ with an AE value of 15.3

[0180] After 14 days, the mean values ​​of both strands differed significantly. Furthermore, there was also a significant shift in the color impression: the significant change in the a-value describes the shift in the color tone from green to red; the significant change in the b-value describes the shift in the color tone from yellow to blue. The control dye thus showed a very unstable dyeing result, with the final dyeing result only becoming apparent after 14 days.

[0181] The color change in the hair from day 0 to day 14 with the inventive coloring was determined with an AE value of only 2, which is virtually imperceptible to the human eye. There was also hardly any change in the color tone itself.

[0182] By initially deactivating the enzyme activity present in indican-containing, indigo-producing plant parts and subsequently adding beta-glucosidase from Trichoderma reesei shortly before application, the dyeing performance of the initially non-dyeing compound (Ind) can be restored. Furthermore, the strong time-dependent color change from an initial turquoise-blue tone to a redder and darker shade is prevented.

Claims

Patent claims 1. Agent for the non-oxidative coloring of keratinic fibers, in particular human hair, containing i) at least one indican-containing component, furthermore ii) at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum and iii) water, iv) characterized in that saponins are present in a total amount of zero to less than 0.01 wt.%, preferably from zero to 0.005 wt.%, particularly preferably from zero to 0.001 wt.%, in each case based on the weight of the ready-to-use coloring agent, wherein the indican-containing component has been pretreated at a temperature in the range of 60°C to 200°C, provided that it comprises plant parts of an indigo-producing plant or an extract from plant parts of an indigo-producing plant.

2. Agent for non-oxidative coloring according to claim 1, characterized in that the indican-containing component i) is selected from crushed parts of an indigo-producing plant and from indican-containing extracts of an indigo-producing plant and mixtures of these indican-containing components i).

3. Agent for non-oxidative coloring according to claim 2, characterized in that the extraction agent used to obtain the at least one indican-containing extract from an indigo-producing plant is selected from water, furthermore C1-C4 alkanols and C2-C4 polyols, in particular ethanol, isopropanol, n-propanol, ethylene glycol, 1,2-propanediol, glycerol and 1,3-butylene glycol, and mixtures of these extraction agents, in particular mixtures of water and at least one C1-C4 alkanol, mixtures of water and at least one C2-C4 polyol, particularly preferably water / ethanol mixtures.

4. Agent according to one of claims 2 or 3, characterized in that the indigo-producing^) plant(s) is / are selected from at least one species of the following genera: - Indigofera, in particular Indigofera tinctoria, Indigo suffruticosa, Indigofera articulata, Indigofera arrecta, Indigofera heterantha “Gerardiana”, Indigofera argentea or Indigofera longiracemosa; - Isatis, especially Isatis tinctoria (woad); - Persicaria, especially Persicaria tinctoria (dyer's knotweed); - Wrightia, especially Wrightia tinctoria; - Calanthe, especially Calanthe veratrifolia; and - Baphicacanthus cusia, synonym Strobilanthes cusia, wherein the indigo producing plant(s) is / are preferably selected from the genus Indigofera and in particular from Indigofera tinctoria.

5. Agent according to one of claims 1 to 4, characterized in that the indican-containing component i) contains, based on its weight, 0.01 - 6 wt.% indican.

6. Agent according to one of claims 1 to 5, characterized in that the pretreatment of the at least one indican-containing component i) is carried out at a temperature in the range from 60 °C to 200 °C by heating, in particular by heating in a solvent, further by treatment with steam or by dry heating, preferably for a period of 1 to 120 minutes, particularly preferably 2 to 60 minutes, extraordinarily preferably 5 to 10 minutes.

7. Agent according to one of claims 1 to 6, characterized in that the extracted indican is separated from the solvent after completion of the extraction procedure and separation of the solid plant parts, so that the indican-containing component i) is in powder form.

8. Agent according to claim 1, characterized in that the indican-containing component i) comprises biotechnologically, petrochemically or oleochemically synthesized indican, wherein it is preferred that the indican-containing component i) comprises, based on its weight, 30-100 wt.% biotechnologically, petrochemically or oleochemically synthesized indican.

9. Agent according to one of claims 1 to 8, characterized in that no 1,4-beta-D-glucanases are contained.

10. Agent for non-oxidative coloring according to one of claims 1 to 9, characterized in that the at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus spp., in particular Aspergillus niger, Aspergillus niger SOI017, Aspergillus niger AS 3.4309, Aspergillus niger HDF 05, Aspergillus oryzae, Aspergillus strain SA 58, Aspergillus terreus EMOO 6-4, and mixtures of Aspergillus niger and Aspergillus oryzae, further selected from Trichoderma spp., in particular Trichoderma reesei, further selected from Penicillium spp., in particular Penicillium simplicissimum H-11, Penicillium citrinum YS40-5 and Penicillium miczynskii, further selected from Pseudomonas, especially Pseudomonas ZD-8, with beta-glucosidases from Trichoderma reesei and Aspergillus niger being particularly preferred.

11. Agent according to one of claims 1-10, characterized in that it contains, based on its weight, at least one beta-glucosidase (EC 3.2.1 .21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum in a total amount of 0.0001 - 1 wt.%, preferably 0.001 - 0.1 wt.%, more preferably 0.002 - 0.009 wt.%, particularly preferably 0.003 - 0.007 wt.%.

12. Agent according to one of claims 1-11, characterized in that it contains, based on its weight, 19.9-95.0 wt.%, preferably 30.0-90.0 wt.%, particularly preferably 50.0-88.0 wt.%, extraordinarily preferably 60.0-85.0 wt.%, of water.

13. Kit for the non-oxidative coloring of keratinic fibers, in particular human hair, with natural dyes, comprising the following compartments which are physically separate from one another: i) a composition (Ind) which contains at least one indican-containing component which, if it comprises plant parts of an indigo-producing plant or is an extract from plant parts of an indigo-producing plant, has been pretreated at a temperature in the range from 60°C to 200°C, and ii) an aqueous composition (E) which contains at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum, iii) wherein saponins are present in a total amount of from zero to less than 0.01% by weight, preferably from zero to 0.005% by weight, particularly preferably from zero to 0.001 wt.-%, in each case based on the weight of the ready-to-use colorant, characterized in that the compositions i) (Ind) and ii) (E) are designed as described in any one of claims 1-12.

14. A method for the non-oxidative coloring of keratin fibers, in particular human hair, comprising the following process steps in the given order: a) Providing a composition (Ind) which contains at least one indican-containing component which, if it comprises plant parts of an indigo-producing plant or is an extract from plant parts of an indigo-producing plant, has been pretreated at a temperature in the range from 60°C to 200°C, and providing an aqueous composition (E) which contains at least one beta-glucosidase (EC 3.2.1.21) from Aspergillus, Trichoderma, Penicillium, Lactobacillus acidophilus, Pseudomonas, Bacillus subtilis or Bifidobacterium bifidum, wherein saponins are present in a total amount of from zero to less than 0.01% by weight, preferably from zero to 0.005% by weight, particularly preferably from zero to 0.001% by weight, in each case based on the weight of the ready-to-use dye, b) mixing the compositions (Ind) and (E) with one another to produce a colorant, c) applying the colorant obtained in step b) to the keratin fibers, wherein the application of the colorant obtained in step b) to the keratin fibers preferably takes place within 1 second to 30 minutes, particularly preferably within 10 seconds to 15 minutes after completion of step b), d) leaving to act for a time of 30 seconds to 60 minutes, preferably 5 to 45 minutes, particularly preferably 15 to 30 minutes, e) rinsing the keratin fibers with water, f) optionally drying the keratin fibers.

15. Dyeing process according to claim 14, characterized in that the colorant obtained in step b) is an agent according to any one of claims 1-12.