Method for extracting high-quality keratin from keratinous material, without chemical products

Abstract

The invention relates to the field of keratin extraction methods. More particularly, the invention relates to a method for extracting keratin, in particular human hair keratin, without chemical products. The method comprises three thermal hydrolysis steps. The keratin obtained is of high quality, the amino acid composition of same being close to that of native keratin, and can be used in applications with a high added value such as cosmetics or medical devices. The biomimicry between the keratin extracted from hair using this extraction method and natural keratin is more than 90%.

Description

PROCESS FOR EXTRACTION OF HIGH QUALITY KERATIN WITHOUT CHEMICALS FROM KERATIN MATERIAL

[0001] The invention relates to the field of keratin extraction processes. More particularly, the invention relates to a process for extracting keratin, in particular of human origin from hair, without chemicals. The keratin obtained is of high quality, its amino acid composition being close to that of native keratin, and can be used in high value-added applications such as cosmetics or medical devices. The biomimicry between the keratin extracted from hair by this extraction process and natural keratin is more than 90%. Field of invention

[0002] Keratin is a protein rich in cysteine-type amino acids. Cysteines have the ability to form disulfide bonds between themselves, which gives keratin a unique helical and fibrous tertiary structure. Human hair is made up of approximately 8% cysteine. Keratin is highly sought after for cosmetic applications, particularly in hair care and skin creams and serums, but also for treating bone and cartilage fragility.

[0003] Keratin is a fibrous protein with a complex structure that gives it remarkable mechanical properties, including strength, flexibility, and durability. The amino acid composition of keratin can vary slightly between species, but in general, it is rich in nonessential amino acids, particularly cysteine, which plays a key role in the formation of disulfide bonds responsible for keratin's stable structure. This structure is based on disulfide bonds and repeating amino acid patterns that vary depending on the location and specific function of keratin in the body.

[0004] Methods for extracting keratin from animal material are known in the state of the art.

[0005] Document EP3984372 describes a process for extracting partially hydrolyzed keratin material from various sources of animal keratin: feathers, wool, hair, nails, etc.

[0006] Document US20190194297 describes a process for extracting keratin from pig hair. The material is first washed in a solution containing a non-ionic detergent. The extracted keratin has a cysteine ​​content of 2.9%.

[0007] Document WO2019 / 209188 describes a process for extracting keratin-beta from human hair using two solvents: GhCrosolan, and CutissentialBehenly 18-MEA, composed of behentrimonium methosulfate, a chemical solvent considered toxic according to the REACH classification. This process takes place over 48 hours. The characteristics of the extracted material are not disclosed. Disadvantages of the state of the art

[0008] Previously described processes do not allow to obtain good quality keratin, i.e., one whose structure is close to that of native keratin and which is highly purified. The use of chemical solvents to clean the material upstream of extraction can leave traces in the finished product and constitutes a waste that must be treated (pollution). In addition, the main ambition of keratin extraction protocols is to efficiently transform organic waste into valuable resources. However, the traditional approach, involving the intensive use of chemicals, either for the initial cleaning of the waste or during the extraction process itself, raises obvious contradictions with the principles of sustainable development and waste recovery.

[0009] The keratins available on the market are either of animal (non-human) origin or of so-called “plant” origin.

[0010] Compared to keratins of animal origin, it is preferable to have keratin of human origin for applications in humans: it will be more effective due to its biological affinity. Indeed, it is known that to improve the biocompatibility of animal or plant keratin for applications in humans, additives or fixatives can be used. Indeed, keratin of human origin has better specificity, complementarity with our human cellular receptors. This is described in the publication WO 2019 / 209188A1. It explains that human keratin, extracted from hair, due to its spatial conformation, will have complete complementarity with cellular receptors in humans. However, depending on the applications, keratins of animal origin can be quite suitable. Diversifying the sources of keratin raw material makes it possible to increase access to this resource.

[0011] As for plant keratin, it doesn't exist strictly speaking. In fact, keratin has only been described in mammals and reptiles. So-called "plant" keratin is actually a structural protein of plant origin (e.g., cellulose) or a protein composition derived from plants.

[0012] It would be desirable to have good quality keratin of human or animal origin and an industrializable process to produce it.

[0013] Faced with the problems set out above, the inventors have developed an innovative process which stands out from those of the prior art in that it allows keratin to be extracted with good yield and without chemicals.

[0014] It is based on the recovery and valorization of keratin from keratinous material of human or animal origin, a material available in large quantities. In a preferred embodiment, this keratinous material is made from human hair, an abundant resource with more than 4,000 tons of waste generated annually in France. In doing so, this process is practically free from any harmful impact on the environment, thus aligning perfectly with the objectives of ecological preservation and waste valorization.

[0015] Thus, the present invention relates to a process for extracting keratin from keratin material defined by the following steps:Washing the keratin material with water in the absence of a chemical productRecovery of the washed material and dryingGrinding of said material to obtain pieces with a size of less than 5 mmMixing of said ground material with deionized water, applying a weight of material / volume of water ratio of between 300 and 800 g / LExtraction reaction in a hermetically sealed thermal reactor in at least 3 steps:20 min to 1 h at a temperature of between 100°C and 160°C20 min to 1 h at a temperature of between 120°C and 200°C30 min to 1 h 30 at a temperature of between 140°C and 210°CRecovery of the supernatantSterilizing filtration through a membrane whose pore diameter is equal to or less than 0.22 µmDrying of keratin Advantages of the invention

[0016] The process of the invention makes it possible to extract the keratin contained in the keratin material with a good yield, while retaining an amino acid composition close to that of native keratin. This is reflected by the high cysteine ​​content of the keratin extracted from human hair, as disclosed in the present invention.

[0017] This process does not use any chemicals, either for washing the keratin material or during the keratin extraction stage. The "extracted keratin" product is therefore free from any toxic residue and is highly natural.

[0018] The inventors have shown that the keratin thus extracted from human hair contains 20 amino acids and has a composition very close to that of native keratin, notably due to its high cysteine ​​content.

[0019] In a particular embodiment of the invention, the keratin is of human origin. Human keratin is more compatible with human biological processes than keratin of animal or “plant” origin, which makes it a source of choice for all applications in humans, whether in cosmetics or in medical devices.

[0020] The process is fast, extraction can be completed in 4 hours, and is industrializable. It allows access to high-quality keratin, particularly human-derived keratin, for sectors that are demanding in terms of the choice of raw materials, such as cosmetics or nutraceuticals. This high quality is demonstrated in particular by the high cysteine ​​content of keratin extracted from human hair, namely 6% - native keratin has a cysteine ​​content of approximately 8%.

[0021] This process provides a "greentech" solution for the recovery of keratinous material. In particular, hair, as waste, is otherwise mostly incinerated. In France, hair represents approximately 4,000 tons, which generates more than 10,000 tons of CO2 per year. This chemically clean process transforms an undervalued resource into a high-value product, unique on the global market.

[0022] The present invention therefore provides the first keratin extraction process capable of producing a high-quality product without chemicals and which is industrializable. In particular, it provides access to high-quality purified human keratin. DETAILED DESCRIPTION OF THE INVENTION

[0023] A first subject of the invention relates to a process for extracting keratin from keratin material defined by the following steps:Washing the keratin material with water in the absence of a chemical productRecovery of the washed material and dryingGrinding of said material to obtain pieces with a size of less than 5 mmMixing of said ground material with deionized water, applying a material weight / water volume ratio of between 300 and 800 g / LExtraction reaction in a hermetically sealed thermal reactor in at least 3 steps:20 min to 1 h at a temperature of between 100°C and 160°C20 min to 1 h at a temperature of between 120°C and 200°C30 min to 1 h 30 at a temperature of between 140°C and 210°CRecovery of the supernatantSterilizing filtration through a membrane whose pore diameter is equal to or less than 0.22 µmDrying of keratin

[0024] “Chemical-free” processing is a processing that does not involve any additional chemical reagent; the extraction is done in water.

[0025] For the purposes of the invention, the term "keratin material" means any material of animal origin containing keratin, such as human hair, fur, wool and feathers. The material may come from a single source or be a mixed material comprising at least two different keratin materials.

[0026] In a particular embodiment of the invention, the keratin material consists of human hair and the keratin extraction process comprises the steps of:Washing the hair with water in the absence of a chemical productRecovery of the washed hair and dryingGrinding said hair to obtain pieces of a size less than 5 mmMixing the ground hair with deionized water, applying a hair weight / water volume ratio of between 300 and 800 g / LExtraction reaction in a hermetically sealed thermal reactor in 3 stages:20 min to 1 h at a temperature of between 100°C and 160°C20 min to 1 h at a temperature of between 120°C and 200°C30 min to 1 h 30 at a temperature of between 140°C and 210°CRecovery of the supernatantSterilizing filtration through a membrane whose pore diameter is equal or less than 0.22 µmDrying of keratin

[0027] In a more particular embodiment, the keratin extraction method comprising the steps of:Washing the hair with water in the absence of a chemical productRecovering the washed hair and dryingGrinding said hair to obtain pieces with a size of less than 5 mmMixing the ground hair with deionized water, applying a hair weight / water volume ratio of between 300 and 800 g / LExtraction reaction in a hermetically sealed thermal reactor in 3 stages:20 min to 1 h at 140°C20 min to 1 h at 180°C30 min to 1 h 30 at 205°CRecovery of the supernatantSterilizing filtration through a membrane whose pore diameter is equal to or less than 0.22 µmDrying of the keratin

[0028] Recovery of the supernatant can be obtained by centrifugation at 4000 rpm at 4°C and can be followed by ultrafiltration.

[0029] Hair is covered in sebum and materials contained in the air (pollution, pollen, etc.) or any other material with which it may come into contact.

[0030] Other keratin materials are also natural materials that have been in contact with possible contaminants.

[0031] It is therefore necessary to carefully wash the keratin material before starting the keratin extraction treatment.

[0032] In the context of the present invention, this washing is done with water, without any chemical product.

[0033] Preferably, the washing comprises several successive baths, in order to eliminate lipid substances and impurities and contaminants of any kind that could affect the quality of the final product.

[0034] The water used for these washes is preferably deionized water (at approximately pH 7). In order to facilitate defatting and the elimination of impurities, the washes are carried out at a temperature preferably between 45°C and 60°C.

[0035] Preferably, the keratin material (such as hair) is rinsed with deionized water between 1 and 3 times prior to said washing step. This rinsing can be done with gentle mechanical agitation.

[0036] In a first particular embodiment, washing the material with water comprises at least one washing cycle by soaking in deionized water for 30 minutes to 1 hour 30 minutes at a temperature preferably between 45°C and 60°C followed by rinsing with deionized water at a temperature preferably between 45°C and 60°C. The number of washing cycles may be 2, 3 or more depending on the level of soiling of the material.

[0037] In a second particular embodiment, the washing with water comprises three washing cycles by soaking in deionized water for 30 minutes to 1 hour 30 minutes at a temperature preferably between 45°C and 60°C followed by rinsing with deionized water at a temperature preferably between 45°C and 60°C.

[0038] After this treatment, the material can be rinsed again with water, 1 to 3 times.

[0039] The number of cycles and the temperature of each cycle can be modulated according to the starting keratin material and its degree of soiling.

[0040] After being washed, the keratin material is recovered, for example by filtration, then dried.

[0041] Drying can be carried out at room temperature or by applying gentle heat (around 45°C) if you wish to reduce the drying time.

[0042] The keratinous material is then ground, for example using a mechanical knife mill or a mechanical ball mill. The pieces of material after grinding must be fairly fine, i.e. less than 5 mm in size, in the order of 0.1 to 5 mm, around 1 mm.

[0043] The ground material is mixed with deionized water. The material weight / water volume ratio is between 300 and 800 g / L. In a preferred embodiment, it is between 500 and 700 g / L. This mixture can be made directly in the reactor where the extraction will be carried out. The material density is quite high without affecting the efficiency of the process. Thus, the process allows for a good extraction yield, which reduces energy costs.

[0044] The extraction is carried out in a hermetically sealed thermal reactor so that the rise in temperature is accompanied by a rise in pressure which contributes to the efficiency of the extraction. The hydrolysis reaction is broken down into at least 3 stages, namely: 20 min to 1 h at a temperature between 100°C and 160°C, preferably between 120°C and 160°C, or even between 130°C and 160°C, 20 min to 1 h at a temperature between 120°C and 200°C, preferably between 140°C and 190°C, or even between 160°C and 190°C, 30 min to 1 h 30 at a temperature between 140°C and 210°C, preferably between 160°C and 210°C, more preferably between 160°C and 210°C, or even between 180°C and 210°C, 190°C and 210°C.

[0045] The time / temperature combination can be adapted by those skilled in the art, but also depending on the starting keratin material.

[0046] In preferred embodiments of the invention, the extraction reaction is carried out as follows:20 min to 1 h at a temperature between 100°C and 160°C, then between 20 min to 1 h at a temperature between 120°C and 200°C, then 30 min to 1 h 30 at a temperature between 160°C and 210°C.20 min to 1 h at a temperature between 120°C and 160°C, then 20 min to 1 h at a temperature between 140°C and 190°C, then 30 min to 1 h 30 at a temperature between 160°C and 210°C.20 min to 1 h at a temperature between 130°C and 160°C, then 20 min to 1 h at a temperature between 160°C and 190°C, then 30 min to 1h30 at a temperature between 180°C and 210°C. 20 min to 1h at a temperature between 130°C and 160°C, then 20 min to 1h at a temperature between 160°C and 190°C, then 30 min to 1h30 at a temperature between 190°C and 210°C.

[0047] These 3 steps constitute 3 temperature rise levels, allowing the progressive unfolding of the keratin and which contributes to making the extraction very efficient. This results in a complete aminogram, as presented in (and commented on below).

[0048] The pressure in the reactor is very high, with an equivalent of 3.6 bars at 140°C at the start of the process and going up to at least 16 bars when the temperature reaches 205°C during the last heating stage.

[0049] At the end of the extraction, the supernatant is recovered. It is preferably maintained at a controlled temperature to preserve the properties of the material it contains, generally a temperature below 15°C, preferably below 10°C, for example in an enclosure at 4°C.

[0050] In a particular embodiment, the product of the extraction reaction is treated to separate the solubilized keratin from the other components of the mixture by applying a centrifugal force, such as centrifugation or using a spin dryer, so as to recover the supernatant containing the keratin.

[0051] Thus, in a particular embodiment, the extraction product is centrifuged for example at 4000 rpm for a period of time ranging from 10 to 30 min, for example for 15 min, at 4°C. The centrifugation step can be repeated to ensure that all debris has been removed.

[0052] The supernatant is then purified by filtration to remove biological contaminants such as bacteria, fungi, and yeast. To ensure sterilization, the supernatant containing keratin is filtered through a membrane with pores of 0.22 microns or less in diameter.

[0053] Optionally, ultrafiltration can be performed on the supernatant before sterilizing filtration.

[0054] Ultrafiltration is a membrane separation method typically used to remove suspended particles such as bacteria, yeasts, and viruses. The size of the particles retained can vary between 1 and 250 nanometers.

[0055] The keratin thus recovered is dried. This drying can be carried out using any suitable technique known to those skilled in the art, such as freeze-drying, spray dryer, etc.

[0056] The keratin extracted by the process according to the invention has an aminogram very close to that of native keratin, which is a marker of its high quality.

[0057] By analyzing human hair keratin extracted using the method of the invention, we observe a biomimicry of more than 90% with the keratin naturally present in the hair. This aminogram shows the presence of 20 amino acids. Remarkably, the amount of cysteine / cystine present is equivalent to 75% of the amount present in native keratin.

[0058] The keratin extraction yield obtained by this process reaches 30 to 40%.

[0059] The present invention will be better understood from the following examples, provided for illustration purposes and in no way to be considered as limiting the scope of the present invention. DESCRIPTION OF FIGURES

[0060] : Aminogram of human hair keratin extracted according to the process according to the invention vs. that of native keratin.

[0061] : Analysis of proteins and peptides present in the keratin sample extracted from human hair using the MALDI technique. This analysis was carried out by CBM-CNRS UPR4301. EXAMPLES

[0062] EXAMPLE 1 - Implementation of the extraction process according to the inventionWashing:

[0063] Raw human hair was obtained from local hair salons.

[0064] In order to remove all dirt, this material was washed 3 times (1h / wash) with deionized water (pH approximately 7) at room temperature in stainless steel tanks under gentle mechanical agitation to remove all traces of impurities. A simple rapid filtration of 20 minutes is then carried out.

[0065] A defatting and disinfection step which consists of soaking the sample 3x (1h at 55°C), alternating with 3 washes with clean, deionized H2O (pH approximately 7) at 55°C is then carried out followed by 3 washes with deionized water (10 min / wash with simple agitation).

[0066] The hair is then dried for two days in the open air on stainless steel trays with simple mechanical stirring 3 times a day / 3 hours in a forced air oven at 45°C to recover the dry hair which is then ground into fine pieces (± 1mm) using a mechanical ball mill.

[0067] This material was then used as raw material for keratin extraction.Extraction:

[0068] Keratin extraction was carried out in a 20-liter airtight reactor. The hair-to-deionized water ratio used was 7.5 kg of hair to 12.5 L of pure water.

[0069] The closed thermal reactor is equipped with gentle continuous stirring and maintains a very high pressure. No chemicals were used for the reaction, the heating rate is 5°C / min and the stirrer is fixed inside allowing good internal stirring of the solution during extraction.

[0070] The following temperature levels were applied and respected:

[0071] 30 min at 140°C, then 30 min at 180°C, then 60 min at 205°C.

[0072] The total duration of the extraction process is approximately 7 hours including the heating, temperature maintenance and cooling phases (4 hours).

[0073] The extracted product is filtered through a 200 μm membrane.

[0074] The whole material is centrifuged twice at 4000 rpm for 30 min at 4°C (to further remove impurities).

[0075] UltraFiltration is carried out: A UF ​​NFS sanitary membrane was installed on the TIA UF / MF / NF / OI skid – 20E028. The membrane was washed with a 4% sodium hydroxide solution (pH = 10) and the temperature was continuously monitored to ensure that it did not exceed 50 °C. Then, the membrane was rinsed with distilled water (circulated in the microfiltration skid) until the permeate and retentate were neutralized to reach a pH = 7. The feed tank was emptied. The solution was poured into the feed tank. The feed flow rate was set at 800 L / h with a recirculation pressure between 10 and 36 bar. Once these were adjusted, permeate collection began. A heating of the medium from 20°C to 36°C was observed, and cooling of the filtration skid compressor was implemented to prevent further temperature rise.

[0076] Only the supernatant is collected and filtered through specific filters. “Ref: MPGP002A1 Description: 0.22 μm membrane filter for particulate and bacteria-free water at the point of dispense for the Milli-Q® IQ, IX and EQ 7 series water purification systems”.

[0077] This involves sterilizing filtration of the liquid extract (supernatant) through a 0.2 µm pore membrane to effectively remove biological contaminants such as bacteria, molds, and yeasts.

[0078] Ultimately, a volume concentration factor of approximately 30% was achieved for an extraction yield of 35%.

[0079] The resulting solution was then lyophilized and / or spray dried to obtain a keratin powder of homogeneous particle size.

[0080] EXAMPLE 2: Characterization of extracted keratin A - Analysis of the aminogram

[0081] Aminograms were performed to obtain the amino acid profile (acid or oxidative hydrolysis for MET and Cystine) by ion / UV chromatography on a dried sample.

[0082] Ion chromatography was used to separate each amino acid, followed by post-column reaction detection. The sample was hydrolyzed prior to analysis, using HCl for 24 h at 110°.

[0083] The result of the aminogram is represented by the. We note the presence of 20 amino acids, as in native keratin. The most represented and present amino acids are glutamic acid, arginine, leucine, serine, threonine and cysteine B - Analysis by SDS-PAGE gel:

[0084] Polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed on a Mini-PROTEAN TGX 4-20% gel (12 wells, 20 μL / well, 8.6 x 6.7cm, Biorad). Samples were prepared according to the 4x Laemmli method (250mM Tris-HCl, pH 6.8, 4% LDS, 40% (w / v) glycerol, 0.02% bromophenol) in the presence of b-mercaptoethanol.

[0085] The samples to be analyzed were diluted to introduce 10, 15, 20 and 25 mg / well respectively. The molecular mass identification standard used is the “Precision Plus Protein Standard” (Biorad ref, #161-0373) containing proteins from 10 to 250 kDa. Migration was performed at 250V, followed by Coomassie blue staining.

[0086] Preliminary results show representative bands of protein molecular masses consistent with those expected: protein substances (peptides and proteins) appear in broad bands below 10 kDa (data not shown).

[0087] C - BCA and Kjeldahl dosage of keratin matter

[0088] As part of the optimization of extraction and quantification processes of keratinous material, a dual analytical approach combining BCA and Kjeldahl assays has been developed in order to obtain a precise and complete characterization of the extracted protein material.

[0089] The BCA (bicinchoninic acid) method is a sensitive colorimetric technique based on the ability of proteins to reduce Cu²⁺ ions to Cu⁺ in an alkaline medium, forming a purple complex absorbing at 562 nm, the intensity of which is proportional to the concentration of soluble peptides. This method exhibits good linearity over a wide concentration range, low inter-protein variability compared to dye-based methods, and compatibility with many denaturing agents or detergents frequently used in the extraction of keratin material. It thus allows the efficient determination of peptides resulting from the partial degradation of keratin, including in complex matrices or after hydrolysis, which is essential for assessing the solubility and quality of the protein extract.

[0090] The Kjeldahl method, on the other hand, is based on the chemical mineralization of organic nitrogen compounds by hot digestion with sulfuric acid, followed by distillation and titration of the released ammonia, allowing the measurement of the total nitrogen content, including both peptides and free amino acids present in our sample. This reference technique offers a comprehensive measurement of protein matter, regardless of the aggregation state or protein structure, and allows the overall yield of keratinous matter extraction to be assessed.

[0091] The joint use of these two methods is of paramount importance: the BCA method specifically targets the soluble peptide fraction, while the Kjeldahl method provides a total measurement of all nitrogenous forms. This analytical complementarity allows for a detailed, reliable and complete characterization of the extracted keratin material, essential for optimizing extraction processes, guaranteeing the reproducibility of results, and ensuring the quality of finished products. Results :

[0092] The keratin concentration measured in the product extracted by the Kjeldahl method is 170.56 mg / mL. This measurement represents the total keratinous material, regardless of its state, as explained previously.

[0093] The concentration measured by the BCA method is 52 mg / mL. This measurement represents the peptide fraction of the soluble keratin material, as explained previously.

[0094] D-Confirmation of the nature of the extracted product by mass spectrometry / MALDI-TOF

[0095] MALDI-TOF analysis is a technique for accurately determining the mass of proteins, peptides, oligonucleotides, polysaccharides, and other molecules in a sample. This technique can determine the exact mass of unknown molecules. It uses a laser to ionize these compounds, which are placed on a special type of target plate coated with a matrix material. The resulting ions are then accelerated in a time-of-flight tube and analyzed to obtain mass spectra that can be used to identify the molecules present in the sample.

[0096] The characteristics of the device used are as follows:

[0097] Instrumental FeaturesMaldi-Tof Autoflex Bruker / UltraFleXtremeBRUKER smartbeanTM -II laser, 355 nmReflectron and linear operation, positive modeAcceleration voltage 20 kVDelayed extraction sourceOperating ConditionsSampleThe sample is dissolved in water 0.1% TFAMatrix10 mg / mlDeposit1 µl of a volume mixtureMatrix:sample:adduct 10:1 or 10:10

[0098] Table 1: Characteristics of the device used for mass spectrometry / MALDI-TOF

[0099] The samples were diluted 100 times and applied in an α-cyano-4-hydroxycinnamic acid matrix with a matrix / sample volume deposition of 10:10, which is correlated by MALDI analyses. The results reveal several signals corresponding to proteins, peptides or other protein substances, identified thanks to their characteristic mass / charge ratio. They confirm the presence of small proteins with sizes less than 1000 Daltons.

Claims

Process for extracting keratin from keratin material defined by the following steps:Washing the keratin material with water in the absence of chemicalsRecovery of the washed material and dryingGrinding of said material to obtain pieces smaller than 5 mmMixing of said ground material with deionized water, applying a material weight / water volume ratio of between 300 and 800 g / LExtraction reaction in a hermetically sealed thermal reactor in at least 3 steps:20 min to 1 h at a temperature between 100°C and 160°C20 min to 1 h at a temperature between 120°C and 200°C30 min to 1 h 30 at a temperature between 140°C and 210°CRecovery of the supernatantSterilizing filtration through a membrane with a pore diameter equal to or less than 0.22 µmDrying of keratin The method of claim 1 wherein said keratin material consists of hair, fur, wool or feathers. Method according to one of the preceding claims in which the hair weight / water volume ratio is between 400 and 700 g / L. Method according to one of the preceding claims in which the steps of the extraction reaction are carried out under the following conditions:20 min to 1 h at a temperature between 120°C and 160°C20 min to 1 h at a temperature between 140°C and 190°C30 min to 1 h 30 at a temperature between 160°C and 210°C Method according to one of the preceding claims in which said keratin material consists of hair. Method according to claim 5 wherein the steps of said extraction reaction are carried out under the following conditions: 20 min to 1 h at 140°C, then 20 min to 1 h at 180°C, then 30 min to 1 h 30 at 205°C. Method according to one of the preceding claims in which said step of washing said keratin material with water comprises at least 2 washing cycles by soaking in deionized water for 30 minutes to 1 hour 30 minutes followed by rinsing with deionized water. Method according to claim 7 in which the washing steps are carried out at a temperature between 45°C and 60°C. Method according to one of the preceding claims in which an ultrafiltration step is further carried out between said recovery of the supernatant and said sterilizing filtration. Method according to one of the preceding claims in which said drying is carried out by lyophilization or spraydryer.

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