PROCESS FOR EXTRACTING HUMAN KERATIN FROM HAIR

Abstract

The invention relates to the field of keratin extraction processes. More particularly, the invention concerns a process for extracting human-derived keratin from hair without the use of 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 by this process and natural hair keratin is over 90%.

Description

Title of the invention: METHOD FOR EXTRACTING HUMAN KERATIN FROM HAIR

[0001] The invention relates to the field of keratin extraction processes. More particularly, the invention concerns a process for extracting human-derived keratin from hair without the use of 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 by this process and the natural keratin of hair is more than 90%. Scope of the invention

[0002] Keratin is a protein rich in cysteine-type amino acids. Cysteines have the ability to form disulfide bonds with each other, which gives keratin a particular helical and fibrous tertiary structure. Human hair is composed of approximately 8% cysteine. Keratin is highly sought after for cosmetic applications, particularly hair care products and creams and serums for topical use, 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 from one species to another, but in general, it is rich in non-essential amino acids, particularly cysteine, which plays a key role in the formation of disulfide bonds responsible for the stable structure of keratin. This structure is based on disulfide bonds and repeating amino acid motifs that vary depending on the location and specific function of keratin in the body.

[0004] Processes for extracting keratin from animal matter are known from the prior art.

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

[0006] US patent 20190194297 describes a process for extracting keratin from pig hair. The material is first washed in a solution containing a nonionic detergent. The extracted keratin has a cysteine ​​content of 2.9%.

[0007] Document WO2019 / 209188 describes a process for extracting beta-keratin from human hair using two solvents: GhCrosolan, and CutissentialBehenly 18-MEA, composed of behentrimonium methosulfate, solvent The chemical is considered toxic according to 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] The processes described above do not allow for obtaining high-quality keratin, namely keratin with a structure close to that of native keratin and which is highly purified. The use of chemical solvents to clean the material prior to extraction can leave traces in the finished product and constitutes waste that must be treated (pollution). Moreover, the main objective 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 clear contradictions with the principles of sustainable development and waste recovery.

[0009] Keratins available on the market are either of animal (non-human) origin or of vegetable origin.

[0010] Compared to keratins of animal origin, human keratin is preferable for applications in humans: it will be more effective due to its biological affinity. Indeed, it is known that additives or fixatives can be used to improve the biocompatibility of animal or plant keratin for human applications. Human keratin exhibits better specificity and complementarity with our human cellular receptors. This is described in publication WO 2019 / 209188A1. It explains that human keratin, extracted from hair, due to its spatial conformation, will have complete complementarity with human cellular receptors.

[0011] As for plant keratin, it does not exist in the strict sense. Indeed, keratin is only described in mammals and reptiles. So-called "plant" keratin is actually a structural protein of plant origin (e.g., cellulose) or a protein compound derived from plants.

[0012] It would be desirable to have good quality human-derived keratin and an industrializable process for producing it. Description of the invention

[0013] Faced with the problems outlined above, the inventors have developed an innovative process that differs from those of the prior art in that it allows the recovery and valorization of keratin from hair, an abundant resource with over 4,000 tons of waste generated annually in France. In doing so, this process virtually eliminates any harmful impact on the environment. thus aligning perfectly with the objectives of ecological preservation and waste recovery.

[0014] Thus, the present invention relates to a process for extracting human keratin from hair defined by the following steps: - Washing hair with water, without using any chemicals - Retrieval of washed and dried hair - Grinding the said hair to obtain pieces smaller than 5 mm - Mix the shredded hair with deionized water, using a hair weight / water volume ratio of between 300 and 800 g / L - Extraction reaction in a hermetically sealed thermal reactor in 3 stages: • 20 min at 1 hour at 140°C • 20 min at 1 hour at 180°C • 30 min to 1 hour 30 min at 205°C - Centrifugation at 4000 rpm at 4°C and recovery of the supernatant - Ultra filtration - Sterilizing filtration through a 0.2 pm pore membrane - Keratin drying Advantages of the invention

[0015] The process of the invention makes it possible to extract the keratin contained in human hair with a good yield, while preserving an amino acid composition close to that of native keratin. This is reflected by the high cysteine ​​content of the extracted keratin.

[0016] This process does not use any chemicals, either for washing the hair or during the keratin extraction step. The "extracted keratin" product is therefore free of any toxic residue and is highly natural.

[0017] The extracted keratin contains 20 amino acids and has a composition very close to that of native keratin, in particular due to its high cysteine ​​content.

[0018] Human keratin is more compatible with human biological processes than keratin of animal or "plant" origin, making it a source of choice for all applications in humans, whether in cosmetics or medical devices.

[0019] Furthermore, the process is rapid, with extraction taking place in 4 hours, and is scalable for industrial production. It provides access to high-quality human-derived keratin for sectors with stringent raw material selection requirements, such as cosmetics and nutraceuticals. This high quality is evidenced in particular by its high cysteine ​​content of 6%, whereas native keratin has a cysteine ​​content of approximately 8%.

[0020] This process provides a "greentech" solution for valorizing hair as waste, which is otherwise mostly incinerated. In France, hair represents approximately 4,000 tonnes, generating over 100,000 tonnes of CO2 per year. This chemically clean process transforms an underutilized resource into a high-value-added product, unique on the global market.

[0021] The present invention therefore provides the first chemical-free and industrially scalable method for extracting high-quality human keratin. DETAILED DESCRIPTION OF THE INVENTION

[0022] A first object of the invention relates to a process for extracting keratin from human hair comprising the steps of: - Washing hair with water, without using any chemicals - Retrieval of washed and dried hair - Grinding of said hair to obtain pieces smaller than 5 mm - Mix the shredded hair with deionized water, using a hair weight / water volume ratio of between 300 and 800 g / L - Extraction reaction in a hermetically sealed thermal reactor in 3 stages: • 20 min at 1 hour at 140°C • 20 min at 1 hour at 180°C • 30 min to 1 hour 30 min at 205°C - Centrifugation at 4000 rpm at 4°C and recovery of the supernatant - Ultra filtration - Sterilizing filtration through a 0.2 pm pore membrane - Keratin drying

[0023] Hair is coated with sebum and substances contained in the air (pollution, pollen, etc.) or any other matter with which it may come into contact. It is therefore necessary to wash it thoroughly before beginning the keratin extraction treatment. In the context of the present invention, this washing is done with water, without any chemicals.

[0024] Preferably, the washing includes several successive baths, in order to remove lipid substances and impurities and contaminants of any kind that could affect the quality of the final product.

[0025] The water used for these washes is preferably deionized water (at pH approximately 7). In order to facilitate the delipidation and elimination of impurities, the washes are carried out at a temperature between 45°C and 60°C.

[0026] Preferably, the hair is rinsed with deionized water between 1 and 3 times prior to the washing step. This rinsing can be done with gentle mechanical agitation.

[0027] In a first embodiment, the hair washing process comprises at least one washing cycle by soaking in deionized water for 30 minutes to 1 hour 30 minutes at a temperature between 45 and 60°C, followed by rinsing with deionized water at a temperature between 45 and 60°C. The number of washing cycles may be 2, 3, or more, depending on the level of soiling of the hair.

[0028] In a second embodiment, the washing of the hair with water comprises three washing cycles by soaking in deionized water for 30' to 1h30 at a temperature between 45 and 60°C followed by rinsing with deionized water at a temperature between 45 and 60°C.

[0029] After this treatment, the hair can be rinsed again with water, 1 to 3 times.

[0030] After being washed, the hair is recovered, for example by filtration, then dried.

[0031] Drying can be carried out at room temperature or by applying gentle heat (around 45°C) if it is desired to reduce the drying time.

[0032] The dry hair is then ground, for example using a mechanical knife grinder or a mechanical ball mill. The pieces of hair after grinding must be quite fine, i.e. less than 5 mm in size, on the order of 0.1 to 5 mm, around 1 mm.

[0033] The ground hair is mixed with deionized water. The hair weight / water volume ratio is between 300 and 800 g / L. In a preferred embodiment, it is between 500 and 700 g / L. This mixing can be done directly in the reactor where the extraction will take place. The material density is quite high without affecting the efficiency of the process. Thus, the process allows for a good extraction yield, which helps to reduce energy costs.

[0034] The extraction is carried out in a hermetically sealed thermal reactor so that the temperature rise is accompanied by a pressure rise, which contributes to the efficiency of the extraction. The hydrolysis reaction is broken down into 3 steps, namely: • 20 min at 1 hour at 140°C • 20 min at 1 hour at 180°C • 30 min to 1 hour 30 min at 205°C

[0035] These 3 steps constitute 3 temperature increases, allowing the progressive unfolding of the keratin and contributing to a highly efficient extraction. This results in a complete amino acid profile, as shown in [Fig. 1] (and discussed below).

[0036] 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.

[0037] The extraction reaction product is centrifuged at 4000 rpm for a period of 10 to 30 minutes, for example for 15 minutes, at 4°C. The supernatant containing the keratin is collected. The centrifugation step can be repeated to ensure that all debris has been removed.

[0038] The supernatant is then purified by ultrafiltration and filtered to remove biological contaminants such as bacteria, fungi, and yeasts. To ensure sterilizing filtration, the supernatant containing keratin is filtered through a membrane with pores of 0.22 µm or less.

[0039] “Ultrafiltration” refers to a membrane separation method typically used to remove suspended particles such as bacteria, yeasts, and viruses. The size of the retained particles can vary between 1 and 100 nanometers.

[0040] The recovered keratin is dried. This drying can be carried out by any suitable technique known to those skilled in the art, such as freeze-drying, spray drying, etc.

[0041] The keratin extracted by the process according to the invention has an amino acid profile very close to that of native keratin, which is a marker of its high quality. A biomimicry of over 90% is observed between the keratin extracted according to the process of the invention and the keratin naturally present in hair. Figure 1 shows this amino acid profile, which displays the presence of 20 amino acids. Remarkably, the amount of cysteine / cystine present is equivalent to 75% of the amount present in native keratin.

[0042] The present invention will be better understood upon reading the following examples, which are provided by way of illustration and shall in no way be considered as limiting the scope of the present invention. DESCRIPTION OF THE FIGURES

[0043] [Fig. 1] [Fig. 1]: Aminogram of keratin from human hair extracted according The process according to the invention versus that of native keratin. EXAMPLES

[0044] EXAMPLE 1 - Implementation of the extraction process according to the invention 1. Washing:#

[0045] Raw human hair was obtained from local hair salons.

[0046] In order to remove all dirt, this material was washed 3 times (Ih / wash) with deionized water (pH approximately 7) at room temperature in stainless steel containers The mixture is gently agitated mechanically to remove all traces of impurities. A simple, quick 20-minute filtration is then carried out.

[0047] A delipidation and disinfection step which consists of soaking the sample 3x (Ih at 55°C), alternating with 3 washes with clean and deionized H2O (pH approximately 7) at 55°C is then carried out followed by 3 washes with deionized water (10 min / wash under simple agitation).

[0048] The hair is then air-dried for two days on stainless steel trays with simple mechanical agitation 3 times a day / 3 h in a forced air oven at 45°C allows the dry hair to be recovered which is then ground into fine pieces (± 1mm) using a mechanical ball mill.

[0049] This material was then used as a raw material for the extraction of keratin. 1. Extraction:#

[0050] Keratin extraction was carried out in a 20-litre hermetic reactor. The hair-to-deionised water ratio used was 7.5 kg of hair to 12.5 L of pure water.

[0051] 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 for good internal stirring of the solution during extraction.

[0052] The following temperature thresholds were applied and respected:

[0053] 30 min at 140°C, then 30 min at 180°C, then 60 min at 205°C.

[0054] The total duration of the extraction process is approximately 7 hours including the heating, temperature maintenance and cooling phases (4h).

[0055] The extracted product is filtered through a 200 qm membrane.

[0056] The entire material is centrifuged twice at 4000 rpm for 30 min at 4°C (to remove further impurities).

[0057] Ultrafiltration is performed: A sanitary UF NFS 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 it did not exceed 50 °C. The membrane was then rinsed with distilled water (circulating within the microfiltration skid) until the permeate and retentate were neutralized to reach a pH of 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 parameters were adjusted, permeate collection began. A temperature rise of 20°C to 36°C was observed, and a cooling system for the filtration skid compressor was put in place to prevent a further temperature rise.

[0058] Only the supernatant is recovered and filtered via specific filters. “Ref: MPGP002A1 Description: 0.22 µm membrane filter for particulate and bacteria-free water at the point of dispensing for the Milli-Q® IQ, IX and EQ 7 series water purification systems.”

[0059] This involves a sterile filtration of the liquid extract (supernatant) through a 0.2 pm pore membrane, which effectively removes biological contaminants such as bacteria, molds and yeasts.

[0060] In the end, a volume concentration factor of approximately 30% was achieved for an extraction yield of 35%.

[0061] The resulting solution was then lyophilized and / or spray dried to obtain a keratin powder of homogeneous particle size.

[0062] EXAMPLE 2: Characterization of extracted keratin A - Aminogram analysis

[0063] Aminograms were carried out to obtain the amino acid profile (acid or oxidative hydrolysis for MET and Cystine) by ion chromatography / UV on a dried sample.

[0064] Ion chromatography was used to separate each amino acid, followed by detection by post-column reaction. The sample was hydrolyzed prior to analysis, using HCl for 24 h at 110°C.

[0065] The result of the amino acid profile is shown in [Fig. 1]. The presence of 20 amino acids is noted, as in native keratin. The most abundant amino acids are glutamic acid, arginine, leucine, serine, threonine, and cysteine. B - Analysis by SDS-PAGE# gel:

[0066] Polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed on a Mini-PROTEAN TGX 4-20% gel (12 wells, 20 mL / well, 8.6 x 6.7 cm, Bio-Rad). Samples were prepared according to the Laemmli 4x method (250 mM Tris-HCl, pH 6.8, 4% LDS, 40% (w / v) glycerol, 0.02% bromophenol) in the presence of β-mercaptoethanol.

[0067] The samples to be analyzed were diluted to introduce 10, 15, 20, and 25 mg / well, respectively. The molecular weight identification standard used was the "Precision Plus Protein Standard" (ref. Biorad, #161-0373), containing proteins from 10 to 250 kDa. The migration was performed at 250 V, followed by Coomassie blue staining.

[0068] Preliminary results show representative bands of protein molecular masses consistent with those expected; they are being confirmed.

[0069] C - Confirmation of the nature of the extracted product by mass spectrometry / MALDI-TOF

[0070] MALDI-TOF analysis is a technique for accurately determining the mass of proteins, peptides, oligonucleotides, polysaccharides, and other molecules in a sample. This technique allows for the precise determination of the 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.

[0071] The characteristics of the device used are as follows:

[0072] [Tables 1] Instrumental Characteristics: MALDI-TOF Autoflex Bruker / UltraFleXtreme BRUKER smartbeanTM-II laser, 355 nm. Reflectron and linear operation, positive mode. Acceleration voltage: 20 kV. Delayed extraction source. Operating Conditions: Sample: The sample is dissolved in 0.1% TFA water. Matrix: 10 mg / ml. Deposition: 1 µL of a volumetric mixture. Matrix: sample:adduct 10:1 or 10:10

[0073] Table 1: Characteristics of the apparatus used for mass spectrometry / MALDI-TOF

[0074] The samples were diluted 100-fold and applied in an α-cyano-4-hydroxycinnamic acid matrix with a matrix / sample volumetric loading ratio of 10:10, which is correlated by MALDI analysis. Preliminary results show several visible signals.

Claims

Demands

1. A process for extracting keratin from human hair comprising the steps of: - Washing the hair with water in the absence of chemicals - Recovering and drying the washed hair - Grinding the hair to obtain pieces smaller than 5 mm - Mixing the ground hair with deionized water, using a hair weight / water volume ratio of between 300 and 800 g / L - Extraction reaction in a hermetically sealed thermal reactor in 3 stages: • 20 min to 1h at 140°C • 20 min to 1h at 180°C • 30 min to 1h30 at 205°C - Centrifugation at 4000 rpm at 4°C and recovery of the supernatant - Ultrafiltration - Sterilizing filtration through a 0.2 µm pore membrane - Drying the keratin

2. A method according to claim 1 wherein said hair washing step comprises three washing cycles by soaking in deionized water for 30' to 1h30 at a temperature between 45°C and 60°C followed by rinsing with deionized water at a temperature between 45 and 60°C.

3. A method according to any one of claims 1 or 2 wherein the hair is rinsed with deionized water between 1 and 3 times prior to said washing step.

4. A method according to any one of the preceding claims wherein the hair weight / water volume ratio is between 500 and 700 g / L.

5. A process according to any one of the preceding claims wherein the steps of the extraction reaction are carried out under the following conditions: 30 min at 140°C, 30 min at 180°C and 1h at 205°C.

6. A method according to any one of the preceding claims wherein said drying is carried out by freeze-drying, or spraydryer.

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