PROCESS FOR EXTRACTION OF HUMAN KERATIN FROM HAIR

Abstract

The invention relates to the field of keratin extraction processes. More particularly, the invention relates to a process for extracting keratin 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 by this extraction process and the natural keratin of the hair is more than 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 relates to a process for extracting keratin 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 by this extraction process and the natural keratin of the hair 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 them, which gives keratin a particular 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 creams and serums for skin 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 between species, 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 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 solvent chemical 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] The previously described processes do not allow obtaining a good quality keratin, namely 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 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 plant origin.

[0010] With respect to keratins of animal origin, it is preferable to have a 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, a keratin of human origin has better specificity, complementarity with our human cellular receptors. This is described in the publication WO 2019 / 209188A1. It is explained there that human keratin, extracted from hair, due to its spatial conformation, will have complete complementarity with cellular receptors in humans.

[0011] As for plant keratin, it does not exist strictly speaking. In fact, keratin is only described in mammals and reptiles. So-called "plant" keratin is in reality 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 human-derived keratin and an industrializable process for producing it. Statement of the invention

[0013] Faced with the problems set out above, the inventors have developed an innovative process which differs from those of the prior art in that it allows the recovery and valorization of keratin from hair, an abundant resource with more than 4000 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 recovery.

[0014] Thus, the present invention relates to a method for extracting human keratin from hair defined by the following steps: - Washing hair with water without chemicals - Recovering washed hair and drying it - Grinding of the said hair to obtain pieces of a size less than 5 mm - Mixing the ground hair with deionized water, applying 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 - Ultra filtration - Sterilizing filtration through a 0.2 pm pore membrane - Drying of keratin Advantages of the invention

[0015] The process which is the subject of the invention makes it possible to extract the keratin contained in human hair 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 extracted keratin.

[0016] This process does not use any chemicals, either for washing the hair or during the keratin extraction stage. The “extracted keratin” product is therefore free from 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, which makes it a source of choice for all applications in humans, whether in cosmetics or in medical devices.

[0019] In addition, the process is rapid, the extraction can be carried out in 4 hours, and is industrializable. It allows access to sectors demanding in terms of the choice of raw materials, such as cosmetics or nutraceuticals, to high-quality human-origin keratin. This high quality is demonstrated in particular by its high cysteine ​​content, namely 6% - native keratin having a cysteine ​​content of approximately 8%.

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

[0021] The present invention therefore provides the first process for extracting high-quality human keratin without chemicals and which is industrializable. DETAILED DESCRIPTION OF THE INVENTION

[0022] A first subject of the invention relates to a method for extracting keratin from human hair comprising the steps of: - Washing hair with water without chemicals - Recovering washed hair and drying it - Grinding of said hair to obtain pieces of a size less than 5 mm - Mixing the ground hair with deionized water, applying 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 - Ultra filtration - Sterilizing filtration through a 0.2 pm pore membrane - Drying of keratin

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

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

[0025] 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 between 45°C and 60°C.

[0026] Preferably, the 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.

[0027] In a first embodiment, washing the hair with water 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, washing the hair with water comprises three washing cycles 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.

[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 you wish to reduce the drying time.

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

[0033] The crushed 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 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 a good extraction yield, which reduces energy costs.

[0034] The extraction is carried out in a hermetically sealed thermal reactor so that the temperature increase is accompanied by a pressure increase which contributes to the efficiency of the extraction. The hydrolysis reaction is broken down into 3 stages, namely: • 20 min to 1h at 140°C • 20 min to 1h at 180°C • 30 min to 1h30 at 205°C

[0035] 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 [Fig.l] (and commented on 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 at least up to 16 bars when the temperature reaches 205°C.

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

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

[0039] By "ultrafiltration" is meant 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 100 nanometers.

[0040] The keratin thus recovered 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 aminogram very close to that of native keratin, which is a marker of its high quality. A biomimicry of more than 90% is observed between the keratin extracted according to the process of the invention and the keratin naturally present in the hair. [Fig. 1] shows this aminogram on which the presence of 20 amino acids can be seen. Remarkably, the quantity of cysteine / cystine present is equivalent to 75% of the quantity present in native keratin.

[0042] The present invention will be better understood from reading 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

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

[0044] EXAMPLE 1 - Implementation of the extraction method 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 (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, quick 20-minute filtration is then carried out.

[0047] A delipidation 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).

[0048] 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.

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

[0050] Keratin extraction was carried out in a 20-litre hermetic reactor. The hair-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 good internal stirring of the solution during extraction.

[0052] The following temperature levels 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 (4 hours).

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

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

[0057] 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, the 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 avoid further temperature rise.

[0058] Only the supernatant is recovered and filtered via specific filters. “Ref: MPGP002A1 Description: 0.22 qm 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”.

[0059] 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.

[0060] Ultimately 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 - Analysis of the aminogram

[0063] 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.

[0064] 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°.

[0065] The result of the aminogram is represented by [Fig.l]. 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:

[0066] Polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed on a Mini-PROTEAN TGX 4-20% gel (12 wells, 20mL / 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.

[0067] 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” (ref Biorad, #161-0373) containing proteins from 10 to 250 kDa. Migration was carried out at 250V, then staining with Coomassie blue.

[0068] Preliminary results show representative bands of protein molecular masses consistent with those expected; they are currently 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 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 covered 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 water 0.1% TFA Matrix 10 mg / ml Deposit 1 pl of a volume mixture Matrix: sample: adduct 10:1 or 10:10

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

[0074] 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. Preliminary results show several visible signals.

Claims

Claims

1. Process for extracting keratin from human hair comprising the steps of: - Washing the hair with water in the absence of chemicals - Recovering the washed hair and drying - Grinding said hair to obtain pieces smaller than 5 mm - Mixing the ground hair with deionized water, applying 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 1 h at 140°C • 20 min to 1 h at 180°C • 30 min to 1 h 30 at 205°C - Centrifugation at 4000 rpm at 4°C and recovery of the supernatant - Ultra filtration - Sterilizing filtration through a 0.2 μm pore membrane - Drying the keratin

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

3. Method according to one of claims 1 or 2 in which the hair is rinsed with deionized water between 1 and 3 times prior to said washing step.

4. Method according to one of the preceding claims in which the hair weight / water volume ratio is between 500 and 700 g / L.

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

6. Method according to one of the preceding claims in which said drying is carried out by lyophilization, or spraydryer.

Images