Method for extracting animal keratin from sheep's wool

Abstract

The invention relates to the field of keratin extraction methods. More particularly, the invention relates to a method for "green" extraction of animal-based keratin from sheep's wool. The aim of this industrialisable method is to produce a chemically pure hydrolysate of animal keratin, from the wool wash to the extracted hydrolysate.

Description

METHOD FOR EXTRACTING ANIMAL KERATINE FROM SHEEP'S WOOL

[0001] The invention relates to the field of keratin extraction processes. More particularly, the invention concerns a "green" extraction process for animal-derived keratin from sheep's wool. The objective of this industrializable process is to produce a chemically clean animal keratin hydrolysate, from the washing of the wool to the extracted hydrolysate. Scope of the invention

[0002] Sheep's wool is an inexpensive and readily available bioresource containing 95% to 98% protein, making it an exceptional potential source of protein for biotechnological applications, particularly due to its high keratin content. However, the solid structure of sheep's wool and its indigestibility are the main obstacles to realizing its potential as a protein source.

[0003] Due to its fibrous nature and complex structure, which gives it remarkable mechanical properties, including strength, flexibility, and durability, keratin is a very valuable protein source. 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. The cysteines in keratin have the ability to form disulfide (SS) bonds with each other, giving keratin a distinctive, stable, helical, fibrous tertiary structure.

[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] Document US20190194297 describes a process for extracting keratin from pig hair. The material is first washed in a solution containing a non-ionic detergent.

[0007] In the context of waste recovery, the use of chemical products poses a problem, particularly in terms of pollution. Disadvantages of the state of the art

[0008] 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). Furthermore, the primary goal 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 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) or plant origin.

[0010] As for plant keratin, it doesn't truly exist. In fact, keratin is only found in mammals and reptiles. So-called "plant" keratin, or "phytokeratin," is actually a structural protein of plant origin (e.g., wheat and almonds) or a protein compound derived from plants.

[0011] It would be desirable to have good quality animal-derived keratin and an industrializable process to produce it, considering a circular economy and a 100% chemically clean process.

[0012] 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 extraction of keratin from sheep's wool and the preparation of hydrolyzed keratin. Wool from sheep raised for meat is an abundant resource, with over 10,000 tons of waste generated annually in France. Currently, this material has no local market due to the relocation of the textile industry outside of France and is mostly incinerated, which has both environmental and financial costs. The process described in this invention helps to solve this problem and, in doing so, virtually eliminates any harmful impact on the environment, thus perfectly aligning with the objectives of ecological preservation and waste recovery.

[0013] Thus, the present invention relates to a process for extracting animal keratin from sheep's wool, defined by the following steps: Washing the wool in aqueous solution and in the absence of chemicals; Recovering the washed wool, wringing and drying it; Grinding the wool to obtain pieces smaller than 5 mm; Mixing the ground wool with deionized water, applying a wool weight / water volume ratio of between 0.1 and 3 kg / L; Extraction reaction in a hermetically sealed thermal reactor in 2 stages: 20 min to 2.5 h at a temperature between 130°C and 160°C; 30 min to 6.5 h at a temperature between 140°C and 210°C; Recovery of the supernatant; Sterilizing filtration through a membrane with a pore diameter equal to or less than 0.22 µm; Drying the keratin

[0014] The process developed here offers an environmentally friendly alternative to state-of-the-art processes using chemicals. By reducing the ecological footprint and eliminating the use of harmful substances, it fits perfectly within a sustainable approach. Advantages of the invention

[0015] The process of the invention allows for the extraction of keratin from sheep's wool with a high yield exceeding 80%. It provides hydrolyzed keratin, available in large quantities and at a reasonable cost, thus meeting the market demand for protein-rich ingredients. The process is distinguished by its extraction efficiency, yielding a particularly concentrated keratin, namely a keratin powder containing over 90% bioactive keratin peptides per 100g.

[0016] This process uses no chemicals, neither for washing the wool nor during the keratin extraction stage. The "extracted keratin" product is therefore free of any toxic residue and is highly natural.

[0017] The keratin obtained by the invention is of high quality, and can be used in high value-added applications such as food supplements for humans (hair, nail, or skin beauty) or animals (pet food), but also in agriculture as a biostimulant, and in biomaterials.

[0018] In vitro cytotoxicity studies have reported no cytotoxic effects of extracted keratin, suggesting that it can be used as a valuable protein ingredient to support health.

[0019] The unique aspect of this process is the natural washing of the wool before keratin extraction. "Natural treatment" refers to washing using natural ingredients. Specifically, the washing can be carried out with a mild detergent or through microbiological means. The conditions of this treatment allow for efficient and rapid cleaning and degreasing of the wool without the use of chemicals. The treated wool can then be directly subjected to thermal treatment for keratin extraction.

[0020] The process is rapid; the extraction relies on a two-step heat treatment. The extraction step typically lasts between 5 and 9 hours and is scalable for industrial production. The process allows access to a variety of sectors in terms of raw material selection, such as cosmetics, nutraceuticals, and agriculture, as well as the rapidly expanding biomaterials sector, particularly bioplastics.

[0021] This process provides a clean solution for the recovery of waste that is otherwise mostly incinerated. In France, this waste represents more than 10,000 tons, generating over 20,000 tons of CO2 per year. This chemically clean process transforms an underutilized resource into a high-value product.

[0022] The present invention therefore provides the first process for extracting high-quality, high-concentration animal keratin, which is chemically clean and industrializable, including the preparation / washing of wool by a process free of any chemical product. DETAILED DESCRIPTION OF THE INVENTION

[0023] A first object of the invention relates to a process for extracting keratin from sheep's wool comprising the steps of: Washing the wool in aqueous solution in the absence of chemicals; Recovering the washed wool, wringing and drying it; Grinding the wool to obtain pieces smaller than 5 mm; Mixing the ground wool with deionized water, applying a wool weight / water volume ratio of between 0.1 and 3 kg / L; Extraction reaction in a hermetically sealed thermal reactor in 2 stages: 20 min to 2.5 h at a temperature between 130°C and 160°C; 30 min to 6.5 h at a temperature between 140°C and 210°C; Recovery of the supernatant; Sterilizing filtration through a membrane having a pore diameter equal to or less than 0.22 µm; Drying the keratin

[0024] Washing in an aqueous solution, without the use of chemicals to clean the wool before keratin extraction, is a treatment that avoids the use of chemical reagents that could alter or denature the keratin. The absence of chemicals also prevents any trace of such products from contaminating the extracted keratin. Furthermore, this process produces no polluting waste since the extraction takes place in water after washing. This washing method ensures clean wool for keratin extraction.

[0025] Sheep's wool is coated with materials from the air (pollution, pollen, etc.), the fields, and also with suint. Suint is a waxy, greasy substance derived from sheep's fat. It is primarily composed of triglycerides, waxes, sterols, esters, squalene, and other lipophilic components. Suint is extracted from raw wool during the washing and degreasing process.

[0026] It is necessary to remove the materials present on the wool before starting the keratin extraction treatment.

[0027] Within the framework of the present invention, the washing of sheep's wool consists of a natural treatment carried out in aqueous solution in the absence of chemical products.

[0028] Two washing methods are presented as an illustration; a person skilled in the art can carry out this washing using alternative protocols.

[0029] The first method involves using a mild cleaner. Typically, the wool is incubated in a warm or hot water bath containing a mild cleaner at a temperature below 90°C. Above 100°C, the keratin structure is altered.

[0030] A "gentle cleaner" refers to a laundry detergent suitable for wool or a soap that does not damage wool and does not generate polluting waste. For example, Marseille soap, obtained through a saponification reaction, is a gentle cleaner.

[0031] Thus, washing can be carried out by incubating the wool in an aqueous solution containing a mild detergent at a temperature between 30°C and 90°C for a period of 15 minutes to 3 hours or more. Preferably, the aqueous solution is water, particularly deionized water or rainwater.

[0032] The second method consists of a microbiological treatment including incubation of the wool in an environment conducive to the natural degradation of impurities, mainly fatty acids, triglycerides, by microorganisms.

[0033] Preferably, this degradation is carried out by endogenous microorganisms present in the wool (fungi, bacteria, mites, etc.). To facilitate the delipidation and elimination of impurities, the wool is incubated in an environment conducive to the growth of fungi and other endogenous microorganisms at a temperature between 25°C and 30°C, preferably between 27°C and 30°C, for 24 to 96 hours.

[0034] Thus, washing can be carried out by incubating the wool in a culture medium adapted to the growth of endogenous microorganisms at a temperature between 25°C and 30°C for a period of 24h to 96h.

[0035] The growth medium suitable for the growth of microorganisms present in sheep's wool can be any medium known to those skilled in the art for this type of culture, including the following media:

[0036] ATCC Medium: 325 Malt Extract Agar (Blakeslee's formula)(used without sagar)

[0037] Malt Extract................................................................20.0 g

[0038] Glucose..........................................................20.0 g

[0039] Peptone.........................................................1.0 g

[0040] DI Water............................................................1000 ml ATCC Medium 336: Broth Medium

[0041] Potato Dextrose Broth (BD 254920)................... 24.0 gDI Water.......................................................... 1000.0 ml ATCC Medium: 312 Czapek's (used without agar)

[0042] NaNO3 ..............................................................3.0 g

[0043] K2HPO4 ........................................................1.0 g

[0044] MgSO4 x 7H2O ................................................0.5 g

[0045] KCl.....................................................................0.5 g

[0046] FeSO4 x 7H2O................................................0.01 g

[0047] Water................................................................900 ml

[0048] Other media, such as Sabouraud's medium (10g peptone, 20g glucose, 15g agar-agar, distilled water, 1L vitamins and growth factors, pH 6), and an aqueous medium based on corn liquor, are also suitable. They contain the nutrients necessary for the growth of microorganisms present in wool (water, glucose, amino acids, and vitamins).

[0049] Alternatively, microbiological washing of wool can be achieved by adding microorganisms to the aqueous solution that constitutes the washing medium. The aqueous solution can then provide a suitable environment for the natural degradation of impurities by the microorganisms, as described previously. The prior art describes such solutions (Adair Jennifer: "A new (yet ancient) way to clean a fleece" Spin-Off, December 17, 2013).

[0050] Thus, washing can be carried out by incubating the wool with exogenous microorganisms in a culture medium adapted to the growth of these microorganisms.

[0051] The wool is then rinsed, preferably with deionized water, between one and three times after the washing step, regardless of the method used. This rinsing can be done, for example, with gentle orbital agitation.

[0052] Preferably, washing and rinsing are repeated in order to remove lipid substances and impurities and contaminants of any kind that could affect the quality of the final product.

[0053] The water used for these washes and rinses is preferably deionized water (at pH approximately 7).

[0054] In one particular embodiment, the rinsing of the wool with water includes at least one soaking cycle in deionized water for 30 minutes to 1 hour 30 minutes at a temperature between 45 and 60°C, followed by a rinse in deionized water at a temperature between 45 and 60°C. The number of wash cycles may be 2, 3, or more, depending on the level of soiling of the wool.

[0055] In another particular embodiment, the rinsing of the wool with water comprises three cycles of 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.

[0056] After this treatment, the wool can be rinsed again with water, 1 to 3 times. After being washed, the wool is recovered, for example by spinning (centrifuge), and then dried.

[0057] Drying can be done at room temperature or by applying gentle heat (around 45°C) if you want to reduce drying time.

[0058] The dried wool is then shredded, for example using a mechanical knife grinder. The pieces of wool after shredding must be quite fine, i.e. less than 5 mm in size, on the order of 0.1 to 5 mm, preferably between 0.5 and 2 mm, around 1 mm.

[0059] For extraction, the shredded wool is mixed with deionized water. The wool weight-to-water volume ratio is between 0.1 and 3 kg / L. In a preferred embodiment, it is between 0.5 and 3 kg / L, or even between 0.8 and 2.5 kg / L, for example, by mixing 1 kg of wool with 2 L of water. This mixing can be done directly in the reactor where the extraction will take place. The material density is quite high without affecting the process efficiency. Thus, the process allows for good extraction yield, which helps reduce energy costs.

[0060] The process of extracting keratin from sheep's wool is carried out in a hermetically sealed thermal reactor, according to the following steps: First step: The sheep's wool is subjected to a temperature between 130°C and 160°C for a period of 30 minutes to 2 hours 30 minutes. Second step: The heat treatment continues at a temperature between 140°C and 210°C for a period of 2 hours 30 minutes to 6 hours 30 minutes.

[0061] The combination of these two steps results in a temperature increase that allows for a very efficient extraction of keratin in a hydrolyzed form. This hydrolysis is partial.

[0062] It is understood that the temperature of the second stage is at least 10°C higher than that of the first stage, even if the ranges shown above overlap. A person skilled in the art knows how to adjust the duration and temperature of each stage using their expertise. Equivalent results can therefore be obtained by increasing the duration and decreasing the temperature, and vice versa.

[0063] In particular embodiments of the invention, the hydrolysis reaction consists of a heat treatment composed of the following 2 steps: 30 min to 2h30 at a temperature between 130°C and 160°C then 2h30 to 6h30 at a temperature between 140°C and 200°C. 30 min to 2h at a temperature between 135°C and 150°C then 1h to 5h at a temperature between 160°C and 200°C. 20 min to 1h30 at 140°C then 30 min to 1h30 at 210°C. 30 min at 140°C then 1h at 210°C.

[0064] After extraction, the supernatant is collected. It is preferably kept 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 a chamber at 4°C.

[0065] 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 centrifuge, so as to recover the supernatant containing the keratin.

[0066] Thus, in a particular embodiment, the extracted product is centrifuged, for example, at 4000 rpm for a period ranging from 10 to 30 minutes, for example for 15 minutes, at 4°C. The centrifugation step can be repeated to ensure that all debris has been removed.

[0067] The supernatant is then purified by filtration to remove biological contaminants such as bacteria, fungi, and yeasts. To ensure sterilization, the keratin-containing supernatant is filtered through a membrane with pores 0.22 microns in diameter or less.

[0068] Optionally, ultrafiltration can be performed on the supernatant before sterilizing filtration.

[0069] 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 250 nanometers.

[0070] The recovered keratin is then dried. This drying can be carried out using any suitable technique known to those skilled in the art, such as freeze-drying, spray drying, etc.

[0071] The extract exhibits a high concentration of keratin. For example, a concentration of 139 mg / mL was measured using the BCA method. After drying, a powder is obtained with a concentration of approximately 90 g of keratin peptides per 100 g (as shown in Figure 1B).

[0072] The keratin extracted by the process according to the invention has a rich amino acid profile, which is a marker of its quality. The amino acid profiles show the presence of numerous amino acids (see comments below).

[0073] The present invention will be better understood by reading the following examples, which are provided by way of illustration and should in no way be considered as limiting the scope of the present invention. DESCRIPTION OF THE FIGURES

[0074] Aminograms of sheep's wool keratin extracted according to the process of the invention. A - after partial drying, around 85%; B - after further drying, around 95%.

[0075] : MALDI spectrum of a sample of the product extracted from sheep's wool produced by the SALSA-ICOA Platform UMR 7311 of the University of Orléans. EXAMPLES

[0076] EXAMPLE 1 - Implementation of the extraction process according to the invention 1- Washing:

[0077] Wash using a mild cleaner

[0078] The sheep's wool was washed by immersion in an aqueous solution of water at 45°C with added liquid Marseille soap. The mixture was incubated for approximately 1 hour with gentle mechanical agitation.

[0079] The wool was then rinsed in a bath of clear water at 45°C. The operation was repeated twice.

[0080] The wool was defatted by immersion and incubation under gentle mechanical agitation in a water bath at a temperature between 45 and 90°C, then rinsed with clear water.

[0081] The wool was then air-dried.

[0082] Clean wool, free from any trace of chemicals and unaltered, was thus obtained. No polluting waste was produced. Washing by microbiological treatment

[0083] The sheep's wool was obtained from local farmers in France.

[0084] In order to eliminate all dirt including suint, the 0.1 kg wool fleece was incubated under orbital shaking at a temperature between 27 and 30°C in 0.5 L in a medium of the following composition previously autoclaved at 120 °C for 20 minutes.

[0085] The culture medium adapted to the growth of endogenous microorganisms of wool (fungi, bacteria, mites…) is here based on corn extract liquor, glucose and classic minerals.

[0086] Preparation of 1 L of culture medium based on liquid Corn Steep (ROQUETTE FRERES solulys L48L) or spray-dried corn liquor: 10.0 g or 5.0 g MgSO4·7H2O, 0.5 g NaNO3, 2.0 g FeSO4·7H2O, 0.02 g KCl, 0.5 g Glucose, 30.0 g K2HPO4, 0.8 g KH2PO4, 0.4 g

[0087]

[0088] Table 1: Incubation medium for sheep's wool

[0089] The wool is incubated at 27°C under agitation (250 revolutions / min for 48 hours). 2- Rinsing

[0090] The wool is then collected and rinsed three times (1 hour per wash) with deionized water (pH approximately 7) at room temperature in stainless steel tanks under gentle orbital agitation to remove all traces of impurities. A simple filtration or quick 20-minute spin cycle is then carried out.

[0091] A disinfection step which consists of soaking the sample 3x (1h at 55°C), alternating with 3 washes with clean and deionized H2O (pH about 7) at 55°C is then carried out followed by 3 washes with deionized water (10 min / wash under simple agitation).

[0092] The wool is then wrung out and dried for two days in the open air on stainless steel trays with simple mechanical agitation, then 3 times a day / 3 hours in a forced air oven at 45°C allows the recovery of dry wool which is then ground into fine pieces (± 1mm) using a mechanical knife grinder.

[0093] This material was then used as a raw material for keratin extraction. 3- Extraction:

[0094] Keratin extraction was carried out in a 20-litre hermetic reactor. The wool-to-deionised water ratio used was 2 kg of sheep's wool to 3.2 kg of pure water.

[0095] The closed thermal reactor is equipped with gentle, continuous stirring and maintains very high pressure. No chemicals were used in the reaction, and the stirrer is fixed internally, ensuring good internal agitation of the solution during extraction.

[0096] The following temperature ranges were applied and respected:

[0097] 30 min at 140°C, then 60 min at 210°C. The reactor pressure is high under these conditions and can reach values ​​exceeding 15 bar.

[0098] The total extraction process takes approximately 7 hours, including heating, temperature holding, and cooling (4 hours). The extracted product is filtered through a 200 μm membrane. The entire material is then centrifuged twice at 4000 rpm for 30 minutes at 4°C (to further remove impurities).

[0099] Ultrafiltration was then performed: A sanitary UF NFS membrane was installed on the TIA UF / MF / NF / OI – 20E028 skid. 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, and 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.

[0100] 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."

[0101] This involves sterile filtration of the liquid extract (supernatant) through a 0.2 µm pore membrane, effectively removing biological contaminants such as bacteria, molds, and yeasts. A final volumetric concentration factor of approximately 30% was achieved, resulting in an extraction yield of 35%. The resulting solution was then spray-dried to obtain a keratin powder with a homogeneous particle size.

[0102] EXAMPLE 2: Characterization of extracted keratin A - Aminogram analysis

[0103] An aminogram was performed to obtain the amino acid profile (acid or oxidative hydrolysis for MET and Cystine) by ion chromatography / UV on a dried sample.

[0104] Ion chromatography was used to separate each amino acid, followed by detection via post-column reaction. The sample was hydrolyzed prior to analysis using HCl for 24 hours at 110°C.

[0105] The amino acid profile results are shown in Figure 1. Figure 1-A shows the presence of 15 major amino acids, while Figure 1-B shows 18 major amino acids, including cysteine ​​and methionine, two sulfur-containing amino acids. Figure 1-B confirms and complements the analysis presented in Figure 1-B (they are derived from independent analyses of separate samples). The most abundant amino acids are glutamic acid, alanine, glycine, leucine, proline, and valine.

[0106] B- BCA and Kjeldahl assay of keratinous material

[0107] As part of the optimization of the 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.

[0108] 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 that absorbs 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 keratin extraction. It thus allows for the efficient quantification 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.

[0109] The Kjeldahl method, on the other hand, relies on the chemical mineralization of nitrogenous organic compounds by hot digestion with sulfuric acid, followed by distillation and titration of the released ammonia. This allows for 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 state of aggregation or protein structure, and allows for the evaluation of the overall yield of keratin extraction.

[0110] The combined 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 precise, reliable, and comprehensive characterization of the extracted keratin material, essential for optimizing extraction processes, guaranteeing the reproducibility of results, and ensuring the quality of finished products. Results :

[0111] The concentration of keratin measured in the product extracted by the Kjeldahl method is between 280 mg / mL and 350 mg / mL. This measurement represents the total keratin material, regardless of its state, as explained previously.

[0112] The concentration measured by the BCA method is 139 mg / mL. This measurement represents the peptide fraction of the soluble keratinous material, as explained previously. C - SDS-PAGE gel analysis:

[0113] Polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed on a Mini-PROTEAN TGX 4-20% gel (12 wells, 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% SDS, 40% (w / v) glycerol, 0.02% bromophenol blue) in the presence of β-mercaptoethanol.

[0114] 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. Bio-Rad, #161-0373), which includes proteins from 10 to 250 kDa. Electron migration was performed at 250 V, followed by Coomassie blue staining.

[0115] Preliminary results show representative bands of protein molecular masses consistent with those expected: protein substances (peptides and proteins) appear in a broad band less than 10kDa (data not shown).

[0116] D-Confirmation of the nature of the extracted product by mass spectrometry / MALDI-TOF

[0117] 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.

[0118] The characteristics of the device used are as follows:

[0119] Instrumental Characteristics: Maldi-TOF: Bruker Autoflex Speed ​​TOF / TOFBRUKER Laser: 355 nm Reflectron operation, positive mode Acceleration voltage ion source 1: 19 kV Reflectron voltage 1: 21 kV Delayed extraction source: 140 ms Operating Conditions Sample The sample is dissolved in 0.1% water TFA 1 / 100 Matrix α-Cyano-4-Hydroxycinnamic acid (CHCA) Deposition Sandwich deposition (Matrix:sample:Matrix) of a volumetric mixture Matrix:sample:Matrix 1:1:1

[0120] Table 2: Characteristics of the instrument used for mass spectrometry / MALDI-TOF.

[0121] The samples were diluted 100-fold and applied to an α-cyano-4-hydroxycinnamic acid matrix using a sandwich deposition method (Matrix1:Sample:Matrix2 or CHCA1:Sample:CHCA2), which was correlated by MALDI analysis. The results revealed several signals corresponding to proteins, peptides, or other proteinaceous substances, identified by their characteristic mass-to-charge ratio. They confirmed the presence of small proteins with sizes less than 1500 Daltons (between 500 and 1500 Daltons).

Claims

A process for extracting keratin from sheep's wool comprising the steps of: Washing the wool in aqueous solution without the use of chemicals; Recovering the washed wool, wringing and drying it; Grinding the wool to obtain pieces smaller than 5 mm; Mixing the ground wool with deionized water, using a wool weight / water volume ratio of between 0.1 and 3 kg / L; Extraction reaction in a hermetically sealed thermal reactor in 2 stages: 20 min to 2.5 h at a temperature between 130°C and 160°C; 30 min to 6.5 h at a temperature between 140°C and 210°C; Recovery of the supernatant; Sterilizing filtration through a membrane with a pore diameter of 0.22 µm or less; Drying the keratin A method according to claim 1 wherein said washing is carried out by incubating said wool in an aqueous solution comprising a mild cleaner at a temperature between 30°C and 90°C for a period of between 15 min and 3 h. A method according to claim 1 wherein said washing is carried out by incubating said wool in a culture medium suitable for the growth of endogenous microorganisms at a temperature between 25°C and 30°C for a period of 24h to 96h. A method according to claim 1 wherein said washing can be carried out by incubating the wool with exogenous microorganisms in a culture medium suitable for the growth of these microorganisms. A method according to any one of claims 2 to 4, wherein said washing of said wool further comprises, after incubation of said wool, at least 2 cycles of rinsing said wool by soaking in deionized water for 30 min 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. A method according to any one of the preceding claims wherein the wool weight / water volume ratio is between 0.8 and 2.5 kg / L. 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 then 1 h at 210°C. 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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