METHOD FOR EXTRACTING ANIMAL KERATINE FROM SHEEP'S WOOL

Abstract

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.

Description

Title of the invention: METHOD FOR EXTRACTING ANIMAL KERATIN FROM SHEEP'S WOOL

[0001] The invention relates to the field of keratin extraction processes. More specifically, the invention relates to 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 its strength, flexibility, and durability, keratin is a very interesting 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) bridges with each other, which gives 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] US patent 20190194297 describes a process for extracting keratin from pig hair. The material is first washed in a solution containing a non-ionic detergent.

[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, a chemical solvent considered toxic according to REACH classification. This process takes place over 48 hours. The characteristics of the extracted material are not disclosed.

[0008] In a context of waste recovery, the use of chemical products poses a problem, particularly in terms of pollution. Disadvantages of the state of the art

[0009] 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 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 waste or during the extraction process itself, raises clear contradictions with the principles of sustainable development and waste recovery.

[0010] Keratins available on the market are either of animal (non-human) origin or of vegetable origin.

[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 or "phytokeratin" is actually a structural protein of plant origin (e.g., wheat and almonds) or a protein compound derived from plants.

[0012] It would be desirable to have good quality animal-derived keratin and an industrializable process for producing it, taking into account a circular economy and a 100% chemically clean process. 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 extraction of keratin from sheep's wool and the preparation of hydrolyzed keratin. Wool from sheep raised for meat constitutes an abundant resource, with more than 50,000 tons of waste generated annually in France. This material currently has no local market due to the relocation of the textile industry outside of France and is mostly incinerated, which has an environmental and financial cost. The process that is the subject of the present 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.

[0014] Thus, the present invention relates to a process for extracting animal keratin from sheep's wool defined by the following steps: - Washing of wool by microbiological treatment in the absence of chemicals, including incubation of the wool in a culture medium adapted to the growth of microorganisms at a temperature between 25°C and 30°C for a period of 24h to 96h; - Collection of washed wool, wringing and drying - Grinding the wool to obtain pieces smaller than 5 mm - Mixing the shredded 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 1 hour 30 min at 140°C • 30 min to 1 hour 30 min at 210°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

[0015] The process developed here offers an environmentally friendly alternative. By reducing the ecological footprint and eliminating the use of harmful substances, it fits perfectly into a sustainable approach. Advantages of the invention

[0016] The process of the invention makes it possible to extract the keratin contained in sheep's wool with a good yield. It provides hydrolyzed keratin, available in large quantities and at a reasonable cost, so as to meet the market demand for protein-rich ingredients.

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

[0018] 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.

[0019] In vitro and in vivo cytotoxicity studies have reported no cytotoxic effects of the extracted keratin, suggesting that it can be used as a valuable protein ingredient that supports health.

[0020] The distinctive feature of this process is that the wool is cleaned by a microbiological treatment prior to keratin extraction. The conditions of this treatment allow for effective and rapid degreasing of the wool without the use of any product. chemical. Wool treated in this way can be directly subjected to a thermal treatment allowing the extraction of keratin.

[0021] The process is rapid, the extraction lasts between 5 and 7 hours, and is industrializable. It allows access to various sectors in terms of the choice of raw materials, such as cosmetics, nutraceuticals and agriculture, but also the biomaterials sector, in particular bioplastics, which is experiencing strong growth.

[0022] This process provides a clean solution for the recovery of waste, which is otherwise mostly incinerated. In France, this waste represents more than 50,000 tonnes, generating over 100,000 tonnes of CO2 per year. This chemically clean process transforms an underutilized resource into a high-value-added product.

[0023] The present invention therefore makes available the first chemically clean and industrializable high quality animal keratin extraction process including the preparation / washing of wool by a microbiological process. DETAILED DESCRIPTION OF THE INVENTION

[0024] A first object of the invention relates to a process for extracting keratin from sheep's wool comprising the steps of: - Washing of wool by microbiological treatment in the absence of chemical products, including incubation of the wool in a culture medium adapted to the growth of microorganisms at a temperature between 25°C and 30°C for a period of 24h to 96h; - Collection of washed wool, wringing and drying - Grinding the wool to obtain pieces smaller than 5 mm - Mixing the shredded 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 1 hour 30 min at 140°C • 30 min to 1 hour 30 min at 210°C - Centrifugation at 4000 rpm at 4°C and recovery of the supernatant - Ultrafiltration using a specific membrane - Sterilizing filtration through a 0.2 pm pore membrane - Keratin drying

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

[0026] It is necessary to remove the matter present on the wool before starting the keratin extraction treatment. In the context of the present invention, this treatment consists of a microbiological treatment comprising incubating the wool in a medium conducive to the natural degradation of impurities, mainly fatty acids and triglycerides, by endogenous microorganisms present in the wool (fungi, bacteria, mites, etc.).

[0027] In order to facilitate the delipidation and elimination of impurities, the incubation of the wool in a medium conducive to the growth of fungi and other endogenous microorganisms is carried out at a temperature between 25°C and 30°C, preferably between 27°C and 30°C, for 24h to 96h.

[0028] 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, in particular the following media:

[0029] ATCC Medium: 325 Malt Extract Agar (Blakeslee's formula) (used without agar)

[0030] Malt Extract............................................................20.0 g

[0031] Glucose.............................................................20.0 g

[0032] Peptone.............................................................1-0 g

[0033] DIWater............................................................1000 ml ATCC Medium 336: Broth Medium

[0034] Potato Dextrose Broth (BD 254920)................24.0 g DI Water....................................................... 1000.0 ml ATCC Medium: 312 Czapek's (utilisé sans agar)

[0035] NaNO3..............................................................3.0 g

[0036] K2HPO4............................................................1.0 g

[0037] MgSO4 x 7H2O.................................................0.5 g

[0038] KC1.....................................................................0.5 g

[0039] FeSO4 x 7H20...................................................0.01 g

[0040] Water................................................................900 ml

[0041] Other media, such as Sabouraud medium (10g peptone, 20g glucose, 15g agar-agar, distilled water, IL vitamins and growth factors, pH 6), or 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).

[0042] The wool is then washed with deionized water between 1 and 3 times after the aforementioned microbiological washing step. This rinsing can be carried out under gentle orbital agitation.

[0043] Preferably, the washing includes several successive steps, in order to remove lipid substances and impurities and contaminants of any kind that could affect the quality of the final product.

[0044] The water used for these washes is preferably deionized water (at pH approximately 7).

[0045] In a first embodiment, washing the wool with water comprises at least one wash 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 wash cycles can be 2, 3 or more depending on the level of soiling of the wool.

[0046] In a second embodiment, the washing of the wool in 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 in deionized water at a temperature between 45 and 60°C.

[0047] After this treatment, the wool can be rinsed again with water, 1 to 3 times.

[0048] After being washed, the wool is recovered, for example by wringing (centrifuge), then dried.

[0049] 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.

[0050] The dried wool is then ground, for example using a mechanical knife grinder. The pieces of wool after grinding 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.

[0051] For the extraction process, the shredded wool is mixed with deionized water. The wool weight / 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 in 2 L of water. This mixing can be done directly in the reactor where the extraction will take place. The density of the material 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.

[0052] 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 2 steps, namely: 20 min to 1 hour 30 min at 140°C • 30 min to 1 hour 30 min at 210°C

[0053] The sequence of these two temperature ramp-up steps allows for a very efficient extraction of keratin in a hydrolyzed form. This hydrolysis is partial. In a preferred embodiment of the invention, the hydrolysis reaction consists of a heat treatment with the following two steps: 30 min at 140°C then 1h at 210°C.

[0054] The extract has a high concentration of keratin: 139 mg / mL. This results in a very rich amino acid profile, as shown in [Fig. 1] (and discussed below).

[0055] 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 210°C.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] The keratin extracted by the process according to the invention has a rich amino acid profile, which is a marker of its quality. Figure 1 shows this amino acid profile, which displays the presence of numerous amino acids.

[0060] 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

[0061] [Fig. 1] [Fig. 1]: Aminogram of sheep wool keratin extracted according to the process according to the invention.

[0062] [Fig.2] [Fig.2]: MALDI spectrum of a sample of the product extracted from wool sheep. EXAMPLES

[0063] EXAMPLE 1 - Implementation of the extraction process according to the invention 1- Washing:

[0064] The sheep's wool was obtained from local breeders.

[0065] 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.

[0066] 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.

[0067] [Tables 1] 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

[0068]

[0069] Table 1: Incubation medium for sheep's wool

[0070] The wool is incubated at 27°C under agitation (250 rpm for 48h).

[0071] The wool is then recovered and washed 3 times (Ih / 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 rapid 20-minute spin cycle is then carried out.

[0072] A disinfection step which consists of soaking the sample 3x (Ih 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).

[0073] 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 h 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.

[0074] This material was then used as a raw material for the extraction of keratin. 2- Extraction:

[0075] Keratin extraction was carried out in a 20-litre hermetic reactor. The wool-deionized water ratio used was 2 kg of sheep's wool to 3.2 kg of pure water.

[0076] The closed thermal reactor is equipped with gentle continuous stirring and maintains a very high pressure. No chemicals were used for the reaction, and the stirrer is fixed inside, allowing for good internal agitation of the solution during extraction.

[0077] The following temperature thresholds were applied and respected:

[0078] 30 min at 140°C, then 60 min at 210°C.

[0079] The total duration of the extraction process is approximately 7 hours, including the heating, holding, and cooling phases (4 hours). The extracted product is filtered through a 200 µm membrane. The entire material is centrifuged twice at 4000 rpm for 30 minutes at 4°C (to further remove impurities).

[0080] Ultrafiltration is then 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 in 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.

[0081] 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.”

[0082] 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.

[0083] EXAMPLE 2: Characterization of extracted keratin A - Aminogram analysis

[0084] An aminogram was carried out in order to obtain the amino acid profile (acid or oxidative hydrolysis for MET and Cystine) by ion chromatography / UV on a dried sample.

[0085] 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.

[0086] The result of the amino acid profile is shown in [Fig. 1]. Fifteen amino acids are present. The most represented and present amino acids are glutamic acid, alanine, glycine, leucine, proline, and valine. B - SDS-PAGE gel analysis:

[0087] Polyacrylamide gel electrophoresis analysis (SDS-PAGE gel) was performed on a Mini-PROTEAN TGX 4-20% gel (12 wells, 8.6 x 6.7 cm, Biorad). 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 [3-mercaptoethanol.

[0088] 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.

[0089] Preliminary results show representative bands of protein molecular masses consistent with those expected; they are being confirmed.

[0090] C - Confirmation of the nature of the extracted product by mass spectrometry / MALDI-TOF

[0091] MALDI-TOF analysis is a technique for precisely determining the mass of proteins, peptides, oligonucleotides, polysaccharides, and other molecules in a sample. This technique allows for the exact 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.

[0092] The characteristics of the device used are as follows:

[0093] [Tables 2] 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

[0094] Table 2: Characteristics of the apparatus used for mass spectrometry / MALDI-TOF.

[0095] 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. Preliminary results show several visible signals.

[0096] MALDI analysis, shown in [Fig. 2], revealed masses in the range below 10 kDa for both samples, with the most intense peak detected at 4372 Da and the highest mass at 8706 Da. No mass was detected in the 10 kDa–200 kDa range; the detected peaks were at 4372.5 Da, 5600 Da, 7663.87 Da, and 8706.7 Da.

[0097] This can be correlated with a distribution of the molecular weights of the peptides and confirms the presence of hydrolyzed keratin. These analyses confirm the presence of peptides of varying molecular weights, in size and structure, ranging from small oligopeptides to longer chains, mainly between 3 and 14 kDa (kilodaltons).

Claims

Demands

1. A process for extracting keratin from sheep's wool comprising the steps of: - Washing the wool by microbiological treatment in the absence of chemicals, including incubating the wool in a culture medium suitable for the growth of microorganisms at a temperature between 25°C and 30°C for a period of 24h to 96h;- Wool recovery and drying - Grinding of the wool to obtain pieces smaller than 5 mm - Mixing of the wool with deionized water, applying a wool weight / water volume ratio between 0.1 and 3 kg / L - Extraction reaction in a hermetically sealed thermal reactor in 2 stages: • 20 min to 1h30 at 140°C • 30 min to 1h30 at 210°C - Centrifugation at 4000 rpm at 4°C and recovery of the supernatant - Ultrafiltration on a specific membrane - Sterilizing filtration through a 0.2 µm pore membrane - Drying of the keratin;

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

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

4. 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.

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 then 1h at 210°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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